April 2013 Volume 22, No 2 - Egyptian Journal of Medical

Transcription

Egyptian Journal of Medical Microbiology, April 2013
Vol. 22, No. 2
INDEX
Pages
Serum sFas, TNF-α and Bcl2 expression following Traumatic Brain Injury
Rawia I Badr M.D., Hatem I Badr M.D., Nabil Aly M.D., Abdelwahab Ibrahim M.D.
Bacteriological and Immunological Study in Diabetic Foot
Nabila A. Hussein; Hamed A. Deraz; Reda A.K. Salem; Ayman Abd-Elrahman M.N, Takwa
Allah M.A. Ghanem; Ola A. Hussein; Manal M. ELgerby; Hoda Abdin ; Ayman A Allam and
Naglaa A. Abd Elwahaab
Evaluation of Serodiagnosis of Tuberculosis in Comparison with Traditional Methods
Ahmed O. Shafik, Mossaad A. Morgan, Sawsan A. Youssef and Shereen H. Ahmed
New Non-Invasive Markers for Evaluation of Fibrosis in Patients with Chronic
Hepatitis C
Randa Mohamed Talaat
Evaluation of Simple Screening Tests in the Diagnosis of Non Fermentative Gram
Negative bacilli, A Prospective Study
Amal A. Wafy, (M.D.); Wageih S. Elnaghy; Ashraf Atef, (M.D.); Atef Taha; Tarek Gamil
Extended Spectrum Beta Lactamases (ESBLs) Detection in Enterobacteriaceae
According to New CLSI Guidelines
Safaa Shawky Hassan
Rapid detection of Extended Spectrum β-lactamase (ESBL) Producing Strains
of Escherichia coli in Urinary Tract Infections Patients in Benha University Hospital
Enas Sh. Khater and Hammouda W. Sherif
Expression of MMTV-like env gene in Egyptian Breast Cancer Patients
Mohamed M. Hafez, Zeinab K. Hassan Mahmoud M. Kamel, Mahmoud N. ElRouby and Abdel
Rahman N.Zekri
A retrospective study of Systemic Lupus Erythematosus (SLE) in Jazan: Clinical and
immunological overview
Maggie Reda Mesbah, Essam Atwa, Mousa Meshi
Application of Pulsed Field Gel Electrophoresis and Ribotyping as Genotypic Methods
Versus Phenotypic Methods for Typing of Nosocomial Infections Caused by
Pseudomonas aeruginosa Isolated from Surgical Wards In Suez Canal University
Hospital
Maii Ahmed Abu-Taleb; Abeer Ezzat El-Sayed Mohamed; Hassan Nasr El-Eslam; Said
Hamed Abbadi; Gehan Saddik El-Hadidy and John J LiPuma
Detection and Identification of Staphylococcus Aureus Enterotoxins in Some Milk
Products and their Handlers
Michael N. Agban and Ahmed S. Ahmed
Role of SHV Genes in Nosocomially Isolated Extended Spectrum β Lactamase Producing
Klebsiellae Pneumonia from Ventilator Associated Pneumonia among ICU Patients Using
PCR Assay
Abeer Ezzat El Sayed; Samia Ragab El Azab
Characterization and Kinetics Study for Rhamnolipids Produced by an Environmental
Pseudomonas aeruginosa isolate
M. Kassem, N. Fanaki, H. Abou-Shleib, F. Dabbous, Y. R. Abdel-Fattah2
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12-22
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41-50
51-56
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Vol. 22, No. 2
Serum sFas, TNF-α and Bcl2 expression following
Traumatic Brain Injury
Rawia I Badr M.D.1, Hatem I Badr M.D.2, Nabil Aly M.D.2, Abdelwahab Ibrahim M.D.2
1
Microbiology and medical Immunology Department , 2Neurosurgery Department,
Faculty of Medicine, Mansoura University, Mansoura, Egypt.
ABSTRACT
Background: Traumatic brain injury (TBI) still represents a leading cause of high morbidity and
mortality. Objective: This study aims to determine serum level of sFas and TNF-α and its relation to
primary outcome (survival/death) of patients following severe TBI and to show variation in Bcl-2
expression and apoptosis percentage after TBI. Patients and Methods: In this Prospective case control
study, twenty five patients who were victims of severe isolated TBI (GCS 3–8) with no indication for
surgical intervention were included in this study. Six healthy volunteers without history of TBI were
included as control group. All of them were subjected to ELISA detection of serum sFas and TNF-α level.
Flow cytometric analysis of Bcl2 expression upon PBMNC and apoptotic percentage was detected.
Results: Serum sFas, TNF-α levels and Bcl2 expression level were significantly higher in TBI patients
and in non-survivors than in control group and survivors respectively. Apoptotic cells percentage was
significantly lower in TBI patients and non-survivors than in control group and survivors respectively.
Conclusion: serum sFas, TNF-α levels can be used as prognostic markers for early selection of patients
at risk of deterioration after severe traumatic brain injury.
Key Words: Traumatic brain injury; apoptosis; soluble Fas; Bcl-2 family.
The intrinsic pathway is initiated by the release
of cytochrome c from the mitochondrial
intermembrane space into the cytosol, leading to
the activation of a cascade of caspases (cysteine
dependent aspartate-specific proteases) and
ultimately to the amplification of the apoptotic
signals.5
In contrast, the extrinsic pathway can be
triggered by engagement of cell surface
transmembrane death receptors Fas/Apo1/CD95, tumor necrosis factor receptor-1 (TNF1), DR3, DR4, and DR5 and their corresponding
ligands.6 This death-inducing signaling complex
(DISC) brings zymogen molecules into
proximity, leading to auto-activation of
caspases, the Fas ligand (FasL) binds to the Fas
receptor,
resulting
in
multimerization,
recruitment of adaptor molecules and the
formation of a caspase-activation complex. The
Fas/FasL is the key regulator of apoptosis.3
Fas receptor (APO-1, CD95) is a type I
transmembrane protein belonging to the nerve
growth
factor
(NGF)/TNF
receptor
superfamily.7 Fas may also occur in a soluble
form (sFas), devoid of a transmembrane region
that may prevent cells from undergoing FasLinduced apoptosis.8 Serum sFas can act as a
decoy for FasL, preventing signaling through
cell surface Fas, and reducing beneficial
regenerative Fas signals as well as pro-apoptotic
signals.6
INTRODUCTION
Traumatic brain injury (TBI) is a complex
process encompassing 2 distinct phases: primary
injury to brain tissue and cerebral vasculature by
virtue of the initial impact and secondary injury
including edema and subsequent brain
swelling.1
Secondary injury mechanisms involve such
diverse pathways as a profound inflammatory
response, excitatory amino acid (glutamate) and
calcium-associated cytotoxicity, or ischemic
events. All of which may lead to acute as well
as delayed progressive cell death.2 Lesions in
the nervous system induce rapid activation of
glial cells and under certain conditions
additional recruitment of granulocytes, T-cells
and monocytes/ macrophages from the blood
stream triggered by upregulation of cell
adhesion molecules, chemokine and cytokines.3
Two different types of cells are visible
following TBI. Type 1 cells show a classic
necrotic pattern that's followed the primary
brain injury, and type 2 cells show a classic
apoptotic pattern on microscopy. Apoptotic
cells have been identified within contusions in
the acute post-traumatic period, and in regions
remote from the site of injury, days and weeks
after trauma.4
Initiation of apoptotic cell death may be
achieved through activation of two distinct
pathways, referred to as intrinsic and extrinsic.
1
Following TBI there is an increased
expression of two main sets of genes. These
genes encode for the caspase family of cysteine
proteases that promote programed cell death
(PCD) and a family of genes that are
homologous to the oncogene Bcl-2.9 The
interaction between Bcl-2 family members that
promote (as Bax) and suppress (such as Bcl-2
and Bcl-xL) apoptosis determines whether cells
undergo PCD.10
Despite the burden of TBI, there are no
optimal long-term outcome predictors for head
injury. The Glasgow coma Scale (GCS) has
become the most widely used scale for assessing
outcome after head injury.11 However, the GCS
scale is increasingly recognized as having
important shortcoming. As early assessment of
patient’s brain damage may be quite difficult
during the stay at the intensive care unit (ICU),
thus, biochemical markers might help both
indicating patients at higher risk for
deterioration
and
guiding
immediate
posttraumatic therapeutic strategies.12, 13
In this study, we aim to determine whether
serum level of both sFas and TNF-α correlates
with primary outcome (survival/death) of
patients following severe TBI and to show the
change, if any, in expression of Bcl-2 and
apoptosis percentage after TBI.
Vol. 22, No. 2
samples were drawn within the first 24 hours of
ICU admission.
ELISA Detection:
One ml venous blood was collected into
tubes; the tubes were immersed into ice and
immediately transported to the laboratory for
processing. Centrifugation was done at 4500
rpm for 5 minutes at 4οC within 30 minutes of
collection, and sera were transferred to plastic
tubes and were stored at -70οC till analysis. All
clinical specimens were stored and processed
identically
to
ensure
uniformity
of
measurements. None of the samples was thawed
before analysis.
a. Measurement of TNFα was carried per
manufacture instructions; EASIA TM ELISA
Kit (Biosource, Nivelles, Belgium).
Briefly, 50 μl incubation buffers were
added to all wells, 200 μl standards, controls,
and samples were added to capture antibody
pre-coated plates and incubated for 2 hours at
room temperature. After washing with buffer,
100 μl of standard zero were added to each well,
50 μl HRP-conjugate were added and incubated
for 2 hours at room temperature. After washing,
200 μl TMB chromogenic solutions were added
to each well and incubated for 30 minutes at
room temperature. Fifty μl of stop solution were
added to each well and the absorbance was
measured at 450 nm, by using ELISA reader
Spectra 111, Austria.
b. Measurement of sFas was carried per
manufacture instructions; RayBio® Human
FAS ELISA Kit was used.
Briefly, 100 μl of Standards, samples were
added to capture antibody pre-coated plates the
wells. After 2.5 hours of incubation, wells were
aspirated and washed. One hundred μl of
Biotinylated antibody were added to each well
and incubated for one hour then, wells were
thoroughly aspirated and washed. One hundred
μl of TMB one step substrate reagent were
added to each well. After 30 minutes incubation,
50 μl of stop solution were added to each well
and the absorbance was measured at 450 nm, by
using ELISA reader Spectra 111, Austria.
Flow cytometric analysis
a .Lymphocyte separation
Five ml of freshly heparinized blood was
mixed with an equal volume of PBS, PH 7.4.
Peripheral blood mononuclear cells (PBMCS)
were separated from whole blood by FicollHypaque density gradient centrifugation. The
lymphocyte sediment was resuspended and
mixed in PBS, centrifuged again for 10 min at
1200 rpm. Fixation was done with ice cold
absolute alcohol and kept at 4OC until analysis.
PATIENTS & METHODS
This Prospective case control study was
done in Microbiology Diagnostics Infection
Control Unit (MDICU) in Microbiology and
Medical
Immunology
department
in
collaboration with Neurosurgery department in
Emergency Hospital, Faculty of Medicine,
Mansoura University. Twenty five patients who
were victims of severe isolated TBI (GCS 3–8)
without previous history of neurological or
psychiatric disease and were transferred to the
ICU within 24 h of TBI with no indication for
surgical intervention were included in this
study. Clinical variables of severe isolated TBI
include: outcome (survival/death), time period
between admission and ICU discharge and
neurological assessment using GCS at the time
of ICU discharge. Upon admission to the trauma
emergency room in emergency hospital, patients
were initially evaluated and resuscitated. All
patients were kept in supine position with 30°
head elevation, sedated and mildly mechanically
hyperventilated (to maintain PaCO2 around 35
mmHg and PaO2 around 100mmHg).
Corticosteroids were not administered. Six
healthy volunteers without history of TBI were
included in this study as control group. Blood
2
Vol. 22, No. 2
Kolmogorov–Smirnov test was performed to
check normal distribution of data. Nonparametric data were analyzed using the Mann–
Whitney U-test for continuous variables. For
correlation analysis, Spearman’s correlation
coefficients were calculated with two-tailed P
value to assess the significance of correlation.
Values of p < 0.05 were considered significant.
b. Staining of Bcl2
100 µl of cell suspension of fixed cells were
incubated with 0.1 mg of purified mouse antibcl-2 (monoclonal antibody) MoAb diluted
1/1000
conjugated
with
fluorescein
isothiocyanate isomer 1 (Clone 100; IgG1
USA), mixed well and incubated at room temp
for 30 minutes, centrifuged at 1200 rpm for 5
minutes. Cells weren’t permeabilized as this is a
surface stain. Cells were resuspended in 0.2ml
of 0.5% Para formaldehyde and data was
acquired by flow cytometer analysis.
c. DNA Staining
After 12 hour of cell fixation, 200 ul of cell
suspension was centrifuged, mixed with
propidium iodide (PI) and filtered through a 30
um pore diameter nylon mesh filter to eliminate
nuclear clumps in another 5 ml tube. The
samples were run in the flow cytometer within 1
hour after the addition of PI.14 PI was used to
determine the total DNA fragmentation as a
subG1 apoptotic cells. The flow cytometer used
in FACS caliber flow cytometer (Becton
Dickinson, Sunnyvale, CA, USA) equipped
with a compact air cooked low power 15 mwat
argon ion laser beam (488nm). The average
number of evaluated nuclei per specimen was
20,000 and the number of nuclei scanned was
120 per second. DNA histogram derived from
flow cytometer was obtained with a computer
program for Dean and Jett mathematical
analysis.15 Apoptosis was studied by staining
the sub G1 Peak by PI.16
Statistical analysis;
Data is computed with the Statistical
Package for the Social Science, Windows 98
version, USA (SPSS 10 software). Variables
with normal distribution were expressed as
mean (±SD). In these variables, the T-test was
applied for group differences. Non-parametric
data were expressed as median and 25th to 75th
percentiles
(interquartile
range).
The
RESULTS
Among TBI patients, the mean age was
29.17±11.28 years with no significant difference
than that of control group 33.83±6.56 years.
Male to female ratio was 17/8. As regard
primary outcome among TBI patients; 14 were
non-survivors while 11 were survivors;
mortality rate was 56% as shown in figure 1.
Figure (1): Comparison of primary outcome
among TBI patient
Difference between survivors and nonsurvivors as regard mean age, male/female ratio
and initial GCS upon admission was shown in
Table 1. No significant difference was detected
between them. Nine out of 11 survivors were
more severely disabled (GCS 5-6) at time of
ICU discharge.
Table (1): Characteristics of survivors and non-survivors among TBI patients
Survivors (n 11)
Non survivors (n 14)
Mean age (years)
30.12 ± 12.32
28.32 ± 11.17
Male/female ratio
7/4
10/4
Mean GCS upon admission
4.12±0.97
4.33±0.65
Mean GCS at discharge from ICU
6.41±1.14
_____
Mean time between admission and ICU discharge (days)
22.16±13.29
_____
Mean time between admission and death(days)
____
9.1±2.41
GCS: Glasgow coma Scale
3
Vol. 22, No. 2
Statistically significant increased level of TNF-α in TBI patients {median= 29.17 (25.65-31.05) pg.
/ml} than that of control group was observed {median= 15.68 (14.85-16.98) pg. /ml} (p=0.0001).
Significantly higher level was detected in non-survivors group {median= 30.85 (29.8-32.88) pg. /ml} as
compared to survivors group {median= 26.2 (22.1-28.2) pg. /ml} (p=0.0001). Values represented in
median (IQR) pg. /ml as shown in figure2.
Figure (2): Comparison of serum TNF-α concentration among all studied groups.
Serum sFas level is increased significantly in TBI group {median= 0.12 (0.1-0.13) pg. /ml} than that
of control group {median= 0.041 (0.04-0.048) pg. /ml} (p=0.0001). Statistically significantly increased
level of sFas is observed in non-survivors group {median= 0.127 (0.12-0.128) pg. /ml} than survivors
group {median= 0.096 (0.091-0.098) pg. /ml} (p=0.0001). Values represented in median (IQR) pg. /ml as
shown in figure 3.
Figure (3): Comparison of serum sFas concentration among all studied groups.
Human cells (PBMNL) stained with Bcl2 anti-apoptotic marker fluorescein isothiocyanate
(FITC), green in color show significant higher level of Bcl2 expression in TBI patients {median= 23.8 %
(13.5-40.1)} when compared to healthy control group {median=12.2 % (10.9-15.4) (p=0.001) as seen in
figure 4 (A-B) and table 2.
Table (2): Expression of Bcl2 and apoptosis percentage among TBI patients and control group.
TBI patients
Control group
P
(n=25)
(n=6)
Bcl2
23.8 (13.5-40.1)
12.2 (10.9-15.4)
0.001
Apoptosis
0.46 (0.0-15.4)
22.4 (15.3-44.2)
0.01
4
Vol. 22, No. 2
A
B
Figure (4): Flow cytometry histogram showing BCL2 expression on PBMNC in TBI patients (A)
and control group (B).
Statistically significant increase in expression of Bcl2 in non-survivors group {median=27.15 %
(23.7-40.1)} than survivors group {median= 16.2% (13.5-19)} (p=0.000) was shown in table 3. Bcl2
expression in all groups’ survivors, non survivors and control was shown in Figure 5. Values represented
in median.
Figure (5): Comparison of Bcl2 expression on PBMNC among all studied groups.
Table (3): Expression of Bcl2 and apoptosis percentage between survivors and non survivors
among TBI patients.
Survivors
Non-survivors
P
(n=11)
(n=14)
Bcl2
16.2 (13.5-19)
27.15 (23.7-40.1)
0.000
Apoptosis
3.31 (1.3-15.4)
0.09 (0.0-0.63)
0.000
DNA cell cycle analysis by modfit programs showing subG1 phase (apoptosis) stained with PI (FL2)
Fluorescent detector red in color, show significant lower percentage of apoptotic cells in TBI patients
{median= 0.46 % (0.0-15.4)} when compared to healthy control group {median= 22.4 % (15.3-44.2)}(p=
0.01) as seen in figure 6 (A-B) and table 2.
5
0
50
100
150
Channels
200
250
Vol. 22, No. 2
0
50
100
Channels
150
200
250
A
B
Figure (6): Flow cytometry histogram showing apoptotic cells in TBI patients
(A) and control group (B).
Percentage of apoptotic cells were significantly decreased in non-survivors group {median= 0.09 %
(0.0-0.63)} than that of survivors group {median= 3.31% (1.3-15.4)} (p= 0.000) as shown in table 3.
Apoptosis percentage in all groups was shown in figure7. Values represented in median.
Figure (7): Comparison of apoptosis percentage among all studied groups.
Among the TBI patients who survived (11/25) (44%), 9 were more severely disabled (GCS5-6).
Those nine patients had significant higher serum level of TNF-α and also significant lower percent of
apoptotic cells than the other 2 (GCS7-8) as shown in table 4.
Table (4): Comparison among survivors group in relation to different parameters.
TNF-α (pg./ml)
sFas (pg./ml)
Bcl2 (%)
Apoptosis (%)
More severely disabled
survivors
(n=9)
26.6
0.096
18.01
1.4
Less disabled
survivors
(n=2)
24.1
0.094
16.2
7.9
P
0.02
>0.05
>0.05
0.02
Apoptosis was negatively correlated to serum levels of both TNF-α, sFas and Bcl2 expression as
shown in Table 5 and figures 8, 9, 10.
6
Vol. 22, No. 2
Table (5): Correlation coefficient of apoptosis and different parameters in TBI patients.
R
P
TNF-α & Apoptosis
-0.71
0.000
sFas & Apoptosis
-0.63
0.001
Bcl2 & Apoptosis
-0.56
0.000
Figure (8): Scatter plot showing that apoptosis percent is negatively correlated with serum TNF-α
level in TBI patients.
Figure (9): Scatter plot showing negative correlation between apoptosis percentage and serum
sFas level in TBI patients.
7
Vol. 22, No. 2
Figure (10): Scatter plot showing that apoptosis percentage is negatively correlated with Bcl2
expression on PBMNC in TBI patients.
survivors than survivors among TBI patients
and associated low percent of apoptotic cells
particularly in non-survivors were detected.
Consequently,
a
pro-inflammatory
mechanism of TNF-α is anticipated as high
TNF-α level was correlated with poor outcome
(mortality rate 56% and 81.8% disability) and
mean time between impact and death of
(9.1±2.41) days. This reflects the importance of
serum TNF-α level as a predictor marker for
imminent events following TBI before clinical
manifestations as also reported by many
authors.19,17 Similarly, TNF-α is one of the
mediators that dramatically increased after TBI
and led to activation, proliferation and
hypertrophy of mononuclear, phagocytic cells
and gliosis.20
In the TBI, the non-survivors had
significantly higher serum levels of both sFas
and TNF-α than survivors. Moreover, survivors
who were more severely disabled (9/11) at time
of discharge, had higher serum TNF-α and sFas
level (GCS 5-6). An association between serum
sFas levels and patient death is detected.21 This
finding is clinically relevant because it might
enable us to predict impending secondary
insults after TBI before the clinical
manifestation of these events. This becomes
even more important when the timing of the
intervention is to be decided.
The current study shows that, the
expression of Bcl2 is significantly increased in
TBI group than in control group and was also
found in non-survivors more than in survivors.
DISCUSSION
Little can be done to treat or reverse the
primary injury that occurs at the time of TBI.
And as the prediction of patients which will
develop subsequent secondary insults depends
mainly upon clinical acumen; the presence of
indicators of impending secondary insults may
be useful as they allow for prevention of more
damage and earlier intervention.17
The present study reveals that, serum sFas
level was significantly increased in TBI group
than in control group and in non-survivors than
in survivors. Similarly, other studies show that
serum sFas concentrations were significantly
increased in the TBI patients when compared
with those of control group. And sFas was
measured for up to 15 days after TBI in all CSF
and serum samples collected.3, 7
In this study, blood samples were preferred
over CSF samples because of the hazards of
spinal tapping in patients with increased intra
cranial tension and the difficulty of getting CSF
samples from healthy volunteers. In addition,
blood samples are simple and safe to be
collected. Also Lenzlinger et al., 2002 7 stated
that, TBI patients’ serum concentrations of sFas
were always higher than that of CSF
concentrations.7
A controversy regarding the dual
pathophysiologic role of TNF-α in TBI is
proposed as both pro and anti-inflammatory
mechanisms have been reported.18 In this study,
significant increased level of TNF-α in non-
8
Increased expression of Bcl-2 is observed in rat
TBI models. Furthermore, Bcl-2 is induced in
neurons in vulnerable regions of brain after TBI
in rats and many neurons lacking Bcl-2 exhibit
apoptotic morphologies.22 This finding suggests
the bad prognostic role carried by increased
level of Bcl-2 in TBI patients.
Western blots detected that Bcl-2 was
minimally present in control patients, while
increased 17-fold in patients after TBI.10 The
changes in expression of Bcl-2 after TBI in
humans are consistent with experimental models
of brain injury of other published series.23,24,25,26
In contrary, several authors found that,
overexpression of Bcl-2 reduces cortical
neuronal loss after TBI.27,28,29 In the same
context; overexpression of Bcl-2 reduces
neurological tissue damage and improves motor
function in rodents after cerebral ischemia.30,31,32
As the apoptotic process is characterized by
extensive morphologic changes involving:
chromatin
condensation
and
regular
fragmentation of genomic DNA into
oligonucleosome fragments of 180 to 200 bp
units, the conventional DNA electrophoresis
technique has technical restrictions. The
technique has low sensitivity and is not suitable
for quantitative estimation. To circumvent these
shortcomings, a flow cytometric approach to
quantitate the cells undergoing apoptosis after
staining of nuclei with PI was used.
A controversy arguing whether PCD could
be a maladaptive response that exacerbates
injury or a physiological response to injury,
participating both in the remodeling of neuronal
circuits or the culling of injured or dysfunctional
cells after injury.10
In this study, percentages of apoptotic cells
were significantly decreased in TBI and nonsurvivors than in healthy control and survivors
respectively. This could be explained by the fact
that, cells undergoing apoptosis die without
membrane rupture and therefore elicit less
inflammatory reactions in contrast to the cells
undergoing necrosis. Consequently, neuronal
apoptosis after TBI may be a protective
response by the brain to remove injured tissue
cells whilst having little effect on remaining
brain tissue.33,34
The conflicting results in this study may be
due to the small number of the patients studied
and different assays used with different
sensitivity.
Regardless of major progress in cerebral
monitoring and imaging techniques, early
selection of patients at risk of deterioration after
severe TBI is difficult. So, the understanding of
secondary brain injury progression at the
Vol. 22, No. 2
cellular level may allow earlier selection of
patients on whom initiation of treatment
modalities may have a significant impact on
outcome and help guide decisions about timing
of interventions.
In conclusion, serum sFas and TNF-α are
valuable prognostic markers which can be used
for early selection of patients with increased risk
of mortality after severe TBI. Moreover, TNF-α
is found to be a promising prognostic factor for
disability after TBI as increased level of TNF-α
in the serum implies significant systemic
effects. Also it should be mentioned that serum
sFas was higher in patients with disability but
without statistical significance. So, further
studies at wider scale are recommended in order
to determine whether the sFas system is the
direct effect of the primary impact or merely the
result of the initiation of secondary injury.
Finally, we recommend that TBI patients with
increased serum level of sFas and TNF-α are
candidate for aggressive therapies such as
decompressive craniotomy, pharmacologically
induced coma or hypothermia.
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‫ ﺑﻌﺪ إﺻﺎﺑﺎت اﻟﺪﻣﺎغ اﻟﺮﺿﻴﺔ‬Bcl2 ‫ وﻧﺴﺒﺔ‬TNF-α ، sFas
‫و ﺗﻬﺪف هﺬﻩ اﻟﺪراﺳﺔ إﻟﻰ ﺗﺤﺪﻳﺪ ﻣﺴﺘﻮى‬. ‫ ( ﺗﻤﺜﻞ ﺳﺒﺐ رﺋﻴﺴﻲ ﻻرﺗﻔﺎع ﻣﻌﺪﻻت اﻟﻤﺮض واﻟﻮﻓﺎ ة‬TBI ) ‫ﻻ ﺗﺰال ﺻﺪﻣﺔ إﺻﺎﺑﺎت اﻟﺪﻣﺎغ‬
‫ وﻧﺴﺒﺔ ﻣﻮت‬Bcl-2 ‫ وﻟﺪراﺳﺔ ﻧﺴﺒﺔ‬TBI ‫ اﻟﻤﻮت( ﻓﻰ اﻟﻤﺮﺿﻰ ﺑﻌﺪ‬/ ‫ وﻋﻼﻗﺘﻪ ﺑﺎﻟﻨﺘﻴﺠﺔ اﻷوﻟﻴﺔ )اﻟﺒﻘﺎء ﻋﻠﻰ ﻗﻴﺪ اﻟﺤﻴﺎة‬TNF-α ‫ و‬sFas
‫ ( ﺑﺪون اﻟﺤﺎﺟﺔ‬GCS 3-8 ) ‫ﺷﺪﻳﺪة‬
TBI ‫ ﺗﻤﺖ هﺬﻩ اﻟﺪراﺳﺔ ﻋﻠﻰ ﺧﻤﺴﺔ وﻋﺸﺮﻳﻦ ﻣﺮﻳﺾ ﺗﻌﺮﺿﻮا ل‬. TBI ‫اﻟﺨﻼﻳﺎ اﻟﻤﺒﺮﻣﺞ ﺑﻌﺪ‬
sFas ‫ ﻟﺘﺤﺪﻳﺪ ﻧﺴﺒﺔ ال‬ELISA ‫ وﻗﺪ ﺗﻢ ﻋﻤﻞ‬.‫ آﻤﺠﻤﻮﻋﺔ ﺿﺎﺑﻄﺔ‬TBI ‫ وأدرﺟﺖ ﺳﺘﺔ ﻣﺘﻄﻮﻋﻴﻦ أﺻﺤﺎء دون إﺻﺎﺑﺔ‬.‫ﻟﻠﺘﺪﺧﻞ اﻟﺠﺮاﺣﻲ‬
‫ وﻧﺴﺒﺔ ﻣﻮت اﻟﺨﻼﻳﺎ‬PBMNC ‫ ﻓﻰ‬Bcl2 ‫ ﻟﻠﻜﺸﻒ ﻋﻦ ﻧﺴﺒﺔ‬Flow cytometric analysis ‫وأﻳﻀﺎ ﺗﻢ ﻋﻤﻞ‬. TNF-α ‫وﻣﺴﺘﻮى‬
‫ أﻋﻠﻰ ﺑﻜﺜﻴﺮ ﻓﻲ اﻟﻤﺮﺿﻰ اﻟﺬﻳﻦ ﺗﻌﺮﺿﻮا ﻟﻺﺻﺎﺑﺔ اﻟﺪﻣﺎﻏﻴﺔ و‬Bcl2 ‫ وﻧﺴﺒﺔ ﻣﺴﺘﻮى‬TNF-α ‫ و‬sFas ‫و آﺎن ﻣﺴﺘﻮى‬.‫اﻟﻤﺒﺮﻣﺞ‬
‫ﻏﻴﺮاﻟﻨﺎﺟﻴﻦ ﻋﻦ اﻟﻤﺠﻤﻮﻋﺔ اﻟﻀﺎﺑﻄﺔ و اﻟﻤﺠﻤﻮﻋﺔ اﻟﻨﺎﺟﻴﺔ ﻋﻠﻰ اﻟﺘﻮاﻟﻲ وأﻳﻀﺎ آﺎﻧﺖ ﻧﺴﺒﺔ ﻣﻮت اﻟﺨﻼﻳﺎ اﻟﻤﺒﺮﻣﺞ أﻗﻞ ﻓﻰ اﻟﻤﺮﺿﻰ اﻟﺬﻳﻦ‬
‫ ﻳﻤﻜﻦ اﺳﺘﺨﺪام ﻣﺴﺘﻮﻳﺎت‬:‫ﺗﻌﺮﺿﻮا ﻟﻺﺻﺎﺑﺔ اﻟﺪﻣﺎﻏﻴﺔ و ﻏﻴﺮاﻟﻨﺎﺟﻴﻦ ﻋﻦ اﻟﻤﺠﻤﻮﻋﺔ اﻟﻀﺎﺑﻄﺔ و اﻟﻤﺠﻤﻮﻋﺔ اﻟﻨﺎﺟﻴﺔ ﻋﻠﻰ اﻟﺘﻮاﻟﻲ اﻻﺳﺘﻨﺘﺎج‬
.‫ آﻌﻼﻣﺎت ﻣﻨﺬرﻩ ﻟﻼﺧﺘﻴﺎر اﻟﻤﺒﻜﺮ ﻟﻠﻤﺮﺿﻰ اﻟﻤﻌﺮﺿﻴﻦ ﻟﻠﺘﺪهﻮر اﻟﺸﺪﻳﺪ ﺑﻌﺪ إﺻﺎﺑﺎت اﻟﺪﻣﺎغ‬TNF-α ‫ و‬sFas
11
Vol. 22, No. 2
Bacteriological and Immunological Study in Diabetic Foot
Nabila A. Hussein; Hamed A. Deraz; Reda A.K. Salem;
Ayman Abd-Elrahman M.N, Takwa Allah M.A. Ghanem; Ola A. Hussein*;
Manal M. ELgerby*; Hoda Abdin* ; Ayman A Allam** and
Naglaa A. Abd Elwahaab***
Internal Medicine, Clinical Pathology*, Microbiology and Immunology **and
Tropical*** Departments, Faculty of Medicine, Zagazig University
ABSTRACT
Background: Diabetes mellitus is increasing rapidly through the world, so the diabetic foot syndrome
become more and more important as a major diabetic complication. Objectives: The aim of study was to
determine the association between the up-regulation of circulating level of IL-6 in diabetic patients with
foot ulcer compared with diabetic patients without foot ulcer. Subjects and methods: The study included
60 subjects, they were divided into three groups; group I included 20 diabetic patients without foot ulcer
syndrome, group II included 20 diabetic patients with diabetic foot ulcer syndrome (DFUS) and group III
included 20 apparently healthy subjects as a control. All subjects were subjected to clinical assessment,
routine laboratory investigations and specific investigations including assay of glycosylated
haemoglobin, serum IL-6 and bacteriological culture and sensitivity for ulcer. Results: There is no
significant difference among the three studied groups regarding the gender, age, duration of the disease
and type of treatment. There was significant difference between group I and other groups regarding
hypertension (p<0.02). There was no significant difference between the three studied groups regarding
WBC. There was a significant difference between the three studied groups as regards neutrophils and
platelets counts [(p=0.009) & (p=0.03)] respectively. There was a highly significant difference between
the three studied groups as regards Hb concentration (p<0.001).There was a highly significant difference
(p<0.001) between group II and both group I and group III as regards ESR (43 ± 17.2, 13±3.9, 11±3),
random blood sugar(RBS) (319.7±47, 238±47.8, 92.5±10.8), glycosylated haemoglobin (HbA1c)
(9.93±1.35, 8.83±1.4, 4.6±0.6), Urea (49.9±37, 52.5±41, 20.6±2.4) and IL-6 (18.9±5.6, 4.9±2.7,
2,77±1).There was positive significant correlations (p<0.001) between IL-6 and levels of RBS (r=0.72),
and HbA1c (r=0.62), respectively. Also, a positive significant correlation between IL-6 and neutrophils%
(r=0.35, p<0.005) was found. The most common isolated microorganisms from foot culture were mixed
gram + ve cocci and gram –ve bacilli representing 60% and lonely gram + ve cocci and gram –ve bacilli
were 20% respectively. Also, it was found that the most gram +ve organism was Staphylococcus aureus
and the most gram –ve organism was E. coli and the most effective antibiotic was Ampicillin-Sulbactam
70% followed by Imipenem 30%. Conclusion: Diabetic patients with foot ulcer were found to have higher
IL-6 level than diabetic patients without foot ulcer and they were prone to complications or mortality.
This assay could facilitate early and accurate diagnosis and greatly aid timely institution of appropriate
treatment.
Keywords: IL-6, diabetic foot ulcer syndrome (DFUS), and HbA1c.
The status of the immune system may be
relevant at several stages in the development of
chronic wounds. Immune activation may
precede the incidence of a diabetic foot ulcer in
the same way that it precedes the manifestation
of type II DM (4, 5).
Interleukin-6 (IL-6) is a multi-functional
cytokine regulating humoral and cellular
responses and playing a central role in
inflammation and tissue injury(6).
The aim of this study was to demonstrate if
acute foot ulcers are associated with an upregulation of circulating level of IL-6 compared
with diabetic patients without foot ulcer.
INTRODUCTION
Diabetes mellitus (DM) is increasing
rapidly through the world, and the diabetic foot
syndrome become more and more important as
a major diabetic complication(1).The life-time
risk of a diabetic patient to develop chronic
diabetic wound has been estimated to reach 1525% (2).
Despite considerable international efforts,
foot ulcers continue to be responsible for high
number of lower- limb amputation that are
associated with substantial decrease in quality
of life and increase risk of mortality(3).
13
Vol. 22, No. 2
thioglycollate broth and Robertson's cooked
meat media anaerobically. In addition, the
specimens from patients with cellulitis and
gangrene were inoculated into lysed blood
agar with vitamin K, neomycin blood agar
and egg yolk agar anaerobically. The
bacterial isolates were identified according
to standard bacteriological methods.
Antibiotic sensitivity was done to bacterial
isolates by disc diffusion method (10).
Assay of glycosylated haemoglobin (HbA1c):
Whole blood was mixed with a lysing
reagent containing a detergent and borate ions.
A preparation of haemolyzed whole blood was
mixed with a weakly binding cation-exchange
resin. The non-glycosylated haemoglobin
(HbAO) binds to the resin leaving (HbA1) free
to be removed by means of resin separator in the
supernate. The percent of HbA1 was determined
by measuring the absorbance values at 415 nm
of the HbA1 fraction and of the total Hb
fraction.
Assay of serum IL-6 (AviBion Human IL-6
ELISA
kit,
Orgenium
Laboratories,
Finland):
Human IL-6 ELISA is in vitro enzymelinked immunosorbent assay for the quantative
measurement of human IL-6 in serum. The
assay employs an antibody specific for human
IL-6 coated on 96-well plate. Standards,
samples and biotinylated anti-human IL-6 were
pipetted into the wells and IL-6 present in a
sample is captured by the antibody immobilized
to the wells and by biotinylated IL-6 specific
detection antibody. After washing away
unbound
biotinylated
antibody,
HRPconjugated steptavidin is pipetted to the wells.
The wells were again washed. Following this
second wash step, TMB substrate solution was
added to the wells resulting in color
development proportional to the amount of IL-6
bound. The stop solution changes the color from
blue to yellow, and the intensity of the color is
measured at 450 nm. The concentrations of IL-6
were extrapolated from a standard curve
constructed from the results of the provided
standard dilutions and corresponding optical
densities.
Statistical analysis:
Data were entered, checked and analyzed
using Epi-Info version 6 and SPP for Windows
version 19. Data were expressed as mean for
quantitative variable numbers and percentages
for categorical variables. Analysis of Variance
(ANOVA) was used for comparison of means of
more than two groups. Fisher's Least Significant
Difference (LSD) test was used to compare the
mean of one group with the mean of another.
SUBJECTS & METHODS
This study was carried out at Internal
Medicine, Clinical Pathology, Microbiology and
Immunology and Tropical Departments,
Zagazig University Hospital. Sixty subjects
were included in the study (40 diabetic patients
and 20 healthy controls). They were divided
into three groups as follows:
Group I: This group included 20 diabetic
patients without foot ulcer with a mean age of
54.4 + 6.5 years. They were 6 males and 14
females.
Group II: It included 20 diabetic patients with
foot ulcer with a mean age of 54.7 + 6.6 years.
They were 10 males and 10 females.
Group III: It included 20 apparently healthy
subjects taken as controls with a mean age of
51.4+5.7 years. They were 10 males and 10
females. They matched well with patients as
regards age and sex.
Written consent from was obtained from
every subject before taking samples after
explaining investigations done for them.
All subjects were subjected to the following:
Clinical assessment:
1- Full medical history.
2- Physical local examination by inspection of
the foot and palpation of peripheral pulses.
Assessment of ulcer according to University of
Texas (7).
Laboratory investigations for all the subjects
including:
1. Complete blood count (CBC) by (SysmexKX 21N-Sysmex Corporation).
2. Erythrocyte sedimentation rate (ESR) by
Westergen method.
3. Random blood sugar (RBS) and Kidney
function tests by Dimension RXL MAX
autoanalyzer (Seimens Medical Solution
Diagnostics, UL, USA).
4. Glycosylated haemoglobin (HbA1c) by
Fast Ion-Exchange Resin Separation
Method(8)
5. Interleukin-6 (IL-6) by Enzyme-Linked
Immunosorbent Assay (EL1SA) using
(AviBion human IL-6 EL1SA kit) (9).
6. Bacteriological culture and sensitivity from
patients with diabetic foot ulcer. At least
three swabs were collected from the ulcers
of every patient. A gram stained smear of
the specimen was examined. The specimens
were cultured on blood agar and
MacConkey agar aerobically and on
Choclate agar at 10% Co2 for 24 hours at
37 ºC.
A swab was inoculated into
14
Vol. 22, No. 2
Table (7-e) shows the LSD of RBS, there
was a highly significant a highly significant
increase in group I and group II compared to
group III (p<0.001).
while, there was a
significant difference between group I when
compared to group II (p<0.01).
Table (7-f) shows the LSD of HbA1c, there
was a highly significant increase in group I and
group II compared to group III (p<0.001)
while, there was a significant difference
between group I when compared to group II
(p<0.01).
Table (5) shows that the most common
isolated microorganisms from foot culture in
group II were both gram +ve cocci and gram –
ve bacilli representing 60%. The most common
isolated gram +ve organism was Staphylococcus
aures (20%) and gram –ve organism was E.coli
(20%). The most active antibiotic was
Ampicillin-Sulbactam 70% followed by
Imipenem 30%.
Macroscopic characters of ulcer show that
45% of ulcers are rounded in shape while 55%
are rounded and irregular, with 1-6 cm in
diameter. As regard edge 80% are punched
while 20% are slooping, with 55% of ulcers
have inflated margins while 45% are
hyperinflated. The stages and grades of ulcers
were as follow: 15% stage A, 35% stage B, 20%
stage C, 30% stage D, 60% grade1, 25% grade 2
and 15% grade 3 (not shown in tables).
Table (6) shows that there was a highly
significant difference between the three groups
as regards IL-6 (p<0.001).
Table (7-g) shows the LSD of IL-6, there
was a highly significant increase in group I and
group II compared to group III (p<0.001) while
while, there was a significant difference
between group I when compared to group II
(p<0.01).
Figure (1) shows significant positive
correlation (p<0.05) between mean value of IL6 and mean value of neutrophils in all studied
groups (r=0.35).
Figure (2) and figure (3) show highly significant
positive correlation (p<0.001) between mean
value of IL-6 and mean value of (RBS and
HbA1c) in all studied groups (r=0.72) (r=0.62)
respectively.
Correlation between variables was done using
correlation coefficient “r”. This test detects if
the change in one variable was accompanied by
a corresponding change in the other variable or
not. Data that is not normally distributed was
presented as median. Student t test was used
when comparing two means. X2 (chi-squared)
(test of significance) is used for difference
between two or more qualitative variable. p <
0.05 was considered statistically significant.
RESULTS
Table (1) shows no significant difference
among the three studied groups regarding the
gender, age, duration of the disease and type of
treatment. Hypertension was more significant in
group I compared to other groups, (p <0.02).
Table (2) shows there was no significant
difference between the three groups as regards
WBC. There was a significant difference
between the three groups as regards Neutrophils
% and platelets counts [(p=0.009) & (p=0.03)]
respectively. There was a highly significant
difference between the three groups as regards
Hb concentration (p<0.004).
Table (7-a) shows the LSD of neutrophil %
was significantly increased in group II
compared to group I and group III (p<0.05),
while there was no significant difference
between group I and group III.
Table (7-b) shows the LSD of Hb
concentration, there was significant increase in
group II compared to group III (p< 0.05), while
there was no significant difference between
group I compared to group II and group III .
Table (7-c) shows the LSD of platelets,
there was a significant increase in group I
compared to group II and group III (p< 0.05),
between group II and group III.
Table (3) shows there was a highly
as regards ESR(p<0.001).
Table (7-d) shows the LSD of ESR, there
was a highly significant increase in group II
compared to group I and group III (p< 0.001),
between group I and group III.
Table (4) shows there was a highly
as regards RBS and HbA1c (p<0.001).
15
Demographic data
Vol. 22, No. 2
Table (1): Demographic data and clinical assessment of the studied groups.
Group I
Group II
Group III
Test
significance
(n=20)
(n=20)
(n=20)
Gender
Male
Female
Age (years)
X ± SD
Range
Duration of the disease
X ± SD
Range
Type of treatment
Insulin
Oral hypoglycemic drugs
Hypertension
6
14
30%
70%
10
10
50%
50%
10
10
50%
50%
of
P
X2 = 2.17
0.33
NS
54.4 ± 6.5
40-62
54.7 ± 6.6
45-65
51.4 ± 5.7
42-65
F = 1.66
0.19 NS
19.3 ± 6.6
2-30
22.7 ± 5.6
15-35
-
t = 1.72
0.09 NS
20
0
1
0
0
1
X2 = 0.53
0.46 NS
18
2
6
90%
10%
30%
100%
0%
5%
0%
0%
5%
2
X = 7.21
0.02 Sig.
Table (2): Comparison between the three studied groups as regarding mean values of CBC.
Group I
Group II
Group III
Parameters
F
P
(n=20)
(n=20)
(n=20)
WBC/10(3) cmm
X ± SD
7.6 ± 1.1
7.97 ± 2.1
6.85 ± 1.2
2.5
0.08 NS
Range
5.6-9.5
5.1-13
5-9.1
ab
Neutrophil %
X ± SD
63.3 ± 6
67.6 ± 6.5
61.4 ± 6.5
5.08
0.009 S
Range
49.8-71.5
50.8-78.2
48.7-68.1
Hb gm/dl
B
X ± SD
11.8 ± 1.3
10.2 ± 1.6
12.4 ± 1.8
9.5
<0.001HS
Range
9-14
6.8-13.2
9.7-15
Platelets /10(3)cmm
A
C
X ± SD
250.2 ± 7.6
334.6 ± 207
327 ± 49.6
KW =
0.03 S
6.81
Range
90-350
53-750
270-420
Median
252
308
320
Table (3): Comparison between the three studied groups as regarding mean values of ESR.
Group I
Group II
Group III
.ESR
KW P
(n=20)
(n=20)
(n=20)
13 ± 3.9
43 ± 17.2 a b
11 ± 3
33.5
X ± SD
8-22
10-72
7-16
0.001 H.S
Range
12
42
10
Median
Table (4): Comparison between the three studied groups as regarding mean values of RBS and HbA1c.
Group I
Group II
Group III
F
P
(n=20)
(n=20)
(n=20)
ab
C
RBS mg/dl
X ± SD
238 ± 47.8
319.7 ± 47
92.5 ± 10.8
172.6
0.001
H.S
Range
190-350
250-420
75-110
Ab
C
HbA1c%
X ± SD
8.83 ± 1.4
9.93 ± 1.35
4.6 ± 0.6
111.3
0.001
H.S
Range
7-12.9
8.2-12.5
3-5.5
16
Vol. 22, No. 2
Table (5): Number, Percentage (%) of isolated microorganisms and drug sensitivity in group II, or
diabetic patients with foot ulcer.
Group II (n = 20)
No
%
Gram
4
20
+ve cocci
4
20
-ve bacilli
12
60
Both
Organism
4
20
Staphylococcus aureus
4
20
E.coli
10
50
Staph. aureus & E.coli
2
10
Staph. aureus &Pseudomonas
Sensitive organisms
14
70
To Ampicillin-Sulbactam
6
30
To Imipenem
Table (6): Comparison between the three studied groups as regarding mean values of IL-6.
Group I
Group II
Group III
P
IL-6
KW
(n=20)
(n=20)
(n=20)
0.001
4.9 ± 2.7
18.9 ± 5.6 a b
2.77 ± 1 c
42.69
X ± SD
H.S
1.18-11.84
8.2-30.51
0.59-4.11
Range
4.65
18.65
3.2
Median
Table (7): LSD of neutrophil, Hb, Platelets, ESR, RBS, HbAlc and IL-6.
Group I
a) LSD of Neutrophil
Group II
< 0.05 S
Group III
NS
b) LSD of Hb
Group II
NS
Group III
NS
c) LSD of Platelets
Group II
< 0.05 S.
Group III
< 0.05 S
d) LSD of ESR
Group II
< 0.001 H.S
Group III
NS
e) LSD of RBS
Group II
< 0.01 S
Group III
< 0.001 H.S
f) LSD of HbA1c
Group II
< 0.01 S
Group III
< 0.001 H.S
g) LSD of IL-6
Group II
< 0.01 S
Group III
< 0.001 H.S
LSD: a= group II vs group I
b= group II vs group III
c= group III
17
Group II
< 0.01 S
< 0.05 S
NS
< 0.001 H.S
< 0.001 H.S
< 0.001 H.S
< 0.001 H.S
Vol. 22, No. 2
90
80
Neutrophil (%l)
70
60
50
40
30
20
r = 0.35, P<0.05
10
0
0
5
10
15
20
25
30
35
IL-6 (pg/ml)
Figure (1): Correlation coefficient (r) and its statistical significance (p)
between mean value of IL- 6 and mean value of Neutrophil in all studied groups.
450
400
RBS (mg/dl)
350
300
250
200
150
100
r = 0.72, P<0.001
50
0
0
5
10
15
20
25
30
35
IL-6 (pg/ml)
between mean value of IL- 6 and mean value of RBS in all studied groups.
14
12
HbA1C (%)
10
8
6
4
2
r = 0.62, P<0.001
0
0
5
10
15
20
25
30
35
IL-6 (pg/ml)
between mean value of IL- 6 and mean value of HbA1c in all studied groups.
18
Vol. 22, No. 2
the control of diabetes in the previous three
months.
This study revealed that both gram+ve
cocci particulary (Staph. aures) and gram –ve
bacilli particulary (E-coli) were the most
common isolated microorganisms in tissue
culture in our patients (group II). This was in
consistent with Lipsky(21), as he found infections
in DFUS were usually polymicrobial,
predominantly comprising aerobic gram +ve
cocci. Gradner and Frante, (22) found that
Staphylococcus aureus was the most common
pathogen found in chronic, non healing DFUS.
Also this previous result was in consistent
with a study showed that infection in patients
who had recently received antibiotics and also
who had deep limb-threating infection or
chronic wounds were usually caused by a
mixture of aerobic gram positive particulary
Staphylococcus aureus, Facultative anaerobic
gram negative (eg. Escherichia coli, proteus
species, klebsiella species), and anaerobic
organisms (23).
This sudy revealed that the most
effective antibiotic was Ampicillin –Sulbactem
followed by imipenem. Many studies of
antibiotic therapy in diabetic foot infection had
been performed, without demonstrating a clear
superiority for any
regimen.
The 2004
guidelines of the infectious disease society of
America list 13 acceptable regimens for
moderate diabetic foot infections Lipsky et
al.(24).
John (25) found the useful single drug
regimens include Ampicillin –Sulbactem,
Pipracillin – Tozobactam, Levofloxacin or
Cefoxitin. For the Penicillin –allergic patients,
Clindamycin and Ciprofloxacin were a useful
combination. Empric Vancomycin should be
reserved for patients with a history of
methicillin- resistant staphylococcus aureus
(MRSA), treatment failure, or severe infection.
Also, he reserved Carbapenems such as
Imipenem-Cilastatin, for more severe infections.
Finally, we described an association
between IL-6 with the severity of ulceration and
grade of infection considering University of
Texas grades.
This study demonstrated that the serum IL6 level in diabetic patients with ulcer was
significantly higher compared to diabetic
patients without ulcer and healthy subjects. This
was in agreement with Weigelt et al. (11). Also
this was in consistent with a study showed that a
parallel up-regulation of IL-6, CRP and
fibinogen were biologically plausible as IL-6
increase the release of acute-phase proteins (26).
Also our results came in accordance with the
DISCUSSION
This study showed no significant difference
in demographic data & clinical assessment
among the studied groups regarding age, sex,
duration of the disease and the type of treatment
except the hypertension which was higher in
diabetic patients without foot ulcer and this
result is consistent with Weigelt et al. (11). On
the other hand, contradictory study showed that
hypertension was associated with appearance of
diabetic foot ulcer (12).
In this study, no significant difference was
found in WBC count between the three studied
groups. This was in agreement with the result of
previous study (13) where patients with moderate
or severe diabetic foot infection presented with
a normal WBC. However, Talebi-Taher et al. (14)
showed that WBC and CRP could be helpful
parameters
in
diagnosing diabetic foot
infection. Also another study showed that
leucocytosis were considered a diagnostic
marker for inflammatory and infectious diseases
(15)
. The insignificant WBC count in this study
may be due to early administration of antibiotics
by the patients.
The current study found that there was a
significant difference in neutrophil % and
platelets count between the three groups while
there was significant decrease in haemoglobin
level in diabetic foot group compared to normal
control. In agreement with these results,
previous study (16) showed that baseline levels of
white blood cell count, polymorphonuclear
leucocyte count, platelets count and decreased
haemoglobin levels were associated with
amputation risk in diabetics with foot ulcers.
In the current study we found that ESR was
higher in diabetic patients with foot ulceration
than diabetic patients without foot ulceration.
Also Fleischer et al. (16, 17) showed that ESR and
CRP levels increased in the presence of
infection and they were the two potentially
valuable biochemical markers. Our resuts
disagreed with Elias and Domurat, (18) who
found that ESR may be elevated in diabetic
patients in the absence of infection.
It has been found that RBS and HbA1c
levels were higher in patients with diabetic foot
ulcer than those without ulcer. This was
matched with a study conducted on 60 diabetic
patients (19). The inadequate control of diabetes
exposes the patients to various complications of
diabetes. A study done by Manda et al. (20)
showed that fasting and postprandial sugar
levels were more in patients without foot ulcers
than those with foot ulcers. HbA1c levels are
more accurate than blood glucose as it reflects
19
result of a study done by Tuttolomondo et al. (27)
who measured serum levels of adiponectin,
resistin and IL-6 in 34 diabetic patients with
foot ulcer and 37 diabetic patients without foot
ulcer. Tthey found that diabetic subjects with
foot ulcer had higher level of IL-6, resistin and
lower adiponectin plasma levels than in diabetic
patients without foot ulcer.IL-6 is a
proinflammatory cytokine. Pro and antiinflammatory processes in different phases of
wound healing so disturbance of the immune
systeminterfere with tissue homeostasisand
wound healing after the manifestation of
ulcersand lead to the chronicnon healing
wounds that are the characteristic of diabetic
foot syndrome(27). On the contrary to our
results, Alexandraki et al. (28) did not find any
significant difference between 167 type 1
diabetic patients and control group as regards
IL-6.
The results of the present study showed a
positive significant correlation between IL-6
with RBS and HbA1c. This was in agreement
with a study found that the level of HbA1c at
baseline showed a significant positive
correlation with high sensitive CRP and IL-6 in
the diabetic group (29). However, contradictory
study did not find significant correlation
between the inflammatory markers (CRP, IL-6,
TNF) and the values of HbA1c (30).
We found that there was a positive
significant correlation between IL-6 and
neutrophil. This result agrees with Asensi et al.
(31)
they found a clear correlation between
higher serum IL-6 level and longer neutrophil
survival.
CONCLUSION
Diabetic subjects with diabetic foot showed
higher IL-6 level in comparison with diabetics
without diabetic foot. There is a significant
positive IL-6 correlation in subjects with
diabetic foot with immuno-inflammatory and
metabolic markers and this correlation further
underline the relationships with inflammatory
background. This study showed the predictive
positive role of IL-6 plasma level towards
diabetic foot presence. The study recommends
to advice uncontrolled diabetic patients to check
IL-6 for its role as expecting factor for
development of inflammatory reaction which
lead to diabetic foot. Also the level of HbA1c
should be checked as there is a positive
correlation between HbA1c and IL-6.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
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‫‪Egyptian Journal of Medical Microbiology, April 2013‬‬
‫‪Vol. 22, No. 2‬‬
‫دراﺳﺔ ﺑﻜﺘﺮﻳﻮﻟﻮﺟﻴﺔ وﻣﻨﺎﻋﻴﺔ ﻓﻰ اﻟﻘﺪم اﻟﺴﻜﺮى‬
‫ﻧﺒﻴﻠﺔ ﻋﻠﻲ ﺣﺴﻴﻦ*‪ ،‬ﺣﺎﻣﺪ ﻋﺒﺪ اﻟﻌﺰﻳﺰ دراز*‪،‬رﺿﺎ ﻋﺒﺪ اﻟﻤﻨﻌﻢ ﺳﺎﻟﻢ*‪ ،‬أﻳﻤﻦ ﻋﺒﺪ اﻟﺮﺣﻤﻦ ﻣﺤﻤﺪ*‪،‬‬
‫ﺗﻘﻮي اﷲ ﻣﺤﻤﺪ ﻋﺒﺪ اﻟﻨﺒﻲ*‪،‬ﻋﻼ ﻋﻠﻲ ﺣﺴﻴﻦ**‪ ،‬ﻣﻨﺎل ﻣﺤﻤﺪ اﻟﺠﺮﺑﻲ**‪ ،‬هﺪي ﻋﺎﺑﺪﻳﻦ ** ‪،‬‬
‫أﻳﻤﻦ ﻋﺒﺪ اﻟﺮﺣﻤﻦ ﻋﻼم***‪ ،‬ﻧﺠﻼء ﻋﺒﺪ اﻟﻤﻨﻌﻢ ﻋﺒﺪ اﻟﻮهﺎب****‬
‫ﻗﺴﻢ اﻟﺒﺎﻃﻨﺔ اﻟﻌﺎﻣﺔ*‪ ،‬ﻗﺴﻢ اﻟﺒﺎﺛﻮﻟﻮﺟﻴﺎ اﻷآﻠﻴﻨﻴﻜﻴﺔ**‪ ،‬ﻗﺴﻢ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻴﺎ و اﻟﻤﻨﺎﻋﺔ***‪ ،‬ﻗﺴﻢ اﻷﻣﺮاض‬
‫اﻟﻤﺘﻮﻃﻨﺔ****‪ ،‬آﻠﻴﺔ اﻟﻄﺐ اﻟﺒﺸﺮي – ﺟﺎﻣﻌﺔ اﻟﺰﻗﺎزﻳﻖ‬
‫ﻣﻘﺪﻣﺔ اﻟﺒﺤﺚ ‪:‬ﻣﺮض اﻟﺴﻜﺮ ﻳﺘﺰاﻳﺪ ﺑﺴﺮﻋﺔ ﻓﻰ ﺟﻤﻴﻊ اﻧﺤﺎء اﻟﻌﺎﻟﻢ وﺑﺎﻟﺘﺎﻟﻰ ﻓﺈن اﻟﻘﺪم اﻟ ﺴﻜﺮى ﺗ ﺼﺒﺢ اآﺜ ﺮ واﻷآﺜ ﺮ اهﻤﻴ ﺔ ﻷﻧﻬ ﺎ‬
‫ﻣﻦ أهﻢ ﻣﻀﺎﻋﻔﺎت ﻣﺮض اﻟﺴﻜﺮ اﻟﺮﺋﻴﺴﻴﺔ‪.‬‬
‫اﻷهﺪاف‪:‬دراﺳﺔ ﻣﺪي ارﺗﺒﺎط ﺗﻘﺮﺣﺎت اﻟﻘﺪم اﻟﺤﺎدة ﻣﻊ ارﺗﻔﺎع ﻣﺴﺘﻮى اﻻﻧﺘﺮﻟﻮآﻴﻦ ‪ ٦‬ﺑﺎﻟﺪم ﻣﻘﺎرﻧﺔ ﻣﻊ ﻣﺮﺿﻰ اﻟ ﺴﻜﺮ دون ﺗ ﺎرﻳﺦ‬
‫ﻗﺮﺣﺔ اﻟﻘﺪم ‪.‬‬
‫اﻟﻤﺮﺿﻰ وﻃﺮق اﻟﺒﺤﺚ‪ :‬ﺷﻤﻠﺖ اﻟﺪراﺳﺔ ﻓﻰ ﺿﻮء هﺬا اﻟﺒﺤﺚ ﺳﺘﻮن ﺷﺨ ﺼًﺎ )‪ ٤٠‬ﻣ ﻦ ﻣﺮﺿ ﻰ اﻟ ﺴﻜﺮي ‪ ٢٠ ،‬ﻣ ﻦ اﻻﺻ ﺤﺎء(‪،‬‬
‫ﺗﻢ ﺗﻘﺴﻴﻤﻬﻢ إﻟﻰ ﺛﻼث ﻣﺠﻤﻮﻋﺎت‪:‬‬
‫اﻟﻤﺠﻤﻮﻋﺔ اﻷوﻟﻰ‪ :‬ﺷﻤﻠﺖ ‪ ٢٠‬ﻣﺮﻳﻀًﺎ ﺑﺎﻟﺴﻜﺮ دون ﻗﺮﺣﺔ ﺑﺎﻟﻘﺪم‪.‬‬
‫اﻟﻤﺠﻤﻮﻋﺔ اﻟﺜﺎﻧﻴﺔ‪ :‬ﺷﻤﻠﺖ ‪ ٢٠‬ﻣﺮﻳﻀﺎ ﺑﺎﻟﺴﻜﺮ ﻣﻊ ﻗﺮﺣﺔ ﺑﺎﻟﻘﺪم‪.‬‬
‫اﻟﻤﺠﻤﻮﻋﺔ اﻟﺜﺎﻟﺜ ﺔ‪ :‬ﺗ ﺸﻤﻞ ‪ ٢٠‬ﺷﺨ ﺼًﺎ أﺻ ﺤﺎء وﺗﻌ ﺮض ﺟﻤﻴ ﻊ اﻟﻤﺮﺿ ﻰ إﻟ ﻰ ﺗ ﺴﺠﻴﻞ اﻟﺘ ﺎرﻳﺦ اﻟﻤﺮﺿ ﻰ اﻟﻜﺎﻣ ﻞ واﻟﻔﺤﻮﺻ ﺎت‬
‫اﻟﺮوﺗﻴﻨﻴﺔ واﻟﻔﺤﻮﺻﺎت اﻟﺨﺎﺻﺔ وﺗﺸﻤﻞ ﻗﻴﺎس ﻧﺴﺒﺔ اﻻﻧﺘﺮﻟﻮآﻴﻦ ‪ ٦‬ﺑﺎﻟﺪم وﻧﺴﺒﺔ اﻟﻬﻴﻤﻮﺟﻠﻮﺑﻴﻦ اﻟﺴﻜﺮى ﺑﺎﻟﺪم‪.‬‬
‫اﻟﻨﺘﺎﺋﺞ‪ :‬ﻋﺪم وﺟﻮد اﺧﺘﻼف اﺣﺼﺎﺋﻰ ﺑﻴﻦ اﻟﻤﺠﻤﻮﻋﺎت ﻓﻰ اﻟﻌﻤﺮ واﻟﻨﻮع واﻟﻤﺪة اﻟﺰﻣﻨﻴﺔ وﻧ ﻮع اﻟﻌ ﻼج ﻟﻤ ﺮض اﻟﺒ ﻮل اﻟ ﺴﻜﺮي‪،‬‬
‫اﺧﺘﻼف آﺒﻴﺮ ﻓﻲ ارﺗﻔﺎع ﺿﻐﻂ اﻟﺪم ﻓﻲ اﻟﻤﺠﻤﻮﻋﺔ اﻷوﻟﻰ ﺑﺎﻟﻤﻘﺎرﻧﺔ ﺑﺎﻟﻤﺠﻤﻮﻋﺎت اﻷﺧﺮى‪.‬‬
‫ﺑﻌﺪ اﺟﺮاء اﻟﺘﺤﻠﻴﻞ اﻻﺣﺼﺎﺋﻰ ﻟﻠﻨﺘﺎﺋﺞ ﺗﻢ اﺳﺘﺨﻼص اﻻﺗﻰ ‪:‬‬
‫ ﻋﺪم وﺟﻮد اﺧﺘﻼف اﺣﺼﺎﺋﻰ ﻓﻲ آﺮات اﻟﺪم اﻟﺒﻴﻀﺎء ﻓﻲ ﺟﻤﻴﻊ اﻟﻤﺠﻤﻮﻋﺎت اﻟﻤﺪروﺳﺔ و آﺎن هﻨﺎك زﻳﺎدة آﺒﻴﺮة ﻓﻲ اﻟﺨﻼﻳ ﺎ‬
‫اﻟﻤﺘﻌﺎدﻟ ﻪ واﻟ ﺼﻔﺎﺋﺢ اﻟﺪﻣﻮﻳ ﺔ ﻓ ﻲ اﻟﻤﺠﻤﻮﻋ ﺔ اﻟﺜﺎﻧﻴ ﺔ ﺑﺎﻟﻤﻘﺎرﻧ ﺔ ﺑﺎﻟﻤﺠﻤﻮﻋ ﺎت اﻷﺧ ﺮى وﻋﻠ ﻲ اﻟﻌﻜ ﺲ هﻨ ﺎك ﻧﻘ ﺺ آﺒﻴ ﺮ ﻓ ﻲ‬
‫ﺗﺮآﻴﺰ اﻟﻬﻴﻤﻮﺟﻠﻮﺑﻴﻦ ﻓﻲ اﻟﻤﺠﻤﻮﻋﺔ اﻟﺜﺎﻧﻴﻪ ﺑﺎﻟﻤﻘﺎرﻧﺔ ﺑﺎﻟﻤﺠﻤﻮﻋﺎت اﻷﺧﺮى‪.‬‬
‫ زﻳﺎدة آﺒﻴﺮة ﻓﻰ ﻣﺴﺘﻮى ﺳﺮﻋﺔ اﻟﺘﺮﺳﻴﺐ ﺑﺎﻟﺪم ﻓﻲ اﻟﻤﺠﻤﻮﻋﺔ اﻟﺜﺎﻧﻴﺔ ﺑﺎﻟﻤﻘﺎرﻧﺔ ﺑﺎﻟﻤﺠﻤﻮﻋﺎت اﻷﺧﺮى‪.‬‬
‫ زﻳ ﺎدة آﺒﻴ ﺮة ﻓ ﻲ ﻣ ﺴﺘﻮى اﻟ ﺴﻜﺮ اﻟﻌ ﺸﻮاﺋﻰ واﻟﻬﻴﻤﻮﺟﻠ ﻮﺑﻴﻦ اﻟ ﺴﻜﺮى ﺑﺎﻟ ﺪم ﻓ ﻲ اﻟﻤﺠﻤﻮﻋ ﺔ اﻟﺜﺎﻧﻴ ﺔ ﺑﺎﻟﻤﻘﺎرﻧ ﺔ ﺑﺎﻟﻤﺠﻤﻮﻋ ﺎت‬
‫اﻷﺧﺮى‪ ،‬زﻳﺎدة آﺒﻴﺮة ﻓﻲ ﻣﺴﺘﻮى اﻟﻴﻮرﻳﺎ واﻟﻜﺮﻳﺎﺗﻴﻨﻴﻦ ﻓﻲ اﻟﻤﺠﻤﻮﻋﺔ اﻟﺜﺎﻧﻴﺔ ﺑﺎﻟﻤﻘﺎرﻧﺔ ﺑﺎﻟﻤﺠﻤﻮﻋﺎت اﻷﺧﺮى‪.‬‬
‫ زﻳﺎدة آﺒﻴﺮة ﻓﻲ ﻣﺴﺘﻮى اﻧﺘﺮﻟﻮآﻴﻦ ‪ ٦‬ﻓﻲ اﻟﻤﺠﻤﻮﻋﺔ اﻟﺜﺎﻧﻴﺔ ﺑﺎﻟﻤﻘﺎرﻧﺔ ﺑﺎﻟﻤﺠﻤﻮﻋﺎت اﻷﺧﺮى‪.‬‬
‫ وﺟﻮد ﻋﻼﻗﺔ ﻃﺮدﻳﺔ ﺑﻴﻦ ﻣﺴﺘﻮى اﻧﺘﺮﻟﻮآﻴﻦ ‪ ٦‬وﻣﺴﺘﻮى اﻟﺴﻜﺮ اﻟﻌﺸﻮاﺋﻰ واﻟﻬﻴﻤﻮﺟﻠﻮﺑﻴﻦ اﻟﺴﻜﺮى ﺑﺎﻟﺪم‪.‬‬
‫ وﺟ ﻮد ﻋﻼﻗ ﺔ ﻃﺮدﻳ ﺔ ﺑ ﻴﻦ ﻣ ﺴﺘﻮى اﻧﺘﺮﻟ ﻮآﻴﻦ ‪ ٦‬وﻣ ﺴﺘﻮى اﻟﺨﻼﻳ ﺎ اﻟﻤﺘﻌﺎدﻟ ﻪ وﻻ ﺗﻮﺟ ﺪ ﻋﻼﻗ ﺔ ﺑ ﻴﻦ ﻣ ﺴﺘﻮى اﻧﺘﺮﻟ ﻮآﻴﻦ ‪٦‬‬
‫وﻣﺴﺘﻮى اﻟﺼﻔﺎﺋﺢ اﻟﺪﻣﻮﻳﺔ‪.‬‬
‫ آﻤﺎ ﻳﻠﻌﺐ ﻋﺎﻣﻞ اﻻﻟﺘﻬﺎب واﻟﻌﺪوى دورا هﺎﻣﺎ ﻓﻲ اﻧﺘﻈﺎم ﻧﺴﺒﺔ اﻟﺠﻠﻮآﻮز ﺑﺎﻟﺪم وﺧﺎﺻ ﺔ ﻋ ﺪوى اﻟﻘ ﺪم ﺣﻴ ﺚ ﺗ ﻢ ﻋﻤ ﻞ ﻣ ﺴﺤﺔ‬
‫ﻣﻦ اﻟﺠﺮح ﻻﻇﻬﺎر اﻟﻤﻴﻜﺮوب اﻟﻤﺆﺛﺮ واﻟﻤﻀﺎدات اﻟﺤﻴﻮﻳﺔ اﻟﺤﺴﺎﺳﺔ ﺿﺪ هﺬا اﻟﻤﻴﻜﺮوب اﻟﻤ ﺆﺛﺮ و آﺎﻧ ﺖ اآﺜ ﺮ اﻟﻤﻴﻜﺮوﺑ ﺎت‬
‫اﻟﻤﺘﺴﺒﺒﺔ هﻰ اﻟﻤﻜﻮرات اﻟﻌﻨﻘﻮدﻳﺔ اﻟﺬهﺒﻴﺔ واﻟﻴﺸﻴﺮﺷﻴﺎ آﻮﻻي‪ .‬و آﺎن اﻷﻣﺴﻠﻠﻴﻦ ﺳﻠﺒﻜﺘﺎم و اﻻﻳﻤﻴﺒﻨ ﻴﻢ ه ﻲ اﻷآﺜ ﺮ ﻓﺎﻋﻠﻴ ﺔ ﻓ ﻲ‬
‫اﺧﺘﺒﺎر اﻟﺤﺴﺎﺳﻴﺔ ﻟﻠﻤﻀﺎدات اﻟﺤﻴﻮﻳﺔ‪.‬‬
‫اﻟﺨﻼﺻﺔ‪ :‬ﺗﻌﺒﺮ هﺬﻩ اﻟﻨﺘﺎﺋﺞ ﻣﺆﺷﺮًا ﻋﻠﻰ وﺟ ﻮد ارﺗﺒ ﺎط ﺗﻘﺮﺣ ﺎت اﻟﻘ ﺪم اﻟ ﺴﻜﺮى اﻟﺤ ﺎدة ﻣ ﻊ ارﺗﻔ ﺎع ﻣ ﺴﺘﻮى اﻻﻧﺘﺮﻟ ﻮآﻴﻦ ‪ ٦‬ﺑﺎﻟ ﺪم‬
‫ﻣﻘﺎرﻧﺔ ﻣﻊ ﻣﺮﺿﻰ اﻟﺴﻜﺮ دون ﺗﺎرﻳﺦ ﻗﺮﺣﺔ اﻟﻘﺪم‪ .‬و‪ -‬ﻧﻨﺼﺢ ﻟﻤﺮﺿﻰ اﻟﺴﻜﺮ ﺑﺎﻟﺪم ﺑﻔﺤﺺ ﻧﺴﺒﺔ اﻻﻧﺘﺮﻟﻮآﻴﻦ ‪ ٦‬وذﻟﻚ ﻟﺪورﻩ ﻓﻰ‬
‫ﺗﻮﻗﻊ ﺣﺪوث اﻟﺘﻬﺎﺑﺎت اﻟﻘﺪم اﻟﺴﻜﺮي‪ .‬آﻤﺎ ﻳﺠﺐ اﻳﻀﺎ ﻣﺘﺎﺑﻌﺔ ﻣﺴﺘﻮى ﻧﺴﺒﺔ اﻟﻬﻴﻤﻮﺟﻠﻮﺑﻴﻦ اﻟﺴﻜﺮى ﻓﻲ ﺣ ﺎﻻت ﻣﺮﺿ ﻰ اﻟ ﺴﻜﺮﻏﻴﺮ‬
‫اﻟﻤﻨﻀﺒﻂ ﻟﻮﺟﻮد ﻋﻼﻗﺔ ﻃﺮدﻳﺔ ﺑﻴﻦ ﻧﺴﺒﺔ اﻟﻬﻴﻤﻮﺟﻠﻮﺑﻴﻦ اﻟﺴﻜﺮى واﻻﻧﺘﺮﻟﻮآﻴﻦ‪٦‬‬
‫‪22‬‬
Vol. 22, No. 2
Evaluation of Serodiagnosis of Tuberculosis in Comparison with
Traditional Methods
Ahmed O. Shafik, Mossaad A. Morgan, Sawsan A. Youssef and Shereen H. Ahmed
Microbiology and Immunology Department - Benha Faculty of MedicineBenha University
ABSTRACT
Tuberculosis is an enormous tool of morbidity and mortality. The vast majority of tuberculosis patients
live in developing countries, where the diagnosis of tuberculosis relies on the identification of acid-fast
bacilli on unprocessed sputum smears using conventional light microscopy. Microscopy has high
specificity in tuberculosis-endemic countries, but modest sensitivity which varies among laboratories
(range 20% to 80%).. Thus, the development of rapid and accurate new diagnostic tools is imperative.
Immune-based tests are potentially suitable for use in low-income countries as some test formats can be
performed at the point of care .In the present study, the diagnostic value of 16-kDa and 38- kDa
mycobacterial antigens was investigated in patients who were diagnosed as open pulmonary tuberculosis.
The humoral immune response was analysed in a group of 60 TB patients, and in control group
consisting of 15 healthy volunteers and 15 subjects with pulmonary diseases other than TB. The
sensitivity, specifity, positive predictive value and negative predictive value of the test were determined at
45%, 93.3%, 93.1% and 45.9%, respectively. In conclusion, the ELISA test has a very good specifity and
an acceptable sensitivity and positive predictive value. It is thought that it could be used in combination
with other methods to increase diagnostic accuracy, especially for culture-negative tuberculosis cases,
which are difficult to diagnose.
Assays based on immunological responses
to M. tuberculosis are preferred to the current
bacteriologic methods of TB diagnosis because
they do not depend on the detection of
mycobacteria only. In recent years, the detection
of antibodies in clinical specimens is getting
increasing attention because: (1) a strong
antibody response is mounted during M.
tuberculosis infection; (2) antibody detection
does not require living cells, unlike assays based
on cell-mediated immunity; and (3) serological
methods for detection of mycobacterium
tuberculosis antigens can be simple, rapid,
inexpensive and relatively non-invasive.(4)
The 38 kDa antigen (also called antigen 5
or antigen 78) is a lipo glycoprotein antigen of
M. tuberculosis. It is one of the most important
antigens of M. tuberculosis. This antigen
induces B- and T-cell responses with high
specificity for tuberculosis and is considered a
prime candidate for the development of new
diagnostic
reagent
for
diagnosis
of
tuberculosis.(5)
The 16-kDa antigen is a cytosolic
regulatory protein (virulence factor); specific to
the M. tuberculosis complex; and is essential for
the survival of the bacilli in the hostile
environment of the host, particularly during
latency. The antigen is undetectable during
exponential growth of M. tuberculosis, but it is
INTRODUCTION
Globally, tuberculosis (TB) accounts for
approximately nine million new cases and three
million deaths every year. Tuberculosis is a
disease of poverty, with 90% of the cases
occurring in developing countries. (1)
Some of the disadvantages of traditional
diagnostic techniques such as detection of acidfast bacilli (AFB) are the lack of sensitivity and
the isolation and growth of the tuberculous
bacillus in culture media are the length of time
(sometimes growth takes several weeks). Recent
molecular biology techniques have made it
possible to diagnose TB in a few hours, but they
are expensive and not readily available in most
hospitals in developing countries. In addition,
these molecular biology techniques have low
sensitivity particularly in smear- and culturenegative patients with pulmonary TB. (2)
The search for rapid and reliable diagnostic
tests for active TB based on the examination of
sputum, blood and other clinical specimens has
been the focus of a number of studies. In
particular, the detection of TB by serological
methods has been a subject of great interest,
particularly with regard to patients who are
unable to produce adequate sputum, and those
who
are sputum smear negative, or are
suspected of having extrapulmonary TB. (3)
23
overproduced during the stationary phase, as
well as in adverse conditions, such as, oxygen
deprivation, nutrition depletion, low pH or
accumulation of toxic by-products. (4)
This study aims at comparison between
traditional methods in TB diagnosis and a
serodiagnostic method based on detection of
lgG antibodies directed against specific
mycobacterium tuberculosis antigens (38 kDa
antigen &16 kDa antigen) using a commercially
available ELISA kit to evaluate its sensitivity
and specificity.
Vol. 22, No. 2
III- Culture:
1. Löwenstein-Jensen (LJ) medium: The
isolated
bacterial
colonies
were
identified by:
• Ziehl Neelsen stain.
• Nitrate reduction test.
• Niacin production test.
2. Manual Mycobacteria Growth Indicator
Tube (MGIT).
B. BLOOD SAMPLES:
They were used to detect lgG antibodies
directed against specific mycobacterium
tuberculosis antigens (38 kDa antigen &16kDa antigen) using commercially available
ELISA kit (PATHOZYME TB COMPLEX
PLUS, Omega Diagnostics)
Methods:
A- Collection of the sputum samples:
Early morning sputum sample was
collected by asking the patient to cough deeply
in a sterile wide screw capped container. Three
successive early morning sputum samples can
be collected if the first sample show negative
staining results.
B- Collection of the serum samples:
A Sample of venous blood was withdrawn
by empty vacutainer and allowed to clot and
retract in a water path at 37° for 15-30 minutes.
Then the clotted blood sample was centrifuged
and clear serum was collected in an eppendorf
tube and stored at –20°C till the time of testing.
C- Decontamination and Concentration;
Sputum processing by 4% NaOH method.(6)
D- Staining:
The smears prepared from the concentrated
specimens were stained by ZiehI-Neelsen
Staining technique according to (Darrow,
1948)(7). Smears were graded according to the
number of AFB per microscopic field
according to (American Thoracic Society,
1981)(8)
E- Culture of sputum samples:
After decontamination, liquefaction and
concentration of the specimens; culture of
sputum samples were carried on:
LJ media according to Kastle and Kubica(9)
The bacterial isolates were identified
according to the following criteria:
a. Rate of growth:
The organisms that grow after 7 days
incubation were considered slow growers.
SUBJECTS, MATERIAL &
METHODS
The study was done at Microbiology &
Immunology Department of Benha Faculty of
Medicine from March (2011) to August (2012)
on 60 patients attending the outpatient clinic of
Benha chest hospital and inpatient Zagazig
Chest hospital. The patients were selected
according to the clinical, radiological and
laboratory data that diagnose them as having
open pulmonary tuberculous infection. Those
with past history of TB, on antituberculous
treatment, HIV positive and new cases not
willing for informed consent were excluded. 30
control subjects were included and consisted of
(15) patients who were admitted to the hospital
with pulmonary diseases other than TB. The
diseases included were pneumonia, chronic
obstructive
pulmonary
disease
and
bronchiectasis. In addition, (15) healthy
voluntary blood donors who came for blood
donation at the Blood Bank of
Benha
University Hospital and neither had history of
any notable infection in the past 2 years, nor had
symptomatic tuberculosis in the lifetime.
All patients under study were subjected to:
• Full history taking including age, sex,
occupation, family and past history of
tuberculosis and history of intake of
antituberculous drugs.
• Clinical examination.
• Radiological examination: plain x-ray
(postero-anterior and lateral views).
SAMPLES:
Early morning sputum sample and blood
samples were collected from shared patients and
the control group and subjected to the
following:
A. SPUTUM SAMPLES:
I- Decontamination and Concentration.
II- Staining: Ziehl Neelsen stained smear.
(10)
b. Niacin accumulation test (11):
A positive test for niacin was indicated by
the appearance of a yellow color in the
test culture and no color in the control
tube.
24
Vol. 22, No. 2
performed according to the manufacturer’s
instructions. In brief, diluted (1:50) serum was
distributed in microtitre wells and incubated for
60 min at 37uC. Unbound serum was removed
by washing with a buffer solution. The wells
were subsequently incubated with peroxidaselabelled antihuman conjugate at 37uC for 30
min. After another wash cycle, peroxidase
substrate
tetramethylbenzidine
containing
hydrogen peroxide was added to the wells and
the colorimetric reaction was prolonged for 15
min in the dark at 37uC until stop reagent was
added. Absorbance values at 450 nm were
recorded. Four standards (with 2, 4, 8 and 16
serounits/ml) were provided to generate a semilog reference curve. Because the sera were
diluted 1:50, the units extrapolated from the
curve were multiplied by 50 to obtain serounits
for result interpretation. According to the
manufacturer’s instructions, a result was
considered positive when the level of antibodies
in a sample was higher than 200 serounits/ml.
absorbance of each well was measured, at
450nm,
using
STAT
FAX-2100
Microplate Reader. Results were expressed as
the number of serological units of specific IgG
per mL
c. Nitrate reduction test (12):
A positive nitrate test was indicated by
the appearance of a blue color in the top
portion of the strip.
Manual Mycobacteria Growth Indicator
Tube (BBL MGIT) (Becton Dickinson,
BD):2 supplements were added to the MGIT
tube before inoculation; BBL MGIT OADC
enrichment (Becton Dickinson, BD) and
MGIT PANTA antibiotic mixture (Becton
Dickinson, BD)
The media were inculated and readed according
to the manufacturer instructions incubated
tubes were incubated at 37°C and examined
daily in a 365nm wavelength UV light source
for orange fluorescence up to 8 weeks of
incubation. When a tube was found to be
positive for bacterial growth, a portion of the
tube content was removed and used to prepare
two smears, one for ZN staining and one for
Gram staining. If AFB were present in the ZN
smear, the tube content was subcultured onto a
slant of LJ medium. If organisms grew on
subculture and were identified as M.
tuberculosis, the BBL MGIT culture was
considered a true positive. However, if AFB
were not seen but the Gram stained smear
showed other bacteria or fungi, the BBL MGIT
culture was considered contaminated.
F- Serological test; the PATHOZYME-TB
Complex Plus (Omega Diagnostics):
In the PATHOZYME-TB Complex Plus
kit, the 38-kDa antigen, which is obtained by
recombinant technology, is mixed with the 16kDa recombinant protein. The test was
RESULTS
There was no statistical significant
difference between study and control groups as
regards age and sex, table (1).
Table (1): Comparison between the study (tuberculous) and control group as regards age and sex.
TB cases
Control group
Test of
p- value
(n=60)
(n=30)
significance
36+11.7
38.5 ± 6.5
1.1
>0.05 (0.27)
Age (mean ± SD)
20 / 33.3%
8 / 26.7%
Sex
Female
0.42
>0.05 (0.52)
(No/%)
40 / 66.7%
22 / 73.3%
Male
Out of the sixty studied patients; 40 (66.7%) were males and 20 (33.3%) were females. Their age
ranged from 19-56 years. The highest rate of tuberculosis was 18 (30%) cases in the age group 25-34
years, table (2).
Table (2): Age group distribution of pulmonary tuberculosis in the studied patients
Age group
Number
(years)
16
15-24
18
25-34
10
35-44
10
45-54
6
55-64
total
60
25
%
26.6%
30%
16.6%
16.6%
10%
100%
Vol. 22, No. 2
As regarding the occupational distribution of the studied patients out of 60 studied patients 42 (70%)
were manual workers, 12 (20%) housewives and 6 (10%) employers.
By analysis of the collected data from the TB cases and controls for some host-related factors for
tuberculosis it was found that there is a significant statistical association between TB infection and
smoking, positive family history& diabetes,table (3).
Table (3): Host-related factors for tuberculosis: comparison of TB cases and controls.
TB cases (60)
Control (30)
Yes
No
Unknown
Yes
No
Unknown
n(%)
n(%)
n(%)
n(%)
n(%)
n(%)
32
28
7
23
Smoking
(53.3) (46.6)
(23.3) (76.7)
28
17
43
2 (6.7)
Family history of TB
(93.3)
(28.3) (71.7)
34
26
8
22
Diabetes
(56.7) (43.3)
(26.7) (73.3)
19
11
27
9 (30)
22 (3.7) 2 (6.7)
Anemia
(63.3)
(18.3)
(45)
4
26
58
Immunosuppressive ttt
2 (3.3)
(13.3) (86.7)
(96.7)
X2
p
6.2
<0.05
4.4
<0.05
6.1
<0.05
3.5
>0.05
1.8
>0.05
All the collected sputum samples form the 60 patints with open pulmonary TB were positive for acid
fast bacilli . All of them were positive when cultured on LJ media and 54 (90%) were positive by MGIT,
while no growth occurred on LJ media from the control samples but 4 (13.3%) were positive by MGIT,
table (4).
Table (4): LJ culture and MGIT results of the sputum samples of the tuberculous and control groups
LJ culture
MGIT
Positive
Negative
Positive
Negative
n(%)
n(%)
n(%)
n(%)
Tuberculous
60 (100)
0 (0)
54 (90)
6 (10)
group (n=60)
Control group
0 (0)
30(100)
4 (13.3)
26 (68.7)
(n=30)
Total
60 (66.7)
30 (33.3)
58 (64.4)
32(35.6)
(n=90)
The mean detection time on LJ media was 28.3 ± 6.3 and that of manual MGIT was 11.9 ± 3.2 and this
difference is statistically significant, table (5).
Table (5): Mean detection times of mycobacterium tuberculosis on LJ media and manual MGIT
LJ media
MGIT
t
p
Detection time
28.3 ± 6.3
11.9 ± 3.2
18.01
<0.001
(mean ± SD)
(days)
Out of the total sputum samples (90), 54 were positive by MGIT and LJ culture , 26 were negative
by both, 4 were negative by LJ culture only and 6 were negative by MGIT only. Using LJ culture as the
gold standard, the sensitivity and specificity of BBL MGIT were 90% and 86.7% respectively. Its
Positive predictive value (PPV) was 93.1% and its Negative predictive value (NPV) was 81.3%, table (6).
26
Vol. 22, No. 2
Table (6): Results of manual MGIT versus LJ culture of the sputum samples of the tuberculous and
control groups
L J culture
Total
Cases(n=60)
Control (n=30)
Positive
Negative
Positive
54
4
58
MGIT
Negative
6
26
32
Total
60
30
90
Antibody levels against 38-kDa and 16-kDa mycobacterial antigens were detected higher than the
cut-off level in the serum of 27(45%) out of the 60 TB cases and in 2 (6.6%) of the control cases,table (7).
Table (7): Results of Pathozyme TB complex plus test (ELISA test) for both TB patients and control
group.
Seropositive
Total
Seronegative n(%)
n (%)
n(%)
Tuberculous group
27 (45%)
33 (55%)
60 (100%)
(n=60)
30 (100%)
2 (6.6%)
28 (93.3%)
Control group (n=30)
Out of the 60 tuberculous cases 33 (55%) cases were seronegative(<200 U/ml),20 (33.3%) cases
were low positive (200-450U/ml) and 7 (11.6%) cases were high positive (> 450 U/ml), figure (1)
From the 30 control subjects only 2 (6.6%) were seropositive(>200 U/ml) and 28 (93.3%) were
seronegative (<200 U/ml), figure (2)
Figure (1): Values of antibody titres for 38-kDa and 16-kDa mycobacterial antigens in the sera of the
studied TB cases(n=60) by the Pathozyme TB complex plus test (ELISA test)
27
Vol. 22, No. 2
Figure (2): Values of antibody titres for 38-kDa and 16-kDa mycobacterial antigens in the sera the
control group (n=30)by the Pathozyme TB complex plus test (ELISA test)
Out of the total serum samples (90), 29 were seropositive and 61 were seronegative. Using LJ
culture as the gold standard, the sensitivity and specificity of Pathozyme TB complex plus test were 45%
and 93.3% respectively. Its Positive predictive value (PPV) was 93.1% and its Negative predictive value
(NPV) was 45.9%, table (8).
Table (8): Results of Pathozyme TB complex plus test (ELISA test) versus LJ culture for both TB
patients and control group.
LJ
Total
Cases (n=60)
Control (n=30)
Positive
Negative
Positive
27
2
29
ELISA
Negative
33
28
61
Total
60
30
90
technique is the most commonly used method
for the rapid diagnosis of TB(13). However, its
sensitivity is suboptimal, as ZN staining is
unlikely to detect samples with <5000
bacilli/ml(14).
In addition, direct microscopic examination
of ZN-stained smears is time-consuming,
especially for low-loaded M. tuberculosis slides.
The development of a simple and inexpensive
test that compares favourably with conventional
procedures in terms of sensitivity would
DISCUSSION
M. tuberculosis remains a major infectious
cause of death in developing countries and has
re-emerged in industrialized countries. TB
control depends on the improved and early
detection of the disease. Rapid identification of
bacilli-positive patients, who are the most potent
source of M. tuberculosis transmission in the
community, is therefore highly recommended.
Direct microscopic examination of clinical
samples by Ziehl-Neelsen (ZN) staining
28
therefore be very helpful in the diagnosis of
TB.(15)
In our study the mean detection times of
mycobacterium tuberculosis from the sputum
samples on MGIT was 11.9±3.2 days compared
to 28.3 ± 6.3 days when using LJ medium. This
difference in detection times is statistically
significant (p<0.001). Many studies have
demonstrated significantly reduced times for
detection of mycobacteria with the use of liquid
media rather than solid egg or agar based
media.(16)
In a study done by Fadzilah et al.(17) for
AFB smear-positive specimens, the mean times
to detection were 11.5± 3.4 days with the
MGIT, and 31.2± 5.8 days with LJ culture. For
AFB smear- negative specimens, the
corresponding times were 13.2±5 days with the
MGIT, and 35.3±5.3 days with LJ culture. The
difference between BBL MGIT and LJ culture
was statistically significant (p<0.0001). In a
recent study done by Pérez-Porcuna et al. (18)
The time of culture positivity in MGIT was a
median of 14 days, 48 days for the LJ cultures,
being 3-fold more rapid in the former than in the
latter (P < 0.0001).
In the present study, using primary LJ
culture as the gold standard, the sensitivity and
specificity of BBL MGIT were 90% and 86.7%
respectively. This agrees with other studies such
as that done by Fadzilah et al. (17) where the
sensitivity and specificity of the MGIT were
90% and 89.6% respectively. The sensitivity of
MGIT in a study done by Chew et al. (19) was
93% and this is also concordant with our results.
Variations in specific antibody responses to
mycobacterial antigens in different human
populations may be linked to human leukocyte
antigen-DR phenotype, and tests designed to
detect responses to a single antigen could show
important geographic variability and limited
sensitivity(20). To overcome these problems,
multiantigen tests have been developed (21).This
combined use of antigens was found to be more
useful in serodiagnosis as it maximizes the
effectiveness of serodiagnosis, but it is not
possible to detect all antibodies as well, as this
could cause low specificity (22). Pottumarthy et
al.(23) indicated that maximum sensitivity was
obtained when seven tests were combined, but
also that specificity fell to 55% in controls.
The test kit used in this study employed two
different protein antigens (38- kDa and 16-kDa
antigens) to detect an IgG response to M
tuberculosis. The combinatory use of the 38kDa and 16-kDa antigens may increase the test
sensitivity compared with the 38-kDa antigen
alone(24). The 38-kDa antigen is the most
Vol. 22, No. 2
frequently studied serological antigen and is
also a core component in different commercial
TB serological tests (25).
Forty five percent of the studied TB
patients were seropositive for 38-kDa and 16kDa antigens (table 14). Seropositivity for the
38-kDa and 16-kDa antigens together was found
by Julian et al.(26) to be 31% in the smearpositive cases and by Imaz et al.(27) to be 58.8%.
Beck et al. (28) detected antibodies against 16kDa and 38- kDa antigens in 50.9% and 59% of
samples of TB patients, respectively. This
diverse antibody response to M. tuberculosis
may be governed by human leukocyte antigen
(HLA) types(29). Houghton et al.(30) pointed out
that the frequency of recognition using the same
recombinant 38-kDa antigen preparation ranged
35–82% with samples from smear-positive
patients from four different geographical areas.
In this study the sensitivity and specificity
of the Pathozyme TB complex plus were 45%
and 93.3% respectively. This low sensitivity
means that the ability of the test to detect those
who are truly infected is low, while the high
specificity means that the test ability to identify
those who are truly negative (not having
infection) is high. The high PPV (93.1%)
indicates that positive results of tests can be
used to confirm the diagnosis but the low NPV
(45.9%) indicates that the test has no value in
the exclusion of TB.
These coincides with previous studies
which reported that the sensitivities of the
ELISA technique based on detection of
antibodies against 38-kDa and 16-kDa antigens
were 59%(31) and 52.5% (22) and the reported
specificities were 98% (31) and 93.33% (22).
Our results were also comparable to those
found by Ben Selma et al.(15) where the
Pathozyme TB complex plus showed a
sensitivity of 43.5% and a specificity of 96.3%.
Also Demkow et al.(31) reported a test sensitivity
of 56%, and a specificity of 99%.
Meena, et al.(32) reported that the
specificities of the test reported previously
coinciding (from 88–100%), but the reported
sensitivities of the test vary (33–89%) for
smear-positive TB patients and (16–54%) for
smear negative TB patients. This variation could
be depending on the phase of the disease and on
the presence of mycobacteria in sputum; it was
much higher in culture positive and in chronic
cases. That could be connected to a higher
antigenic load and to the persistent stimulation
of the immune system(31). This explains the
results obtained by Imaz et al.(27) where the
sensitivities of the test were 29% in smear-
29
negative patients and 58.8% in smear-positive
patients.
In a study done by senol, et al.(33), the
sensitivity, specificity, positive predictive value,
and the negative predictive value of the
Pathozyme TB complex plus were found to be
25%, 90%, 66.7%, and 60%, respectively. This
low level of sensitivity and negative predict
value low could be explained by the involved
study group who were children. The data from
the literature indicate that none of the available
serodiagnostic tests for TB have an adequate
sensitivity and specificity under various clinical
conditions to be useful for diagnosing TB in
children. (31)
2.
3.
4.
5.
CONCLUSION
Although MGIT is more costly and
laborious, it has distinct advantages over the
conventional LJ culture with respect to faster
growth. It is safe, and simple to use and does
not require exogenous gas or radioactive
elements compared with other mycobacterial
cultures.
Nevertheless, due to LJ culture
positive but BBL MGIT negative specimens, a
combination of solid and liquid culture systems
is still required for the highest recovery of
mycobacteria from clinical specimens.
The ELISA test (Pathozyme TB complex
plus) is simple, easy to perform, has a very good
specificity and an acceptable sensitivity and
positive predictive value. To replace culture, the
‘gold standard’ recommended by the WHO, the
sensitivity and specificity of a satisfactory
serological test should be higher than 80% and
95% respectively. So this test has a good
specificity and a lower level of sensitivity than
recommended. But this sensitivity is
comparable to the compromised sensitivity of
the direct microscopic examination of clinical
samples by Ziehl-Neelsen staining technique
(the most commonly used method for rapid
diagnosis in limited resources labs). So, this test
is useful in diagnosis, although its use alone is
not recommended as a single tool to confirm or
to rule out TB. It could be used in combination
with other methods to increase diagnostic
accuracy, especially for culture-negative
tuberculosis cases and extrapulmonary TB
which are difficult to diagnose.
6.
7.
8.
9.
10.
11.
12.
13.
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‫‪Vol. 22, No. 2‬‬
‫ﺗﻘﻴﻴﻢ اﻟﺘﺸﺨﻴﺺ اﻟﻤﺼﻠﻲ ﻟﻠﺴﻞ ﺑﺎﻟﻤﻘﺎرﻧﺔ ﻣﻊ اﻟﻄﺮق اﻟﺘﻘﻠﻴﺪﻳﺔ‬
‫أ ‪ .‬د‪ .‬أﺣﻤﺪ ﻋﻤﺮ ﺷﻔﻴﻖ و أ‪.‬د‪.‬ﻣﺴﻌﺪ ﻋﺒﺪ اﻟﻔﺘﺎح ﻣﺮﺟﺎن و أ‪ .‬د‪ .‬س وﺳﻦ ﻋﺒﺪ اﻟﺮﺣﻤﻦ ﻳﻮﺳﻒ و د‪ .‬ﺷﻴﺮﻳﻦ ﺣﻠﻤﻲ أﺣﻤﺪ‬
‫ﻗﺴﻢ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻰ واﻟﻤﻨﺎﻋﺔ ‪ -‬آﻠﻴﺔ ﻃﺐ ﺑﻨﻬﺎ – ﺟﺎﻣﻌﺔ ﺑﻨﻬﺎ‬
‫إن اﻟﺒﺤﺚ ﻋﻦ ﻃﺮق ﺳﺮﻳﻌﺔ وﻓﻌﺎﻟﺔ ﻟﺘﺸﺨﻴﺺ ﻣﺮض اﻟﺴﻞ أﺻﺒﺢ ﻣﺤﻮر اهﺘﻤﺎم اﻟﻜﺜﻴﺮ ﻣﻦ اﻟﺪراﺳﺎت وﺧﺎﺻﺔ ﺗﻠﻚ اﻟﻄﺮق‬
‫اﻟﺘﻲ ﺗﻌﺘﻤﺪ ﻋﻠﻰ اﻟﺒﺤﺚ ﻋﻦ اﻷﺟﺴﺎم اﻟﻤﻀﺎدة وﻻ ﺳﻴﻤﺎ ﻓﻲ اﻷﺷﺨﺎص اﻟﺬﻳﻦ ﻳﺼﻌﺐ اﻟﺤﺼﻮل ﻋﻠﻰ ﻋﻴﻨﺎت اﻟﺒﺼﺎق ﻣﻨﻬﻢ أو ﺗﻜﻮن‬
‫ﻧﺘﻴﺠﺔ اﻟﻔﺤﺺ اﻟﻤﺠﻬﺮي ﺳﺎﻟﺒﺔ أو ﺗﻜﻮن اﻻﺻﺎﺑﺔ ﺑﻬﻢ ﺧﺎرج اﻟﺮﺋﺔ ‪.‬‬
‫ﺗﻬﺪف اﻟﺪراﺳﺔ إﻟﻲ اﻟﻤﻘﺎرﻧﺔ ﺑﻴﻦ اﻟﻄﺮق اﻟﺘﻘﻠﻴﺪﻳﺔ ﻓﻲ ﺗﺸﺨﻴﺺ اﻟﺴﻞ اﻟﺮﺋﻮي وﻃﺮﻳﻘﺔ اﻳﺠﺎد اﻻﺟﺴﺎم اﻟﻤﻀﺎدة ﻻﻧﺘﺠﻴﻨﺎت‬
‫ﻣﺴﺘﺨﻠﺼﺔ ﻣﻦ ﻣﻴﻜﺮوب اﻟﺪرن) ‪ (38 kDa & 16kDa‬ﺑﺎﺳﺘﺨﺪام أﺣﺪ اﻻﺧﺘﺒﺎرات اﻟﻤﻌﺘﻤﺪة ﻋﻠﻰ ﺗﻘﻨﻴﺔ اﻻﻣﺘﺼﺎص اﻟﻤﻨﺎﻋﻲ‬
‫اﻟﻤﺮﺗﺒﻂ ﺑﺎﻻﻧﺰﻳﻢ )اﻟﻴﺰا‪ ( ELISA /‬وهﻮ ) ‪ ( Pathozyme TB complex plus‬ﻟﻤﻌﺮﻓﺔ ﻣﺪى ﺣﺴﺎﺳﻴﺘﻪ ودﻗﺘﻪ ﻓﻰ‬
‫اﻟﺘﺸﺨﻴﺺ‪.‬‬
‫ﺗﻢ إﺟﺮاء هﺬﻩ اﻟﺪراﺳﺔ ﻋﻠﻲ ﻣﺮﺿﻲ ﻋﻴﺎدات ﻣﺴﺘﺸﻔﻲ اﻟﺼﺪر ﺑﺒﻨﻬﺎ وﻣﺴﺘﺸﻔﻲ اﻟﺼﺪر ﺑﺎﻟﺰﻗﺎزﻳﻖ وﻓﺤﺼﺖ اﻟﻌﻴﻨﺎت ﺑﻘﺴﻢ‬
‫اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻲ واﻟﻤﻨﺎﻋﻪ ﺑﻜﻠﻴﺔ ﻃﺐ ﺑﻨﻬﺎ ﻓﻲ اﻟﻔﺘﺮﻩ ﻣﻦ ﺷﻬﺮ ﻣﺎرس‪ ٢٠١١‬اﻟﻲ اﻏﺴﻄﺲ ‪. ٢٠١٢‬وﻗﺪ ﺷﻤﻠﺖ اﻟﺪراﺳﺔ ‪60‬‬
‫ﻣﺮﻳﻀﺎ ﻣﻨﻬﻢ ‪ ٤٠‬ذآﺮا و ‪ ٢٠‬اﻧﺜﻰ وﺗﺮاوﺣﺖ اﻋﻤﺎرهﻢ ﺑﻴﻦ ‪ ١٩‬اﻟﻲ ‪ ٥٨‬ﻋﺎم وﻟﻘﺪ ﺗﻢ اﺧﺘﻴﺎراﻟﺤﺎﻻت اﻟﻤﺸﺘﺒﻪ إﺻﺎﺑﺘﻬﺎ ﺣﺪﻳﺜﺎ‬
‫ﺑﺎﻟﺪرن اﻟﺮﺋﻮى اﻟﻨﺸﻂ ﺑﻨﺎء ﻋﻠﻰ اﻟﺘﺎرﻳﺦ اﻟﻤﺮﺿﻰ واﻟﻔﺤﺺ اﻹآﻠﻴﻨﻴﻜﻰ واﻟﻔﺤﺺ ﺑﺎﻷﺷﻌﺔ واﻟﻔﺤﺺ اﻟﻤﻌﻤﻠﻲ‪ .‬وﺗﻢ اﺳﺘﺒﻌﺎد اى‬
‫ﺣﺎﻟﺔ ﻳﺸﺘﺒﻪ اﺻﺎﺑﺘﻬﺎ ﺑﻤﺮض ﻧﻘﺺ اﻟﻤﻨﺎﻋﺔ اﻟﻤﻜﺘﺴﺐ أو ﺳﺒﻖ وأن أﺻﻴﺒﺖ ﺑﺎﻟﺪرن أوﺗﺄﺧﺬ اﻷدوﻳﺔ اﻟﻤﻌﺎﻟﺠﺔ ﻟﻠﺪرن أو رﻓﻀﺖ‬
‫اﻟﻤﺸﺎرآﺔ ﻓﻰ اﻟﺒﺤﺚ‪ .‬آﻤﺎ ﺿﻤﺖ اﻟﺪراﺳﺔ ﻣﺠﻤﻮﻋﺔ ﻣﻘﺎرﻧﺔ )ﻗﻴﺎﺳﻴﺔ( وﺷﻤﻠﺖ ‪ ١٥‬ﻣﺮﻳﻀﺎ ﺑﺄﻣﺮاض ﺻﺪرﻳﺔ أﺧﺮى ﻋﺪا اﻟﺪرن‬
‫ﺗﻢ ﺣﺠﺰهﻢ ﺑﻘﺴﻢ اﻻﻣﺮاض اﻟﺼﺪرﻳﺔ ﺑﺎﻟﻤﺴﺘﺸﻔﻲ اﻟﺠﺎﻣﻌﻰ ﺑﺒﻨﻬﺎ و‪ ١٥‬ﺷﺨﺺ ﻣﻦ اﻷﺻﺤﺎء ﻟﻴﺲ ﻟﻬﻢ ﺗﺎرﻳﺦ ﻣﺮﺿﻲ ﺑﺎﻻﺻﺎﺑﺔ‬
‫ﺑﺎﻟﺪرن ﻣﻦ اﻟﺬﻳﻦ ﺣﻀﺮوا ﻟﻠﺘﺒﺮع ﺑﺎﻟﺪم ﺑﺎﻟﻤﺴﺘﺸﻔﻰ اﻟﺠﺎﻣﻌﻰ ﺑﺒﻨﻬﺎ‪.‬‬
‫وﻗﺪ ﺧﻀﻊ آﻞ ﻣﺮﺿﻲ اﻟﺪراﺳﺔ ﻟﻼﺗﻲ‪:‬‬
‫ اﺧﺬ اﻟﺘﺎرﻳﺦ اﻟﻤﺮﺿﻲ ﻣﺘﻀﻤﻨﺎ‪ :‬اﻟﻌﻤﺮ‪ -‬اﻟﻨﻮع – اﻟﻤﻬﻨﺔ – اﻟﺘﺎرﻳﺦ اﻟﻌﺎﺋﻠﻲ واﻟﻤﺮﺿﻲ وﺗﺎرﻳﺦ اﻻﺻﺎﺑﺔ ﺑﺤﺎﻟﻪ ﻣﻤﺎﺛﻠﺔ و‬‫ﺗﺎرﻳﺦ اﻟﻌﻼج ﺑﺄدوﻳﺔ ﺿﺪ اﻟﺪرن‪.‬‬
‫ اﻟﻔﺤﺺ اﻻآﻠﻴﻨﻴﻜﻲ‪.‬‬‫ اﻟﻔﺤﺺ ﺑﺎﻷﺷﻌﺔ‪ :‬اﺷﻌﻪ ﻋﻠﻰ اﻟﺼﺪر )ﻣﻨﻈﺮ ﺟﺎﻧﺒﻲ و أﻣﺎﻣﻲ ﺧﻠﻔﻲ(‬‫ﺗﻢ ﺗﺠﻤﻴﻊ ﻋﻴﻨﺔ ﺑﺼﺎق ﺻﺒﺎﺣﺎ )ﻋﻴﻨﻪ اﻟﺼﺒﺎح اﻟﺒﺎآﺮ( وﻋﻴﻨﺔ دم ﻣﻦ اﻟﻤﺸﺎرآﻴﻦ ﻓﻲ اﻟﺪراﺳﺔ ‪.‬‬
‫وﺧﻀﻌﺖ اﻟﻌﻴﻨﺎت ﻟﻸﺗﻲ‪:‬‬
‫أوﻻ( ﻋﻴﻨﺎت اﻟﺒﺼﺎق‪:‬‬
‫‪ .١‬ﺗﺮآﻴﺰ وﺗﻄﻬﻴﺮ ﻋﻴﻨﺎت اﻟﺒﺼﺎق ﺑﻄﺮﻳﻘﺔ ﺑﺘﺮوف‪.‬‬
‫‪ .٢‬اﻟﻔﺤﺺ اﻟﻤﺠﻬﺮى ﻟﻤﺴﺤﺎت اﻟﺒﺼﺎق ﺑﺎﺳﺘﺨﺪام ﺻﺒﻐﺔ زﻳﻞ ﻧﻴﻠﺴﻮن‪.‬‬
‫‪ .٣‬زرع اﻟﻌﻴﻨﺎت ﻋﻠﻰ ﻣﺴﺘﻨﺒﺖ ﻟﻔﻨﺸﺘﻴﻦ ﺟﻨﺴﻦ اﻟﺨﺎص ﺑﻤﻴﻜﺮوب اﻟﺪرن واﻟﺘﻌﺮف ﻋﻠﻰ ﺳﻼﻻت اﻟﺪرن اﻟﻤﻌﺰوﻟﺔ ﺑﻮاﺳﻄﺔ‬
‫اﻟﻔﺤﺺ اﻟﻤﺠﻬﺮى ﻟﻤﺴﺤﺎت اﻟﺒﺼﺎق ﺑﺎﺳﺘﺨﺪام ﺻﺒﻐﺔ زﻳﻞ ﻧﻴﻠﺴﻮن و اﻟﺘﻔﺎﻋﻼت اﻟﺒﻴﻮآﻴﻤﻴﺎﺋﻴﺔ وهﻲ اﺧﺘﺒﺎر اﺧﺘﺰال‬
‫اﻟﻨﻴﺘﺮات و اﺧﺘﺒﺎر اﻧﺘﺎج اﻟﻨﻴﺎﺳﻴﻦ‪.‬‬
‫‪ .٤‬زرع اﻟﻌﻴﻨﺎت ﻋﻠﻰ ﻣﺴﺘﻨﺒﺖ ‪MGIT‬‬
‫‪.٥‬‬
‫ﺛﺎﻧﻴﺎ( ﻋﻴﻨﺎت اﻟﺪم‪:‬‬
‫اﺳﺘﺨﺪﻣﺖ ﻋﻴﻨﺎت اﻟﺪم ﻟﻔﺼﻞ اﻟﻤﺼﻞ ﻣﻨﻬﺎ و اﻟﺒﺤﺚ ﻋﻦ اﻷﺟﺴﺎم اﻟﻤﻀﺎدة ﻻﻧﺘﺠﻴﻨﺎت ﻣﺴﺘﺨﻠﺼﺔ ﻣﻦ ﻣﻴﻜﺮوب اﻟﺪرن ) &‬
‫‪(38 kDa 16kDa‬‬
‫وﻣﻦ ﻧﺘﺎﺋﺞ اﻟﺒﺤﺚ وﺟﺪ ان ﻣﺘﻮﺳﻂ اﻟﻮﻗﺖ اﻟﺬي ﺗﻢ ﺑﻪ ﻓﺼﻞ ﻣﻴﻜﺮوب اﻟﺪرن ﺑﻌﺪ زراﻋﺘﻪ ﻋﻠﻰ ﻣﺴﺘﻨﺒﺖ ال ‪ MGIT‬هﻮ‬
‫‪ 11.9 ± 3.2‬ﻳﻮم ﻣﻘﺎرﻧﺔ ب ‪ 28.3 ± 6.3‬ﻳﻮم ﻋﻨﺪ اﺳﺘﺨﺪام ﻣﺴﺘﻨﺒﺖ ﻟﻔﻨﺸﺘﻴﻦ ﺟﻨﺴﻦ ‪ .‬وآﺎﻧﺖ ﺣﺴﺎﺳﻴﺔ ﻣﺴﺘﻨﺒﺖ ال ‪MGIT‬‬
‫‪ 90%‬ودرﺟﺔ ﺧﺼﻮﺻﻴﺘﻬﺎ آﺎﻧﺖ ‪ . 86.7%‬ووﺟﺪ ان ‪ %٤٥‬ﻣﻦ ﻣﺮﺿﻰ اﻟﺪرن ﺗﺤﺖ اﻟﺪراﺳﺔ آﺎﻧﺖ ﻧﺘﺎﺋﺠﻬﻢ اﻳﺠﺎﺑﻴﺔ ﻣﻊ‬
‫اﻟﺘﺸﺨﻴﺺ اﻟﻤﺼﻠﻲ ﻟﻠﺒﺤﺚ ﻋﻦ اﻷﺟﺴﺎم اﻟﻤﻀﺎدة ﻻﻧﺘﺠﻴﻨﺎت ) ‪ (38 kDa & 16kDa‬ﺑﺎﺳﺘﺨﺪام ﺗﻘﻨﻴﺔ اﻻﻟﻴﺰا وآﺎﻧﺖ ﺣﺴﺎﺳﻴﺔ‬
‫اﻟﺘﺸﺨﻴﺺ اﻟﻤﺼﻠﻲ ) ‪ %٤٥ ( Pathozyme TB complex plus‬ودرﺟﺔ ﺧﺼﻮﺻﻴﺘﻪ آﺎﻧﺖ ‪. %٩٣.٣‬‬
‫وﻳﺴﺘﻨﺘﺞ ﻣﻦ اﻟﺒﺤﺚ ان ﻣﺴﺘﻨﺒﺖ ال ‪ MGIT‬ﻣﻦ اﻟﻄﺮق اﻟﺴﺮﻳﻌﺔ واﻵﻣﻨﺔ ﻟﻌﺰل ﻣﻴﻜﺮوب اﻟﺪرن ﻣﻘﺎرﻧﺔ ب ﻣﺴﺘﻨﺒﺖ ﻟﻔﻨﺸﺘﻴﻦ‬
‫ﺟﻨﺴﻦ ‪ .‬وان اﻟﺘﺸﺨﻴﺺ اﻟﻤﺼﻠﻲ ) ‪ ( Pathozyme TB complex plus‬ذو ﺧﺼﻮﺻﻴﺔ ﻋﺎﻟﻴﺔ ﻟﻜﻦ ﺣﺴﺎﺳﻴﺘﻪ أﻗﻞ ﻣﻦ اﻟﻤﺴﻤﻮح‬
‫ﺑﻪ ﻟﺘﺸﺨﻴﺺ اﻟﺪرن وﻟﻜﻦ ﻳﻤﻜﻦ اﺳﺘﺨﺪاﻣﻪ ﻣﻊ اﻻﺧﺘﺒﺎرات اﻻﺧﺮى ﻟﺰﻳﺎدة دﻗﺔ اﻟﺘﺸﺨﻴﺺ ﺧﺎﺻﺔ ﻓﻰ ﺣﺎﻻت اﻟﺪرن ﺧﺎرج اﻟﺮﺋﺔ‬
‫ﺣﻴﺚ ﻳﺼﻌﺐ ﺗﺸﺨﻴﺺ ﺗﻠﻚ اﻟﺤﺎﻻت ﺑﺎﻟﻄﺮق اﻟﻤﺨﺘﻠﻔﺔ اﻟﻤﻌﺮوﻓﺔ‪.‬‬
‫‪32‬‬
Vol. 22, No. 2
New Non-Invasive Markers for Evaluation of
Fibrosis in Patients with Chronic Hepatitis C
Randa Mohamed Talaat
Molecular Diagnostics Department, Institute of Genetic Engineering and
Biotechnology, Minofiya University
ABSTRACT
Background HCV is a leading cause of chronic liver diseases, cirrhosis and hepatocellular carcinoma.
Liver fibrosis and the end-stage of liver fibrosis, 'Cirrhosis, represent the final common pathway of
virtually all chronic liver diseases. During this process different biochemical markers associated with
connective tissue turnover are released into the blood for example increased level of
procollagen III N-terminal (PIII-NP), decreased serum level of matrix metalloproteinase (MMP- 1) ,
increased levels of 7S fragment of type IV collagen, hyaluronic acid, gelatinase A, and tissue inhibitor
metalloproteinases. Methods This study was carried out on 50 patients with evidence of chronic hepatitis
C, they were (42) males and (8) females. All cases were selected from Out patient clinic of the
Hepatology unit of Research Institute for Tropical Medicine..The patients were divided according to the
stage of fibrosis into 5 groups from F0 to F4 according to metavir stage.serum. MMP-1, MMP-2 and HA
levels determined using enzyme-linked immunosorbent assay technique. Results Our retrospective study
determines serum level of Metalloproteinase -1(MMP-1), Metalloproteinase -2 ( MMP-2), and
Hyaluronic acid (HA) as non invasive markers of liver fibrosis in chronic hepatitis C and to correlate
their serum levels with the stage of fibrosis assessed by histopathological staging of liver biopsy. HA level
increased significantly with progression of fibrosis whereas Serum level of MMP-1 and MMP-2 had no
statistical significant change with progressive fibrosis. Conclusions Serum level of HA can be used as an
independent predictor of significant fibrosis, while other studied markers are dependent predictors of
significant fibrosis.
Keywords: Liver fibrosis; Hyaluronic acid; Metalloproteinase -1; Metalloproteinase -2
INTRODUCTION
MATERIAL & METHODS
Infection with hepatitis C virus (HCV) is
characterized by a disturbingly high propensity
to progress to persistent infection leading to
chronic liver disease which, in a proportion of
patients, may evolve into cirrhosis, liver failure
and hepatocellular carcinoma(6), Liver fibrosis
is a dynamic process i.e. the net result of
apposition of new fibrillar extracellular matrix
(ECM) associated with more or less effective
degradation and remodeling(12). In addition, it
bears the key distinction between fibrosis (the
static evidence) and fibrogenesis (the dynamic
process)(7) For the last century, liver biopsy has
been considered as the gold standard of
assessing the stage and grade of chronic liver
disease(13). The aim of the present study is the
evaluation of fibrogenic seromarkers: MMP-1,
MMP-2, and HA as non invasive markers of
liver fibrosis in chronic hepatitis C and to
correlate their serum levels with the stage of
fibrosis assessed by histopathological staging of
liver biopsy
This study was carried out on 50 patients
with evidence of chronic hepatitis C, they were
(42) males and (8) females. All cases were
selected from Out Patient Clinic of the
Hepatology Unit of Research Institute for
Tropical M edicine (2008). The patients were
divided into 5 groups from F0 to F4 Inclusion
criteria were:-Adult subjects aged 21- 70 years
old,
both genders,subjects positive for anti- HCV
and HCV-RNA.and it was confirmed by
PCR.Exclusion criteria were:-subjects with
active schistosomiasis,subjects who had
received specific antiviral therapy prior to
enrollment in this study, subjects suffering from
other liver diseases not related to HCV e.g.
autoimmune hepatitis, sclerosing cholangitis.
Patients were subjected to the following
investigations:Routine
laboratory
investigations including complete blood count
by D-cell 60 (Diagon) No 1AC7AAA2893,
Liver profile tests: prothrombin time,
prothrombin concentration by Thrombostat No
471364, serum albumin, AST, ALT, alkaline
33
phosphatase, and serum bilirubin by Olympus
AU400 – 8077174HAMBURG,Hepatitis viral
markers: HCV-Ab & HBsAg by ELISA
technique by Awareness technology INC stat
fax 2100,stat fax 2600,stat fax 2200 ,HCV-RNA
by PCR technology extraction by QIACUBE
from Qiagen, Master mix by MX3000 from
stratagene, Assessment of serum matrix
metalloproteinase-1 (MMP-1).by using ELISA
technique by calbiochem kit.by Awareness
technology INC stat fax 2100, stat fax 2600, stat
fax 2200, Assessment of serum matrix
metalloproteinase-2 (MMP- 2).by using Elisa
technique by calbiochem kit by Awareness
technology INC stat fax 2100, stat fax 2600. stat
fax 2200; Assessment of serum hyalouronic
acid.by using Elisa technique by hyaluronic acid
test kit by Awareness technology INC stat fax
2100, stat fax 2600, stat fax 2200,Liver biopsy
and
histopathological
examination
for
necroinflammatory grading and fibrosis staging
applying the METAVIR scoring system and
patients were divided into groups according to
METAVIR stage into (F0, F1, F2, F3 and F4).
Assessment
of
serum
matrix
metalloproteinase-1 (MMP-1): by MMP-1
ELISA kit which was a non isotopic
immunoassay for the in vitro quantitation of
Table (1): Comparison between the studied
F0
F1
(no. 10)
(no. 10)
Mean ± SD
Mean ± SD
24.8 ± 4.66
32.5 ± 5.29
Age
Vol. 22, No. 2
human MMP-1 protein in tissue culture media
and serum.(14)
Assessment
of
serum
matrix
metalloproteinase-2 (MMP-2): by MMP-2
ELISA kit which was a non isotopic
immunoassay for the in vitro quantitation of
human MMP-2 protein in tissue culture media,
serum and plasma(3).
Assessment of serum Hyaluronic acid (HA):
by hyaluronic acid test kit; using an enzymelinked binding protein assayed for the
determination of HA in the human serum or
plasma.
RESULTS
The patients were divided according to the
stage of fibrosis according to METAVIR, from
the histopathological examination of liver
specimens to 5 groups.
Group (1)10 patients F0 : No fibrosis
Group (2)10 patients F1 : Stellate enlargement
of portal tracts without septae formation
Group (3) 12 patients F2 : enlargement of portal
tracts with rare septae formation
Group (4)10 patients F3: Numerous septae
without cirrhosis
Group (5) 8 patients F4 : Cirrhosis
groups according to age
F2
F3
(no. 12)
(no. 10)
Mean ± SD Mean ± SD
37.7 ± 7.70
44.8 ± 6.06
F4
(no. 8 )
Mean ± SD
57.7 ± 6.2
Pvalue
< 0.01
There is highly statistical significant difference between studied groups regarding age.
Table (2) Comparison between the studied groups regarding gender
Gender F0
F2
F3
F1
(no. 12)
(no. 10)
(no. 10)
(no. 10)
No
%
No
%
No %
No
%
Female
Male
3
7
30
70
1
9
10
90
2
10
16.7
83.3
0
10
0.0
100
F4
(no. 8)
No %
Pvalue
2
6
< 0.05
25
75
The previous table and fig(1) showed a statistically significant difference between the studied
groups(p<0.05)
34
F0
F1
F1
Vol. 22, No. 2
F2
F3
120
100
80
60
40
20
0
Female
Male
Fig(1): Relation between the studied groups as regard gender
Table (3) Comparison between the studied
fibrogenic F0
F1
(no. 10)
markeres (no. 10)
Mean ± SD
Mean ± SD
3.66 ± 2.28
10.4 ± 8.96
MMP-1
190.0 ± 77.5
443.1 ± 122.8
MMP-2
10.02 ± 6.20
24.54 ± 19.14
HA
groups as regard fibrogenic markers .
F2
F3
F4
(no. 12)
(no. 10)
(no. 8)
Mean ± SD
Mean ± SD
Mean ± SD
6.44 ± 4.84
4.55 ± 4.90
3.15± 2.66
506.7 ± 172
524.8 ± 106.7 554.2 ± 112.7
49.64 ± 18.2
182.9 ± 98.2
255.1 ± 118.0
Pvalue
< 0.05
< 0.01
< 0.01
Table(3) showed that F1 revealed a significant increase in MMP-1 compared with other groups
(<0.05) with the mean (10.4 ± 8.96).
F4 showed a significant increase in MMP-2 compared with other groups (<0.01) with the mean
(554.2 ± 112.7.)Also.
F4 showed a significant increase in HA compared with other groups (<0.01) with the mean
(255.1±118.0).
Table (4): Comparison between the studied groups as regard laboratory investigations
F0
F1
F2
F3
F4
(no. 10)
(no. 10)
(no. 12)
(no. 10)
(no. 8)
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD
Hb g/dl
13.8 ± 1.31
14.67 ± 0.71
14.34 ± 0.94
14.74 ± 0.73
11.93 ± 0.96
Platelets
281.3 ± 77.77
241.5 ± 25.79
222.0 ± 44.03
211.9 ± 40.43
107.3 ± 13.94
WBCs
6286.0±1733.6 6140.0±1121.7
6116.6±1050.3 6330.0±2046.1 4575.0±1102.9
Albumin g/dl
4.23 ± 0.221
4.31 ± 0.288
4.25 ± 0.281
3.75 ± 0.206
2.47 ± 0.446
PT Sec
12.63 ± 0.969
13.13 ± 0.601
13.67 ± 0.524
13.20 ± 0.879
14.72 ± 0.837
ALP U/L
147.7 ± 267.7
72.0 ± 13.70
66.8 ± 13.12
61.7 ± 20.07
127.7 ± 38.44
ALT U/L
96.2 ± 65.6
91.3 ± 45.4
85.5 ± 28.2
70.8 ± 24.5
45.7 ± 6.2
AST U/L
62.0 ± 28.9
71.1 ± 31.1
61.4 ± 23.5
72.3 ± 27.3
80.6 ± 14.6
Total bilirubin 0.889 ± 0.299
0.744 ± 0.168
0.925 ± 0.328
0.944 ± 0.214
5.28 ± 2.65
Mg/dl
Direct bilirubin 0.241 ± 0.103
0.203 ± 0.055
0.276 ± 0.132
0.243 ± 0.081
2.25 ± 1.16
Mg/dl
PCR
160299± 2050
259822± 14273 315927± 19672 467307±29530 544000±21603
35
Pvalue
< 0.01
< 0.01
> 0.05
< 0.01
< 0.01
> 0.05
> 0.05
> 0.05
< 0.01
< 0.01
>0.05
Vol. 22, No. 2
Table (5): Correlation between METAVIR stage and fibrogenic Markers in all groups
METAVIR Stage
r value
rvalue
rvalue
r value
r value
F0
F1
F2
F3
F4
Metalloproteinase -1
0.231
0.233
0.247
-0.510
0.276
Metalloproteinase -2
-0.100
-0.110
-0.028
-0.480
0.11
Hyaluronic acid
0.35*
0.35*
0.36*
-0.422*
0.67*
P value
>0.05
>0.05
<0.05
Table (5)showed correlation between METAVIR stage and fibrogenic markers in all groups.,
Hyaluronic acid showed the significant positive correlation with METAVIR stage (p<0.05)
Table (6): Correlation between fibrogenic markers and laboratory investigations in group 1
Hyaluronic acid
Laboratory investigations
MMP1
MMP2
r Value
r Value
r Value
0.408
-0.320
0.095
Hb
0.079
-0.478
-0.300
WBCs
-0.241
0.228
-0.316
Plat
-0.519
0.360
-0.570
Alb
0.360
0.181
-0.399
PT
-0.445
0.123
-0.229
ALT
-0.291
-0.026
-0.061
AST
0.554
-0.369
0.288
ALP
-0.206
-0.459
0.659
T Bil
-0.294
-0.393
0.546
D Bil
P<0.05 *; P<0.001 **; No star=no significance p>0.05
Table (6) showed correlation between fibrogenic markers and laboratory investigations in group (1).
There was found no significant correlation between fibrogenic markers and laboratory investigations in
group 1 (p>0.05)
Hyaluronic acid
MMP1
MMP2
r Value
r Value
r Value
0.076
0.426
0.254
Hb
0.288
0.311
0.215
WBCs
Plat
Alb
0.150
0.080
0.274
0.275
-0.448
-0.038
PT
ALT
0.130
-0.226
-0.012
-0.371
0.249
0.101
AST
0.384
-0.380
0.214
ALP
T Bil
-0.348
0.383
0.251
-0.299
0.044
-0.345
D Bil
0.557
-0.336
-0.336
P<0.05 *; P<0.001 **; No star=no significance p>0.05.
There was found no significant correlation between fibrogenic markers and laboratory investigations in
group (2) (p>0.05)
36
Vol. 22, No. 2
Hyaluronic acid
MMP1
MMP1
r Value
r Value
r Value
-0.170
0.074
0.553
Hb
0.069
-0.097
0.402
WBCs
0.449
-0.160
0.281
Plat
0.261
-0.304
0.224
Alb
0.594
-0.011
-0.439
PT
-0.573
0.065
-0.185
ALT
-0.530
0.119
0.049
AST
0.189
0.177
-0.260
ALP
0.323
-0.016
0.025
T Bil
0.320
-0.074
0.040
D Bil
No significant correlation between fibrogenic markers and laboratory investigations in group (3) (p>0.05)
Hyaluronic acid
MMP1
MMP1
r Value
r Value
r Value
-0.554
-0.090
-0.126
Hb
-0.045
-0.185
-0.024
WBCs
0.206
0.255
-0.166
Plat
0.040
-0.269
-0.689
Alb
-0.298
0.162
-0.162
PT
0.084
-0.110
0.292
ALT
AST
0.770
-0.199
ALP
-0000.186
T Bil
-0.171
D Bil
P<0.05 * ; P<0.001 **; No star=no significance p>0.05
-0.384
0.048
0.36
0.088
-0.025
0.152
0.452
0.439
There was no significant correlation between fibrogenic markers and laboratory investigations in group
(4) (p>0.05)
Hyaluronic acid
Laboratory
MMP1
MMP1
r Value
r Value
r Value
investigations
0.034
0.170
0.062
Hb
0.002
0.398
0.177
WBCs
0.026
0.392
-0.087
Plat
0.500
-0.707
0.556
Alb
0.435
-0.315
0.260
PT
0.250
-0.286
-0.004
ALT
0.016
-0.261
0.472
AST
-0.230
0.294
-0.021
ALP
0.220
0.121
0.149
T Bil
0.186
0.074
0.126
D Bil
37
Vol. 22, No. 2
Table (10) showed correlation between fibrogenic markers and laboratory investigations in group
(٥). No significant correlation between fibrogenic markers and laboratory investigations in group (٥)
(p>0.05)
Table (11): Correlation between fibrogenic markers and PCR in all groups
Hyaluronic Acid
MMP1
PCR of F0
R value
0.436
-0.174
PCR of F1
R value
-0.343
0.673
MMP2
0.353
-0.430
Table (11) showed correlation between
fibrogenic markers and PCR in all groups. No
significant correlation between fibrogenic
markers and PCR in all groups .
PCR of F2
R value
0.429
0.330
PCR of F3
R value
-0.265
0.008
PCR of F4
R value
-0.276
0.219
-0.223
0.204
-0.121
MMP-1 did not appear until the patients were in
advanced stages of fibrosis.In the present study,
there was no statistical significant change in
serum MMP-2 level with active necroinflammation or progressive fibrosis.These
results came in disagreement with(3) who
reported that regular determinations of both
TIMP-1 and MMP-2 in patients of chronic
hepatitis C may be used as indicators of
increasing fibrosis and the development of
cirrhosis.
Conclusions
HA level increased significantly with
progression of fibrosis, also had positive strong
correlation with significant fibrosis.Serum level
of MMP-1 had no statistical significant change
with progressive fibrosis,Serum level MMP-2
had no statistical significant change with
progressive fibrosis and The serum level of HA
can be used as an independent predictor of
significant fibrosis, while other studied markers
are dependent predictors of significant fibrosis.
DISCUSSION
In the present study, the age of patients
ranged between 21-70 years. There was positive
correlation between age and fibrosis stage. This
was in agreement where there was an evidence
of both continuous exposure and Cohort effects
reflecting a continuing pattern of infection with
different risk factors, which explained the
marked rise of viral hepatitis prevalence,
especially HCV, in older ages in Egypt, and that
more aggressive liver disease was detected in
older patients(1,4,2). In the current study, HA
level increased significantly with progression of
fibrosis, also had positive strong correlation
with significant fibrosis . This was supported
by(5) who reported that HA was accurate in
predicting significant fibrosis, severe fibrosis
and cirrhosis.These results come in agreement
with(9) who reported that a good correlation
between serum HA and different stages of
hepatic fibrosis. So serum HA may be used as a
useful marker of hepatic fibrosis.Many authors
have explored these markers as a potential
noninvasive tool to predict fibrosis changes.
Most of them evaluated HA in adult HCV
patients, and many applied a combined panel of
TIMP-1, PIIINP and HA(10). In the current
study, serum level of MMP-1 had no statistical
significant change with increasing necroinflammatory activity or with progressive
fibrosis and no correlation between MMP-1
concentration and METAVIR grades and
stages.These results disagreed with(14) who
established that serum levels of MMP-1 had a
declining tendency with the severity of liver
fibrosis and inflammation and abnormal serum
REFERENCE
1.
2.
3.
38
Abdel-Wahab MF, Zakaria S, kamel M
(1994): High seroprevalence of hepatitis C
virus infection among risk groups in Egypt.
Am. J. Trop. Med. Hyg.; 51(5):563-547.
Darwish M, Faris R, Clemens J (1996):
High seroprvalence of hepatitis A, B, C and
D viruses in residents in an Egyptian
village in the Nile Delta. A pilot study. Am.
J. Trop. Med. Hyg.; 54(6): 554-558.
El-Gindy I, El-Rahman AT, El-Alim MA
(2003): Diagnostic potential of serum
matrix metalloproteinase-2 and tissue
inhibitor of metalloproteinase-1 as noninvasive markers of liver fibrosis in patients
with HCV- related chronic liver disease.
Egypt. J. Immunol.; 10(1):27-35.
4.
5.
6.
7.
8.
9.
Vol. 22, No. 2
10. Parkes J, Guha IN, Roderick P (2011).
Enhanced Liver Fibrosis (ELF) test
accurately identifies liver fibrosis in
patients with chronic hepatitis C. J Viral
Hepat.;18:23–31.
11. Poynard T, Imbert-Bismut F, Ratziu V
and et al., (2002): Biochemical markers of
liver fiborsis in patients infected by
Hepatitis C virus: Longitudinal validation
in a randomized trial. J Viral Hepat., 9:128133.
12. Poynard T, Yuen MF, Ratziu V and Lai
CL (2003): Viral hepatitis C. Lacet;
362(9401): 2095-100.
13. Roosi P, Bertani T, Baio P (2003):
Hepatitis Cvirus related cryoglobulinemic
glomerulonephritis . Long-term remission
after antiviral therapy.Kidney Int;63:223641.
14. Zhang BB, Min Cai W, Weng HL and et
al., (2003): Diagnostic value of platelet
derived growth factor–BB, transforming
growth
factorα1,
matrix
metalloproteinase-1 and tissue inhibitor of
metalloproteinase-1 in serum and peripheral
blood mononuclear cells for hepatic
fibrosis. World J Gastroenterol; 2490-2496.
El-Sayed MN, Gomatos PJ, Rodier GR
(1996): Seropervalence survey of Egyptian
tourism workers for hepatitis B virus, HCV,
HIV, Treponema pallidum infection:
association of HCV infection with specific
regions of Egypt. Am J Trop med Hyg;
55(2): 179-84.
Halfon P, Bourliere M, Pénaranda G
(2005): Accuracy of hyalurolnic acid level
for predicting liver fibrosis stager in
patients with chronic hepatitis C. Comp.
Hepatol. 4: 6.
Houghton M (2000): Strategies and
prospects for vaccination against hepatitis C
viruses. Curr top microbiol immuno;
242:327-329.
Massimo Pinzani, Krista Rombouts,
Stefano Colagrande (2005): Fibrosis in
chronic liver disease: diagnosis and
management. Journal of Hepatology ; 42,
S22-S36.
Myers RP, Tainturier MH, Ratziu Vand
et al., (2003):Prediction of liver
histological lesions with biochemical
markers in patients with chronic hepatitis
B. J Hepatol ;39:222-230.
Nath NC, Rahman MA, Khan MR
(2011): Mymensingh Med J. Oct;
20(4):614-9.
39
Vol. 22, No. 2
Evaluation of Simple Screening Tests in the Diagnosis of Non
Fermentative Gram Negative bacilli, A Prospective Study
Amal A. Wafy, (M.D.)(1); Wageih S. Elnaghy(1); Ashraf Atef, (M.D.)(2);
Atef Taha(3); Tarek Gamil(4)
(1)
Medical Microbiololgy & Immunology Department, Faculty of Medicine,
Tanta University; (2)Orthopaedic Department, Faculty of Medicine, Tanta University;
(3)
Internal Medicine Department, Faculty of Medicine, Tanta University;
(4)
Urology Department, Faculty of Medicine, Tanta University
ABSTRACT
Carbapenemases are B-lactamases which include serine B-lactamases and metallo B-lactamases (MBLs).
Their production is the most important mechanisms of microbial resistance to [beta]-1actam antibiotics.
Modified Hodge test is a phenotypic method for detection of carbapenemases. EDTA disk synergy (EDS)
test is used for detection of MBLs. AmpC disk test is the commonly used tests for detection of AmpC
[beta]-lactamases. Detection of genes coding for carbapenem resistance by PCR, usually give reliable
and satisfactory results, but this method is of limited practical use for daily application in clinical
laboratories because of the cost. This study was conducted over a period of ten months (July 2012 to
April 2013) at the Medical Microbiology & Immunology Department, Faculty of Medicine, Tanta
University. A total of 110 Acinetobacter species and 120 Pseudomonas species were included in this
study. These organisms were isolated from specimens like aspirated synovial fluid from knee infective
arthritis, sputum, tracheal aspirate, pus, urine, blood, pleural fluid and ascitic fluid of patients admitted
to Internal Medicine, Chest, ENT, Orthopaedic and Urology Departments, Faculty of Medicine, Tanta
University. The Acinetobacter species and Pseudomonas species were identified and screened for
meropenem resistance by Kirby-Bauer method. The meropenem resistant strains were subjected to
modified Hodge test for detection of carbapenemases. EDTA disk synergy (EDS) test was done with
simultaneous testing of two different [beta]-lactams (meropenem and ceftazidime), for detection of
metallo-[beta]-lactamases in the meropenem resistant isolates. AmpC disk test was also done for the
meropenem resistant strains for detection of AmpC [beta]-lactamases. Of the 110 clinical isolates of
Acinetobacter species, 82 were A. baumannii, while 28 were A. lwoffii. Among the 120 Pseudomonas
isolates screened, 91 were Ps. aeruginosa, while the remaining 29 were other Pseudomonas spp. Forty
two (51.0%) A. baumannii, 8 (31.8%) A. lwoffii, 29 (31.8%) Ps. aeruginosa and 8 (27.6%) Pseudomonas
spp. were found resistant to meropenem. Among the 29 meropenem resistant Ps. aeruginosa, 13 (44.8%)
were AmpC [beta]-lactamase producers, 15 (51.7%) were MBL producers by EDTA disk synergy test, but
only 10 (34.4%) were positive for carbapenemases by modified Hodge test. Of the 8 meropenem resistant
Pseudomonas spp., 5 (62.5%) were AmpC [beta]-lactamase producers, 2 (25.0%) were MBL producers
by EDTA disk synergy test, but only 1 (12.5%) was positive for carbapenemases by modified Hodge test.
Among the 42 meropenem resistant A. baumannii, 32 (76.2%) were AmpC [beta]-lactamase producers, 3
(7.1%) were MBL producers, but only 2 (4.8%) was positive for carbapenemases by modified Hodge test.
Of the 8 meropenem resistant A. lwoffii, 3 (37.5%) were AmpC [beta]-lactamase producers, and 2 ( 25.0
%) were positive for MBL that were detected only using EDTA-ceftazidime combination and no
carbapenemases were detected by modified Hodge test. EDS is a more sensitive diagnostic method for
detection of MBLs. The modified hodge test is not considered a useful screening test for carbapenemases
as many MBL producing isolates were not detected by this test, while EDS is a more sensitive diagnostic
method for detection of MBLs. AmpC B- lactamase is a major factor for carbapenemases resistance
among the isolates in the hospital.
enzymes mutate continuously in response to the
heavy pressure of antibiotic use leading to
development of newer [beta]-lactamases with a
broad spectrum of activity. Carbapenemases are
B-lactamases which include serine B-lactamases
and metallo B-lactamases (MBLs). The latter
require metal ion zinc for their activity, which is
inhibited by metal chelators like EDTA and
INTRODUCTION
One of the most important mechanisms of
microbial resistance to [beta]-1actam antibiotics
(penicillins, cephalosporins, monobactams, and
carbapenems) is hydrolysis by [beta]lactamases. Genes coding for [beta]-lactamase
41
Vol. 22, No. 2
Genotypic methods
namely PCR and
iso–electric
focusing
(IEF) have
been
evaluated in different settings by various
workers globally(2,3,7,10,11,12,13). From India,
however, only two studies have evaluated PCR
for detection of MBL encoding gene among
Acinetobacter
baumannii(2,3). Detection of
genes coding for carbapenem resistance by
PCR, usually give reliable and satisfactory
results(8), but this method is of limited practical
use for daily application in clinical laboratories
because of the cost. Thus, a simple and
inexpensive testing method for screening of
carbapenemase
producers
is
necessary.
Therefore this study was undertaken to detect
MBLs and AmpC [beta]-lactamases in clinical
isolates of nonfermentative Gram-negative
bacteria. Clinical usefulness of the various
methods for detection of carbapenemases and
MBLs as a screening test was also evaluated.
thiol–based
compounds
but
not
by
sulbactam, tazobactam and clavulanic acid . The
carbapenemases
especially
transferrable
metallo-[beta]-lactamases (MBLs) are the most
feared because of their ability to hydrolyze
virtually all drugs in that class, including the
carbapenems. There are several mechanisms for
carbapenem resistance such as the lack of drug
penetration due to mutation in porins, loss of
certain outer membrane proteins and efflux
mechanisms(1).
In addition to their resistance to all [beta]lactams, the MBL producing strains are
frequently resistant to aminoglycosides and
fluoroquinolones. However, these usually
remain susceptible to polymyxins. Unlike
carbapenem resistance due t several other
mechanisms, the resistance due to MBL and
other carbapenemase production has a potential
for rapid dissemination(2). Consequently, the
rapid detection of carbapenemase production is
necessary to initiate effective infection control
measures to prevent their dissemination. Further
compromising therapeutic options among the
seven types of MBL genes described throughout
the world ,bla-IPM and bla –VIM are the most
common and have been reported from India
responsible
for
MBL
too(2,3). Genes
production may be chromosomal or plasmid
mediated and pose a threat of horizontal transfer
among other Gram -ve bacteria(4).
Modified Hodge test is a phenotypic
method for detection of carbapenemases(3).
Various methods like, EDTA disk synergy
(EDS) test(3), MBL E-test(1), EDTA-based
microbiological assay (4) are used for detection
of MBLs. But EDS test is a relatively simple
and sensitive method for MBL detection.
However, there are no established standard
phenotypic methods for screening of specific
serine carbapenemases. AmpC disk test(5) and
three-dimensional extract method(6) are the
commonly used tests for detection of AmpC
[beta]-lactamases.
Recently, CLSI has recommended modified
Hodge test for detection of carbapenemases
activity in Enterobacteriaceae, but not in nonfermenters(7).
Different
phenotypic techniques for
detection of MBL producers using different
chelators have been described by various
authors (5,6,7,8 Ref. )however, no standard
guidelines are available and no single method
has been found to be perfect (9)Ref. as they
have shown discoordinating results depending
on the employed methodology, B-lactam
substrate and MBL inhibitors used and bacterial
genus tested(6).
MATERIAL & METHODS
This study was conducted over a period of
ten months (July 2012 to April 2013) at the
Medical
Microbiology
&
Immunology
Department, Faculty of Medicine, Tanta
University . A total of 110 Acinetobacter
species and 120 Pseudomonas species were
included in this study. These organisms were
isolated from specimens like aspirated synovial
fluid from septic knee arthritis which is
confirmed by clinical manifestations in the form
of fever, anorexia, local hotness, redness &
inability to move the joint that is confirmed by
cytological study of this specimen e.g. increased
pus cells + decreased glucose content +
increased protein content, pus, urine, blood from
feverish leukaemic patients, ascitic fluid of
hepatic patients admitted to Internal Medicine,
Orthopaedic and Urology Departments, Faculty
of Medicine, Tanta University which were sent
to the microbiology laboratory for routine
culture identification and sensitivity testing.
The
Acinetobacter
species
and
Pseudomonas species were identified based on
standard bacteriological techniques(16,17). All
these isolates were screened for meropenem
resistance by Kirby-Bauer disc diffusion
method according to CLSI guidelines(18).
Modified Hodge test: The meropenem
resistant strains were subjected to modified
Hodge test for detection of carbapenemases(19).
suspension of
An
overnight
culture(18)
Escherichia coli ATCC 25922 adjusted to 0.5
McFarland standard(12) was inoculated using
a sterile cotton swab on the surface of a
Mueller-Hinton agar (MHA) (HI-MEDIA,
42
Mumbai, India). After drying, 10 lag
meropenem disk (HI-MEDIA, Mumbai, India)
was placed at the center of the plate and
the test strain was streaked from the edge of
the disk to the periphery of the plate in four
Vol. 22, No. 2
different directions.
The
plate
was
incubated overnight at 37ºC. The presence
of a 'cloverleaf shaped' zone of inhibition due
to carbapenemase production by the test
strain was considered as positive (Fig. 1).
Fig. (1): Modified Hodge test. Positive strain shows a "cloveraleaf shaped" zone of inhibition due to
carbapenemase production, while the negative strain shows an undistorted zone of inhibition
surface of a MHA plate. A 10 [micro]g
meropenem disk or 30 [micro]g ceftazidime
disk (HI-MEDIA, Mumbai, India) was placed
on the agar. A blank disk (6 mm in diameter,
Whatmann filter paper no. 1) was kept on the
inner surface of the lid of the MHA plate and 10
[micro]l of 0.5 M EDTA is added to it. This
EDTA disk was then transferred to the surface
of the agar and was kept 10 mm edge-to-edge
apart from the meropenem or ceftazidime disk.
After incubating overnight at 37ºC, the presence
of an expanded growth inhibition zone is
positive for this test between the two
disks was nterpreted as positive for MBL
production as in figure (2).
EDTA disk synergy (EDS) test was done
with
simultaneous
testing
of two
different[beta]-lactams
(meropenem
and
ceftazidime), for detection of metallo-[beta]lactamases in the meropenem
resistant
isolates (20).
A 0.5 M EDTA solution was prepared by
dissolving 186.1 g of disodium EDTA.
2[H.sub.2]O (REACHEM, Chennai, India) in
1,000 ml of distilled water. The pH was
adjusted to 8.0 by using NaOH (HI-MEDIA,
Mumbai, India) and was sterilized by
autoclaving(19).
An overnight liquid culture of the test
isolate was adjusted to a turbidity of 0.5
McFarland standard(18) and spread on the
Fig. (2): EDTA disc synergy test. Positive strain shows a synergistic zone of inhibition between
ceftazidime or meropenem disc and EDTA disc, while the negative strain sows no synergistic zone of
inhibition
43
Amp C disk test: AmpC disk test was also
done for the meropenem resistant strains for
detection of AmpC [beta]-lactamases (21). On a
MHA plate, culture of E. coli ATCC 25922 was
made from an overnight culture suspension
adjusted to 0.5 McFarland standard (18). A 30
lag cefoxitin disk was kept on the surface of the
agar. A blank disk (6 mm in diameter,
Whatmann filter paper no. 1) was moistened
with sterile saline and inoculated with a few
Vol. 22, No. 2
colonies of the test strain. The inoculated disk
was then placed beside the cefoxitin disk almost
touching it. The plate was incubated overnight
at 37[degrees]C. A flattening or indentation of
the cefoxitin inhibition zone in the vicinity of
the disk with test strain was interpreted as
positive for the production of AmpC [beta]lactamase. An undistorted
zone
was
considered as negative as in figure (3).
Fig. (3): AmpC disc test. Indentation of the cefoxitin zone of inhibition is seen in the vicinity of the disk
with positive strain, while there is an undistorted zone of inhibition near the negative strain.
RESULTS
Of the 110 clinical isolates of Acinetobacter
species, 82 were A. baumannii, while 28 were
A. lwoffii. Among the 120 Pseudomonas isolates
screened, 91 were Ps. aeruginosa, while the
remaining 29 were other Pseudomonas spp.
Fig. (5): Ps. aeruginosa colonies on
nutrient agar plate.
Forty two (51.0%) A. baumannii, 8 (31.8%)
A. lwoffii, 29 (31.8%) Ps. aeruginosa and 8
(27.6%) Pseudomonas spp. were found resistant
to meropenem (table1)
Among the 29 meropenem resistant Ps.
aeruginosa, 13 (44.8%) were AmpC [beta]lactamase producers, 15 (51.7%) were MBL
producers by EDTA disk synergy test, but only
10 (34.4%) were positive for carbapenemases
by modified Hodge test. Two isolates were
positive for both MBL and AmpC [beta]lactamase. One was positive for carbapenemase
Fig (4): Acinetobacter growth on
Chromagar plate.
44
by modified Hodge test, but was negative for
MBL and AmpC [beta]-lactamase by EDTA
disk synergy test and AmpC disk lest
respectively. Of the 15 MBL producers, 7 were
detected by simultaneously testing with both
meropenem and ceftazidime in EDS, 7 were
detected
only
using
EDTA-ceftazidime
combination and 1 was positive by EDTAmeropenem combination alone.
Of the 8 meropenem resistant Pseudomonas
spp., 5 (62.5%) were AmpC [beta]-lactamase
producers, 2 (25.0%) were MBL producers by
EDTA disk synergy test, but only 1 (12.5%)
was positive for carbapenemases by modified
Hodge test. Of the 2 MBL producers, one was
detected by simultaneously testing with both
meropenem and ceftazidime in EDS, while the
other was detected only using EDTA-
Vol. 22, No. 2
ceftazidime combination. Among the 42
meropenem resistant A. baumannii, 32 (76.2%)
were AmpC [beta]-lactamase producers, 3
(7.1%) were MBL producers, but only 2 (4.8%)
was positive for carbapenemases by modified
Hodge test. Among the 3 MBL producers, one
was detected by simultaneously testing with
both meropenem and ceftazidime in EDS
and 2 were detected only using EDTAceftazidime combination.
Of the 8 meropenem resistant A. lwoffii,
3 (37.5%) were AmpC [beta]-lactamase
producers and 2 ( 25.0 %) were positive for
MBL that were detected only using EDTAceftazidime
combination and
no
carbapenemases were detected by modified
Hodge test.
.
Table (1): shows different isolated bacterial species
Bacteria
Acinetobacter
110
Number of isolates
Baumanni
Species
82
Number of species
74.5
%
42
Merpenem. Resistant
51.2%
%
Lwoffii
28
25.5
8
28.6%
Pseudomonas
120
Aeruginosa
91
31.8
29
31.8%
Others
29
27.6
8
27.6%
Table (2): .Results of modified Hodge test, EDTA disc synergy test & AmpC disc test
Bacteria
Ps. aeruginosa
Pseudomonas spp.
A. baumannii
A. lwoffii
No. of
meropenem
resistant isolates
29
8
42
8
No. of positives (%)
Modified
EDTA disc
Hodge test
synergy test
10(34.4)
15(51.7)
1(12.5)
2(25)
2(4.8)
3(7.1)
0(0)
2(25)
AmpC disc
test
13(44.8)
5(62.5)
32(76.2)
3(37.5)
type MBL in Japan and Italy ,respectively(11).
Clinical isolates of these strains have been
identified worldwide. In India ,MBL Producing
pseudo. aeruginosa was 1st reported in 2002 .
The current CLSI document(14) has no
guidelines for detecting MBLS, how ever ,it has
recommended modified Hodge test for the
detection of carbapenemases but in members of
enterobactericae only.
In the current study, high prevalence of
meropenem resistance is detected among
A.baumannii. Sinha M. & Srinivasa H. have
reported 14.0 % resistance to carbapenems with
150 Acinetobacter species at Bangloze, India(22).
Whereas 64% was noted as meropenem
DISCUSSION
MBL.S. have been identified from clinical
isolates in members of Enterobacteriacea ,
P.aeruginosa and Acenitobacter spp. Over the
past few years. Strains producing these enzymes
have been responsible for nosocomial out
breaks that have been accompanied by serious
& prolonged infections. MBLS are powerful
carbapenemases that have a broad spectrum of
antibacterial activity and can hydrolyze a wide
variety of B-lactams, including penicillins,
cephalospoins and carbapenems. Since the
initial isolation of carbapenem resistant A.
baumanii producing bla-IMP-1 and bla-vim -1
45
Vol. 22, No. 2
According to Indian study, in the Indian
scenario, MBL production may not have an
imipenem role in carbapenem resistance among
Acinetobacter species.
Akirnci et al. (2005) found that the highest
imipenem resistance rate was detected in
Acinetobacter species (67.4)%followed by Ps.
aeruginosa (48.3%)(32).
A synergistic zone of inhibition must have
been normally present between EDTA and
Ceftazidime due to MBL production that was
not detected with two M BL producing Ps.
aeruginosa isolates. While these (2) isolates
were also simultaneously producing AmpC βlactamase.So, it is clear that both EDTA
meropenem
and
EDTA
Ceftazidime
combination must be used simultaneously to
detect all MBL producers that may be missed by
using either of them separately .
In addition, one of the meropenem resistant
Ps. aeruginosa was detected to be positive for
carbapenemase by modified – Hodge test while
was negative for MBL & AmpC β- lactamase .
So, rather than MBL, other classes of
carbapenemases (class A or D) may be the
causes of Ps. aeruginosa meropenem
resistance.(35).
Nathisuwan et al. (2001) found that the
phenotypic confirmatory test , though useful ,
may not be accurate .Whereas Giamarellou H.
(2005) found that the double disc test was the
easiest and most cost – effective for application
by clinical laboratories(33,34).
Among AMPC B-lactamase (44.8%) and
(76.2%) of the meropenem resistant of Ps.
aeruginosa and Acinetobacter baumannii
respectively were identified. So, AmpC Blactamase could be considered to have an
important contributory role in meropenem
resistance among isolates in our study similar to
other studies(23, 27,28) .
This appears to be more likely with A.
baumannii which in our study did not show
production of MBL or other carbapenemses in
spite of being meropenem resistant.
It has been emphasized that determination
of the correct mechanism of carbapenem
resistant is of utmost importance to decide the
appropriate therapeutic regimen of the
carbapenem resistant nonfermenters .
In several previous studies, I.V colistin
combined in the rifampicin and imipenem was
recommended for
carbapenem resistant
isolates packing MBLs , while combination of
both colistin and rifampicin (with or without
tigecycline) was recommended(27,28) .
Colistin is a polypeptide anitibiotic
belonging to polymyxin group, and is being
resistant among A. baumamii isolates in
Australia (24) and similarly in Brooking, New
York surveillance(23).
In our study, our result was similar to
Australian & New York studies. The
indiscriminate use of carbapenems have resulted
in the increase in carbapenems resistant
Acinetobacter species.
EDTA disk synergy test detected MBL
production in(5) Pseudomonas species and(2)
Acinetobacter species, respectively .EDTA disk
synergy test is evaluated to be better in MBL
diagnosis than the modified Hodge test .
Similar result have been reported in other
studies (20,25).
In our study, the increased sensitivity of
MBL producers to EDTA ceftazidime
combination was reported which was not
detected by EDTA meropenem combination, the
main reason is due to the ceftazidime production
of a marked inhibitory effect with EDTA
.Similar results were reported in Tokyo , Japan.
Observation have been made by Patricia
et al.(7) and Zerrine et al.(13) who advocate
addition of lower concentration of EDTA disc
also along with higher Concentration disc to
avoid undesirable or confusing results due to
sensitivity of isolates to higher concentration of
EDTA. Lee et al. (2003) reported that using
EDTA double disc synergy test improved the
screening of MBL producing isolates. This
method is suitable for routine use in the clinical
microbiology laboratories and useful for
monitoring of these emergent isolates(31).
Among the Ps. aeruginosa about (31.9%)
meropenem resistance was reported in our
study. A surveillance study from Latin America
has reported 40% carbapenem resistant Ps.
aeruginosa isolates, whereas an Indian study
done at a tertiary care hospital in Puducherry
10.9% carbapenem resistance was detected(36).
In our study, carbapenem resistance among
Ps. aeruginosa was lesser than the latin
America surveillance study . Moreover, arising
trend in the carbapenem resistance is clearly
detected among the non-fermenters. Also 51.7
% of the carbapenem resistance among Ps.
aeruginosa was detected to be attributable to
MBLs production.
A Korea report documented 11.4% of
imipenem-resistant
Pseudomonas
isolates
produced MBLs(37). While an Indian study has
reported
75%
of
impenem
resistant
Pseudomonas species(25).
Among meropenem resistant A.baumannii
isolates, MBL production was 7.1%, which was
lesser in comparison to 14.2% of imipenem
resistant in Korea(37).
46
increasingly used for the treatment of multi drug
– resistant gram-ve bacterial infections(29). In
vitro colistin has shown an excellent activity
against a variety of gram –ve bacilli while in
vivo hasn't shown serious toxicity(30) prolonged
I .V. administrate .
In our study, drawbacks were summarized as
follow:
The small sample size & the failure in
evaluation of the clinically useful detection
of
carbapenemases.
Our study was
restricted to carbapenemases detection and
the comparison of efficacy of different
technique for carbapenemases detection .
Moreover, further studies are required to
evaluate the clinically useful method in
carbapenemases detection .
carbapenem resistance in Acinetobacter
baumannii . India J Med Microbiol
2001;29:269-74.
4. Varaiya A, Kulkarni N M, Bhhalekar P ,
Dogra J. Incidence of metallo- βlactamase
producing Pseudomonas
aeruginosa in ICU patients. India J Med
Res 2008;127:398-402.
5. Franklin C, Liolios L, Peleg AY. Pelegg
Phenotypic detection of carbapenemsusceptible metallo- β- lactamase producing
Gram-negative bacilli in the clinical
laboratory . J Clin Microbiol 2006;44:313944.
6. Picão RC, Andrade SS, Nicoletti AG,
Campana EH, Moraes GC, Mendes
RE,et
al.
Metallo- β- lactamase
detection:Comparative
evaluation
of
double-disk synergy versus combined disk
tests for IMP, GIM-,SIM-,SPM-,orVIMproducing isolates.J Clin
Microbiol
2008;46:2028-37 .
7. Marchiaaro P, Mussi MA, Ballerini V ,
Pasteran F , Vila AJ,et al. Limansky.
Sensitive EDTA- based microbiological
assays for detection of metallo- βlactamase in nonfermentative Gramnegative bacteria. J Clin Microbiol
2005;43:5648-52.
8. Jain S,Rai S, Prasad KN, Dhole TN,
Maurya A .Detection of carbapenemase
enzymes
in
clinical
isolates
of
Pseudomonas aeruginosa by remodified
Hodge test and other phenotypic methods.
Eur Soc Clin Microbiol Infect Dis
2011:Abstract p768.
9. Singh SP , Shariff M, Barua T , Thukral
SS.Comparative evaluation of phenotypic
tests for identification of metallo- βlactamase producing clinical isolates of
Pseudomonas aeruginosa. Indian J Med
Res 2009;129:713-5.
10. Mostachio AK, van der Heidjen I, Rossi
F, Levin As , Costa SF . Costa. Multiplex
PCR for rapid detection of genes encoding
oxacillinases and metallo- β- lactamase in
carbapenem
resistant
Acintobacter
spp.JMed Microbiol 2009;58:1522-4.
11. Lee K, Lee WG, Uh Y Ha GY, Cho J,
Chong
Y
;Korean
Nationwide
Surveillance of Antimicrobial Resistance
Group. VIM-and IMP-Type Metallo- βlactamasePseudomonas
spp.and
Acintobacter spp.in Korean Hospitals.
Emerg infect dis 2003;9:868-71.
12. Ellington NJ,Kistler J, Livermore
DM,Woodford N . Multiplex PCR for
rapid detection of genes encoding acquired
CONCLUSION
MBL production is evaluated to play
an important role in carbapenem resistance
mechanism among Pseudomonas species ,
whereas it may not have major role in
carbapenem resistance among Acinetobacter
species. The modified hodge test is not
considered a useful screening test for
carbapenemases as many MBL producing
isolates were not detected by this test, while
EDS is a more sensitive diagnostic method for
detection of MBLs. Ceftazidime is evaluated to
be better substrate for EDS in comparison to
meropenem but both must be used
simultaneously for the detection of all MBL
producers. Carbapenemases other than MBL
may
be
responsible
for
carbapenem
resistance in these non fermenters.
In addition, AmpC B- lactamase is a major
factor for carbapenem resistance among the
isolates in the hospital.
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‫اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻲ‬
‫ﺗﻌﺘﺒﺮ اﻧﺰﻳﻤﺎت اﻟﻜﺎرﺑﺎﺑﻴﻨﺎﻣﻴﺰ ﻣﻦ أهﻢ اﺳﺒﺎب ﻣﻘﺎوﻣﺔ اﻟﺒﻜﺘﻴﺮﻳﺎ ﻟﻠﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﺔ ﻣ ﻦ اﻟﻨ ﻮع اﻟﺒﻴﺘ ﺎ ﻻآﺘ ﺎم‪ .‬و ﺗ ﺸﺘﻤﻞ ﺗﻠ ﻚ‬
‫اﻻﻧﺰﻳﻤﺎت ﻋﻠﻰ ﻧﻮﻋﻴﻦ ‪ :‬اﻟﻨﻮع اﻻول هﻮ اﻟﻤﻴﺘﺎﻟﻮﺑﻴﺘﺎ ﻻآﺘ ﺎﻣﻴﺰ و اﻟﻨ ﻮع اﻟﺜ ﺎﻧﻰ ه ﻮ اﻟﺒﻴﺘﺎﻻآﺘ ﺎﻣﻴﺰ اﻟﻤﺤﺘ ﻮى ﻋﻠ ﻰ اﻟ ﺴﻴﺮﻳﻦ و ﻳﻌ ﺪ‬
‫اﺧﺘﺒ ﺎر ه ﻮدج اﻟﻤﻌ ﺪل ه ﻮ اﻟﻄﺮﻳﻘ ﺔ اﻟﻈﺎهﺮﻳ ﺔ ﻟﻠﻜ ﺸﻒ ﻋ ﻦ اﻟﻜﺎرﺑ ﺎﺑﻴﻨﻴﻤﻴﺰ و ﻳﻠﻴ ﻪ اﺧﺘﺒ ﺎر ﻗ ﺮص اﻟﺘ ﺎزر ﻣ ﻊ اﻻدﻳﺘ ﺎ ﻟﻠﻜ ﺸﻒ ﻋ ﻦ‬
‫اﻟﻤﻴﺘﺎﻟﻮﺑﻴﺘﺎﻻآﺘﺎﻣﺒﺰ ﺛﻢ اﺧﺘﺒ ﺎر ﻗ ﺮص اﻷﻣ ﺐ ﺳ ﻰ ﻟﻠﻜ ﺸﻒ ﻋ ﻦ اﻟﺒﻴﺘﺎﻻآﺘ ﺎﻣﻴﺰ ﻣ ﻦ ﻧ ﻮع اﻷﻣ ﺐ ﺳ ﻰ‪ .‬ﻳﻌﺘﺒ ﺮ اﻟﺒﺤ ﺚ ﻋ ﻦ اﻟﺠﻴﻨ ﺎت‬
‫اﻟﻤ ﺴﺌﻮﻟﺔ ﻋ ﻦ اﻟﻤﻘﺎوﻣ ﺔ ﻟﻠﻜﺎرﺑ ﺎﺑﻴﻨﻤﺰ ﺑﻄﺮﻳﻘ ﺔ ﺗﻔﺎﻋ ﻞ اﻟﺒﻠﻤ ﺮة اﻟﻤﺘﺴﻠ ﺴﻞ "ﺑ ﻲ ﺳ ﻰ ار" ﻣ ﻦ اﻟﻄ ﺮق اﻟﺘ ﻰ ﻟﻬ ﺎ ﻧﺘ ﺎﺋﺞ ﻣﺆآ ﺪة و ﻟﻜ ﻦ‬
‫اﻻﺳﺘﺨﺪام اﻟﻌﻤﻠﻰ اﻟﻴﻮﻣﻰ ﻟﻬﺎ ﺻﻌﺐ ﻧﻈﺮا ﻟﻠﺘﻜﻠﻔﺔ اﻟﻤﺎدﻳ ﺔ اﻟﻤﺮﺗﻔﻌ ﺔ‪ .‬ﻟﻘ ﺪ ﺗﻤ ﺖ ﺗﻠ ﻚ اﻟﺪراﺳ ﺔ ﻟﻤ ﺪة ﻋ ﺸﺮة أﺷ ﻬﺮ اﺑﺘ ﺪاء ﻣ ﻦ ﻳﻮﻟﻴ ﻮ‬
‫‪ ٢٠١٢‬و ﺣﺘﻰ أﺑﺮﻳﻞ ‪ ٢٠١٣‬ﺑﻘﺴﻢ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻴﺎ اﻟﻄﺒﻴﺔ و اﻟﻤﻨﺎﻋﺔ‪ -‬آﻠﻴﺔ اﻟﻄﺐ – ﺟﺎﻣﻌﺔ ﻃﻨﻄﺎ و اﺷﺘﻤﻠﺖ اﻟﺪراﺳﺔ ﻋﻠ ﻰ ‪١١٠‬‬
‫ﻋﺰﻟ ﺔ ﻣ ﻦ اﻻﺳ ﻴﻨﻴﺘﻮﺑﺎآﺘﺮ و ‪ ١٢٠‬ﻋﺰﻟ ﺔ ﻣ ﻦ اﻟﺒﻜﺘﻴﺮﻳ ﺎ اﻟﺰاﺋﻔ ﺔ‪ .‬و ﻗ ﺪ ﺗ ﻢ ﻗ ﺼﻞ ﺗﻠ ﻚ اﻟﺒﻜﺘﻴﺮﻳ ﺎ ﻣ ﻦ ﻋﻴﻨ ﺎت ﻣ ﺴﺤﻮﺑﺔ ﻣ ﻦ اﻟ ﺴﺎﺋﻞ‬
‫اﻟﺴﻴﻨﻮﻗﻴﺎﻟﻰ ﻣﻦ ﺣﺎﻻت اﻟﺘﻬﺎب ﺗﻘﻴﺤﻰ ﻟﻤﻔﺼﻞ اﻟﺮآﺒﺔ و ﻋﻴﻨﺎت ﺻﺪﻳﺪ و ﺑﻮل و دم و ﻋﻴﻨﺎت ﻣﻦ اﻟ ﺴﺎﺋﻞ اﻟﺒﺮﻳﺘ ﻮﻧﻰ ﻣ ﻦ ﻣﺮﺿ ﻰ‬
‫ﻣﺪﺧﻠﻴﻦ ﺑﺄﻗﺴﺎم اﻟﺒﺎﻃﻨﺔ اﻟﻌﺎﻣﺔ و ﺟﺮاﺣﺔ اﻟﻌﻈﺎم و اﻟﻜﺴﻮر وﺟﺮاﺣ ﺔ اﻟﻤ ﺴﺎﻟﻚ اﻟﺒﻮﻟﻴ ﺔ و اﻟﺘﻨﺎﺳ ﻠﻴﺔ و اﻟﻌﻘ ﻢ – آﻠﻴ ﺔ اﻟﻄ ﺐ – ﺟﺎﻣﻌ ﺔ‬
‫ﻃﻨﻄﺎ‪ .‬ﻟﻘﺪ ﺗﻢ اﻟﺘﻌﺮف ﻋﻠﻰ أﻧﻮاع اﻟﺒﻜﺘﻴﺮﻳﺎ ﻣ ﻦ اﺳ ﻴﻨﻴﺘﻮﺑﺎآﺘﺮ و اﻟﺰاﺋﻔ ﺔ و ﺗﺤﺪﻳ ﺪ اﻻﻧ ﻮاع اﻟﻤﻘﺎوﻣ ﺔ ﻟﻠﻤﻴ ﺮوﺑﻴﻨﻢ ﺑﻄﺮﻳﻘ ﺔ اﻟﻜﻴﺮﺑ ﻰ‪-‬‬
‫ﺑﺎور‪ .‬ﺗﻢ ﺗﻌﺮﻳﺾ اﻻﻧﻮاع اﻟﻤﻘﺎوﻣﺔ ﻟﻠﻤﻴﺮوﺑﻴﻨﻢ ﻻﺧﺘﺒﺎر هﻮدج اﻟﻤﻌﺪل ﻟﻠﺒﺤﺚ ﻋﻦ اﻟﻜﺎرﺑﺎﺑﻴﻨﻴﻤﻴﺰ و اﺧﺘﺒﺎر ﻗﺮص اﻟﺘﺎزر ﻣﻊ اﻻدﻳﺘﺎ‬
‫ﻣﺴﺘﺨﺪﻣﺎ اﻟﻤﻴﺮوﺑﻴﻨﻢ و ﺳﻴﻔﺘﺎزﻳﺪﻳﻢ ﻟﻠﺒﺤﺚ ﻋﻦ اﻟﻤﻴﺘﺎﻟﻮﺑﻴﺘﺎﻻآﺘﺎﻣﻴﺰ‪ .‬ﺗﻢ ﻋﻤﻞ اﺧﺘﺒﺎر ﻗﺮص اﻻﻣ ﺐ ﺳ ﻰ ﻟ ﻨﻔﺲ اﻻﻧ ﻮاع ﻟﻠﺒﺤ ﺚ ﻋ ﻦ‬
‫اﻣﺐ ﺳﻰ ﺑﻴﺘﺎﻻآﺘﺎﻣﻴﺰ‪ .‬وآﺎﻧﺖ اﻟﻨﺘﺎﺋﺞ هﻰ ﻣﻦ ﺑﻴﻦ ال‪ ١١٠‬ﻋﺰﻟﺔ اﺳﻴﻨﻴﺘﻮﺑﺎآﺘﺮ ﺣﻮاﻟﻰ ‪ ٨٢‬ﻣﻦ ﻧﻮع اﻻﺳﻴﻨﻴﺘﻮﺑﺎآﺘﺮ ﺑﺎوﻣ ﺎﻧﻰ ﺑﻴﻨﻤ ﺎ‬
‫‪ ٢٨‬ﻧﻮع آﺎﻧﺖ ﻣﻦ ﻧﻮع اﻻﺳﻴﻨﻴﺘﻮﺑﺎآﺘﺮ ﻟﻮﻓﻴﻲ‪ .‬و ﻣ ﻦ ﺑ ﻴﻦ ال ‪ ١٢٠‬ﻋﺰﻟ ﺔ ﻟﻠﺰاﺋﻔ ﺔ وﺟ ﺪ ان ‪ ٩١‬ﻣ ﻦ ﻧ ﻮع اﻟﺰاﺋﻔ ﺔ اﻟﺰﻧﺠﺎرﻳ ﺔ ﺑﻴﻨﻤ ﺎ‬
‫ال‪ ٢٩‬اﻟﺒﺎﻗﻴﺔ ﻣﻦ اﻻﻧﻮاع اﻻﺧﺮى ﻟﻠﺰاﺋﻔﺔ‪.‬‬
‫وﺟﺪ ان ‪ ٤٢‬ﻋﺰﻟﺔ ﻣﻦ اﻻﺳﻴﻨﻴﺘﻮﺑﺎآﺘﺮ ﺑﺎوﻣﺎﻧﻰ و ‪ ٨‬ﻋﺰﻻت ﻣﻦ اﻻﺳﻴﻨﻴﺘﻮﺑﺎآﺘﺮ ﻟﻮﻓﻴﻲ و ‪ ٢٩‬ﻋﺰﻟﺔ ﻣ ﻦ اﻟﺰاﺋﻔ ﺔ اﻟﺰﻧﺠﺎرﻳ ﺔ‬
‫و ‪ ٨‬ﻋﺰﻻت ﻣﻦ اﻻﻧﻮاع اﻻﺧﺮى ﻟﻠﺰاﺋﻔﺔ آﺎﻧﺖ ﻣﻘﺎوﻣﺔ ﻟﻠﻤﻴﺮوﺑﻴﻨﻢ‪ .‬ﻣﻦ ﺑﻴﻦ ال ‪ ٢٩‬ﻋﺰﻟ ﺔ ﻟﻠﺰاﺋﻔ ﺔ اﻟﺰﻧﺠﺎرﻳ ﺔ اﻟﻤﻘﺎوﻣ ﺔ ﻟﻠﻤﻴ ﺮوﺑﻴﻨﻢ‬
‫– ‪ ١٣‬ﻋﺰﻟ ﺔ آﺎﻧ ﺖ ﻣﻨﺘﺠ ﺔ ﻟﻼﻣ ﺐ ﺳ ﻰ ﺑﻴﺘﺎﻻآﺘ ﺎﻣﻴﺰ – ‪ ١٥‬ﻋﺰﻟ ﺔ آﺎﻧ ﺖ ﻣﻨﺘﺠ ﺔ ﻟﻠﻤﻴﺘﺎﻟﻮﺑﻴﺘﺎﻻآﺘ ﺎﻣﻴﺰ ‪ ١٠ -‬ﻋ ﺰﻻت آﺎﻧ ﺖ ﻣﻨﺘﺠ ﺔ‬
‫ﻟﻠﻜﺎرﺑﺎﺑﻴﻨﻴﻤﻴﺰ‪ .‬ﻣﻦ ﺑﻴﻦ ال ‪ ٨‬ﻋﺰﻻت ﻟﻠﺰاﺋﻔﺔ ﻣﻦ اﻻﻧﻮاع اﻻﺧﺮى اﻟﻤﻘﺎوﻣﺔ ﻟﻠﻤﻴﺮوﺑﻴﻨﻢ – ‪ ٥‬ﻋﺰﻻت آﺎﻧﺖ ﻣﻨﺘﺠﺔ ﻟﻼﻣ ﺐ ﺳ ﻰ ﺑﻴﺘ ﺎ‬
‫ﻻآﺘ ﺎﻣﻴﺰ – ‪ ٢‬ﻋﺰﻟ ﺔ آﺎﻧ ﺖ ﻣﻨﺘﺠ ﺔ ﻟﻠﻤﻴﺘﺎﻟﻮﺑﻴﺘﺎﻻآﺘ ﺎﻣﻴﺰ – ﻋﺰﻟ ﺔ واﺣ ﺪة آﺎﻧ ﺖ ﻣﻨﺘﺠ ﺔ ﻟﻠﻜﺎرﺑ ﺎﺑﻴﻨﻴﻤﻴﺰ‪ .‬ﻣ ﻦ ﺑ ﻴﻦ ال ‪ ٤٢‬ﻋﺰﻟ ﺔ‬
‫ﻟﻼﺳ ﻴﻨﻴﺘﻮﺑﺎآﺘﺮ ﺑﺎوﻣ ﺎﻧﻰ اﻟﻤﻘﺎوﻣ ﺔ ﻟﻠﻤﻴ ﺮوﺑﻴﻨﻢ – ‪ ٣٢‬ﻋﺰﻟ ﺔ آﺎﻧ ﺖ ﻣﻨﺘﺠ ﺔ ﻟﻼﻣ ﺐ ﺳ ﻰ ﺑﻴﺘ ﺎ ﻻآﺘ ﺎﻣﻴﺰ – ‪ ٣‬ﻋ ﺰﻻت ﻣﻨﺘﺠ ﺔ‬
‫ﻟﻠﻤﻴﺘﺎﻟﻮﺑﻴﺘﺎﻻآﺘﺎﻣﻴﺰ – ﻓﻘﻂ ﻋﺰﻟﺘﺎن آﺎﻧﺖ ﻣﻨﺘﺠﺔ ﻟﻠﻜﺎرﺑﺎﺑﻴﻨﻴﻤﻴﺰ‪ .‬ﻣﻦ ﺑﻴﻦ ال ‪ ٨‬ﻋﺰﻻت ﻟﻼﺳ ﻴﻨﺒﺘﻮﺑﺎآﺘﺮ ﻟ ﻮﻓﻴﻲ اﻟﻤﻘﺎوﻣ ﺔ ﻟﻠﻤﻴ ﺮوﺑﻴﻨﻢ‬
‫– ‪ ٣‬ﻋ ﺰﻻت آﺎﻧ ﺖ ﻣﻨﺘﺠ ﺔ ﻟﻼﻣ ﺐ ﺳ ﻰ ﺑﻴﺘﺎﻻآﺘ ﺎﻣﻴﺰ – ﻋﺰﻟﺘ ﺎن آﺎﻧﺘ ﺎ ﻣﻨﺘﺠﺘ ﺎن ﻟﻠﻤﻴﺘﺎﻟﻮﺑﻴﺘﺎﻻآﺘ ﺎﻣﻴﺰ و ﻟ ﻢ ﺗﻮﺟ ﺪ ﻋ ﺰﻻت ﻣﻨﺘﺠ ﺔ‬
‫ﻟﻠﻜﺎرﺑﺎﺑﻴﻨﻴﻤﻴﺰ‪.‬‬
‫ﻳﻌﺘﺒﺮ اﺧﺘﺒﺎر ﻗﺮص اﻟﺘﺎزر ﻣﻊ اﻻدﻳﺘﺎ ﻣﻦ اﻻﺧﺘﺒﺎرات اﻟﺘﺸﺨﻴﺼﻴﺔ اﻻآﺜﺮ ﺣﺴﺎﺳﻴﺔ ﻟﻠﺒﺤﺚ ﻋﻦ اﻟﻤﻴﺘﺎﻟﻮﺑﻴﺘﺎﻻآﺘ ﺎﻣﻴﺰ‪ .‬ﻻ ﻳﻌ ﺪ‬
‫اﺧﺘﺒﺎر هﻮدج اﻟﻤﻌﺪل ﻣﻔﻴﺪا ﻟﻠﺒﺤﺚ ﻋﻦ اﻟﻜﺎرﺑﺎﺑﻴﻨﻴﻤﻴﺰ ﺣﻴﺚ ان ﻣﻌﻈﻢ اﻟﻌﺰﻻت اﻟﻤﻨﺘﺠﺔ ﻟﻠﻤﻴﺘﺎﻟﻮﺑﻴﺘﺎﻻآﺘﺎﻣﻴﺰ ﻟﻢ ﺗﺸﺨﺺ ﺑﻪ ﺑ ﻞ آ ﺎن‬
‫اﺧﺘﺒﺎر ﻗﺮص اﻟﺘ ﺎزر ﻣ ﻊ اﻻدﻳﺘ ﺎ اآﺜ ﺮ ﺣ ﺴﺎﺳﻴﺔ و دﻗ ﺔ ﺗﺸﺨﻴ ﺼﻴﺔ ‪ .‬وﺟ ﺪ ان اﻧ ﺰﻳﻢ اﻣ ﺐ ﺳ ﻰ ﺑﻴﺘ ﺎ ﻻآﺘ ﺎﻣﻴﺰ ه ﻮ اﻟﻌﺎﻣ ﻞ اﻟﺮﺋﻴ ﺴﻰ‬
‫ﻟﻠﻤﻘﺎوﻣﺔ ﻟﻠﻜﺎرﺑﺎﺑﻴﻨﻢ ﻣﻦ ﺑﻴﻦ اﻟﻌﺰﻻت ﻓﻰ اﻟﻤﺴﺘﺸﻔﻴﺎت‪.‬‬
‫‪49‬‬
Vol. 22, No. 2
Extended Spectrum Beta Lactamases (ESBLs) Detection in
Enterobacteriaceae According to New CLSI Guidelines
Safaa Shawky Hassan
Clinical Pathology Department, National Cancer Institute,
Cairo University, Egypt.
ABSTRACT
Introduction: The production on ESBLs can lead to life-threatening infections with Increased morbidity,
mortality and healthcare-associated costs. Objectives To investigate the value of different simple disc
diffusion screening methods of ESBLs in comparison to semiautomated microbiology machines. Material
and methods: Three commercially available microbiology identification and susceptibility testing systems
were evaluated and compared with regard to their ability to presumptively or definitely detect ESBL
production in Enterobacteriaceae. Themethods tested were disc diffusion screening tests using selected
antimicrobial agents (Kirby-Bauer antibiotic testing) , combined disk test and MicroScan ESBL plus
ESBL confirmation panels (Dade Behring Inc., West Sacramento, Calif.). Disk diffusion screening and
combined discs were evaluated against the result of microScan ESBL plus ESBL confirmation panels.
Results: Disk diffusion screening tests revealed 61 suspected ESBL. Reported ESBL by Combined ESBL
Confirmatory test disks were 70. Confirmed ESBL by microscan were 79. All confirmed ESBL by
microscan were resistant to Cefpodoxime and ceftazidime in disc diffusion method. Three test agreements
reported in 58 isolates. Conclusions: Cefpodoxime (CPO) and ceftazidime(CAZ) show the highest
agreement with microscan for ESBL detection followed by cefotaxime( CTX) & ceftriaxone (CRO)so we
can rely upon them in detecting ESBL confirmed by microscan in our routine daily work.
Key words: Enterobacteriaceae, ESBL,CLSI guidelines.
CAZ, aztreonam, CTX, and ceftriaxone. The
use of several antimicrobial agents increases the
sensitivity of ESBL detection(5). Confirmatory
testing should be performed on organisms in
which the ESBL screen may indicate ESBL
production. Phenotypic confirmatory testing
involves testing the E. coli, K. pneumoniae, or
K. oxytoca isolates against CAZ and CTX alone
and in-combination with clavulanic acid
(CTX/CA and CAZ/CA, respectively).
The Clinical and Laboratory Standards
Institute (CLSI) and the European Committee
on Antimicrobial Susceptibility Testing
(EUCAST)
recently
changed
their
recommendations concerning the interpretation
and reporting of in vitro drug susceptibility
testing (DST) results and brighten the
importance in clinical microbiology lab.(1).
The aim of this study:
To investigate the value of different simple
disc diffusion screening methods of ESBL (disc
diffusion screening test& Combined ESBL
Confirmatory test disks) in comparison to
semiautomated
microbiology
machines
(MicroScan ESBL plus
ESBL confirmation)
according to new CLSI guidelines.
INTRODUCTION
The prevalence of extended-spectrum βlactamase (ESBL) production in strains of the
family Enterobacteriaceae such as Escherichia
coli, Klebsiella spp. and Enterobacter spp., has
been increasing continuously during the past
decade in Europe and worldwide(1).
The
production of ESBLs can lead to lifethreatening infections with increased morbidity,
mortality and healthcare-associated costs(2).
Extended-spectrum beta-lactamase (ESBL)
production
in
members
of
the
Enterobacteriaceae can confer resistance to
extended-spectrum cephalosporins, aztreonam,
and penicillin. As such, the accurate detection of
ESBL producers is essential for the appropriate
selection of antibiotic therapy(3).
The clinical microbiology laboratory has
the task of screening and confirming isolates for
ESBL production. This is a challenge for the
laboratory to detect ESBL-containing gramnegative bacilli because they can appear
susceptible in vitro to certain beta-lactam antimicrobial agents yet result in clinical treatment
failure(4). Currently the CLSI documents
recommend screening of ESBL production in E.
coli, K. pneumoniae, and Klebsiella oxytoca by
using the antimicrobial agents cefpodoxime,
51
Vol. 22, No. 2
agents
(Kirby-Bauer antibiotic testing),
combined disk test and MicroScan ESBL plusESBL confirmation panels (Dade Behring Inc.,
West Sacramento, Calif.),we consider the
microscan detection of ESBL as the reference
method.
1- For susceptibility testing using the disc
diffusion screening method according to
Kirby–Bauer was used. Antibiotic discs (Becton
Dickinson, Franklin Lakes, NJ, USA) were
selected, and results were interpreted according
to the 2011 guidelines of EUCAST and
CLSI(5,6).
Each Klebsiella pneumoniae, K. oxytoca, or
Escherichia coli isolate should be considered a
potential ESBL-producer if the test results are as
follows:
MATERIAL & METHODS
Study design:
Three commercially available microbiology
identification and susceptibility testing systems
were evaluated and compared with regard to
their ability to presumptively or definitely detect
ESBL production in Enterobacteriaceae.
One hundred and thirteen samples collected
from National cancer institute-Cairo University
from August 2010 to August 2011 from adult
cancer patients. Different sample types were
collected and included in this study; 26 pus, 29
urine, 18 sputum, 26 blood, 6 throat swab, 5
Drain,3 others (pleural effusion – mouth swabcatheter).
The methods tested were disc diffusion
screening test using selected antimicrobial l
Table 1: Interpretation of disc diffusion screening test according to CLSI guidelines(7).
Disk diffusion
MICs
cefpodoxime < 22 mm
cefpodoxime > 2 µg/ml
ceftazidime < 22 mm
ceftazidime > 2 µg/ml
aztreonam < 27 mm
aztreonam > 2 µg/ml
cefotaxime < 27 mm
cefotaxime > 2 µg/ml
ceftriaxone < 25 mm
ceftriaxone > 2 µg/ml
2- Combined ESBL Confirmatory test disks:
The disk diffusion method with CTX and CAZ
disks alone and in combination with CA was
used to detect ESBL production. An ESBL
producer had a ≥5-mm-zone size difference
between the CTX/CA or CAZ/CA disks
compared to disks without the CA.(8)
3- MicroScan ESBL plus ESBL confirmation:
The MicroScan panel was performed in
accordance with the guidelines of the
manufacturer. Strain characterization and
antimicrobial susceptibility testing with
MicroScan WalkAway-96 system were
performed with Neg/BP/Combo NM31 panels,
according to manufacture’s instructions. Panels
were read following 16 to 20 h of incubation.
Confirmatory testing uses both CTX and CAZ,
alone and in combination with CA. ESBL
production was determined by a ≥3
twofoldconcentration decrease in MICs of either CAZ
or CTX in the presence of a fixed concentration
of CA versus the MIC when tested alone.
Subsecquently strains were considered as
ESBL-positive or –negative in accordance with
CLSI recommendations(9).
RESULTS
Bacterial isolates in 113 sample are as
follow: The clinical isolates include 52 E. coli
isolates and 34 Klebseilla (32 K. pneumoniae
isolates 1 klebseilla ozaena, 1 klebseilla
oxytoca), 21 enterobacter, 4 proteus, 1 Kluvera
ascorbata, 1 Morganella morganii.
Results by each method were as follow:
I Disk diffusion screening tests results using
cefpodoxime, ceftazidime, aztreonam,
cefotaxime,
ceftriaxone
(Kirby-Bauer
antibiotic testing) revealed 61 isolates
resistant to all these antimicrobial agents
(suspected ESBL were 61). Resistance to
CTX & CRO were 75 while resistance to
Cefpodoxime and ceftazidime were 79.
II (II)Results
of
Combined
ESBL
Confirmatory test disks detected ESBL in
70 isolates.
III Results of Microscan Confirmed ESBL
were 79 isolates.
Result of each method compared to microscan
result:
I Confirmed Esbl by microscan and Disk
diffusion screening tests were 58.
II Confirmed ESBL by microscan and
combined ESBL confirmatory test disks
positive were 60.
52
III Confirmed Esbl by microscan with
resistance to CTX & CRO were 75.
IV Confirmed ESBL by microscan 79,
Cefpodoxime and ceftazidime resistant
isolates were 79. i.e all confirmed ESBL by
Vol. 22, No. 2
V
microscan were resistant to Cefpodoxime
and ceftazidime in disc diffusion method.
Three test agreements reported in 58
isolates.
Table 2 shows Interpretation of result of different used methods
Microscan
Combined disks
ESBL
Not ESBL Total
ESBL
Not
Total
ESBL
79
34
113
70
43
113
(69.9%)
(30.1%)
(100%)
(61.9%)
(38.1%)
(100%)
Disk diffusion screening
ESBL Not
Total
ESBL
61
52
113
(54%) (46%) (100%)
Table 3 shows Microscan versus other test methods to detect the most test which give the most close
result to microscan and show the three test agreement.
Microscan
Microscan ESBL
Microscan ESBL Microscan ESBL Microscan vs
ESBL vs +ve
vs dds**
vs R*
vs R*
combined disc vs
combined disc
CTX&CRO
CPO&CAZ
dds**
79
60
79
58
79
(76%)
100%
(73.4%)
(100%)
(100%)
R* Resistant -dds ** disk diffusion screening
75
(95%)
79
(100%)
79
(100%)
79
(100%)
58
(73%)
Figure 1: The results of different methods compared to microscan detection of ESBL in
enterobacteriacae pathogens.
MS: Microscan- CD: Combined disc – DDS: Disk diffusion screening
only for optimal patient management but also
for immediate institution of appropriate
infection control measures to prevent the spread
of these organisms(12).
The phenotypic confirmation of ESBL
production is recommended by CLSI(5). Isolates
which are screen positive for ESBL production
should be confirmed by testing with CTX and
CAZ alone and in combination with CA. Both
CTX and CAZ with and without CA are tested
to ensure detection of ESBL production.
DISCUSSION
In 2010 CLSI changed their guidelines
concerning ESBL detection and interpretation.
Reporting of penicillins and cephalosporins as
resistant, independent of in vitro results, is no
longer recommended(10,11) However, detection
of ESBL is still considered useful or even
mandatory for epidemiological purposes.(5,6)
Correct identification of ESBL-positive
Enterobacteriaceae in due time is mandatory not
53
Although CAZ currently detects most ESBLs in
the United States, the use of only one drug
combination will not detect all ESBL producers.
Furthermore; ESBLs of the CTX-M group are
increasing and spreading througho-ut the world.
These enzymes are more active against CTX
than against CAZ(13,14,15,16).
In this study all confirmed ESBL by
Microscan were resistant to cefpodoxime and
ceftazi-dime in disc diffusion method.The
sensitivity of screening for ESBLs in enteric
organisms can vary depending on which
antimicrobial agents are tested. The use of more
than one of the five antimicrobial agents
suggested for screening will improve the
sensitivity of detection. Cefpodoxime and
ceftazidime show the highest sensitivity for
ESBL detection(7) and this is in agreement with
our study. The use of semiautomated systems
for
identification
and
antimicrobial
susceptibility testing of gram-negative rods is
now common practice in many laboratories(8).
The performance of these systems with respect
to ESBL identification in comparison to
conventional methods such as the disc
approximation method (DAM), DDS, and Etest
ESBL has been studied previously(17,18,19,20,21,22).
Using the standard disk diffusion as screening
test
for
identifying
ESBL
producer,
cefopodoxime was found to be the most
efficient antimicrobial agent in screening
isolates as potential ESBL producer followed by
cefatazidime in Klebseilla spp. Isolates(23) and
this is in agreement with our finding. While the
presence of the ESBL genes generally was
associated with varying degrees of resistance to
cephalosporins, the presence of a particular
genotype could not predict the susceptibility
pattern to a particular drug with the exception of
blaSHV, which was associated with resistance
to ceftazidime. Similarly, isolates that had
blaCTX-M were more sensitive to ceftazidime
than those without(24). The susceptibility of
blaCTX-M containing isolates to ceftazidime
has been documented by other authors who
suggest that ceftazidime can be used in the
treatment of community-acquired UTIs due to
CTX-M ESBLs[25]. This presents a possible
clinical application of genotyping ESBLs and
for empiric therapy in
UTIs suspected to be
due to ESBL-producing E. coli. Half of the K.
pneumoniae isolates carried blaSHV which
predicted resistance to ceftazidime, making it
unsuitable for use as treatment in this species(24).
And this explains why there is difference in
cephalosporin susceptibility testing in our
finding.
Our investigation differs from those of
Vol. 22, No. 2
other researchers in that : instead of evaluating
a single method, we compared side-by-side the
three methods (one semiautomated and two
manual phenotypic methods that are currently
commercially available, easy to perform,&
cheap), we used isolates that were consecutively
collected from routine clinical specimens
instead of using a well-characterized strain
collection of challenge strains with known βlactamase types; and investigators were blinded
to whether the isolate was an ESBL producer or
not. Our study design is therefore suitable to
optimally reflect daily clinical practice.
In conclusion: In the present study, we
investigated the new CLSI guidelines
decreasing the MIC values for cephalosporins in
simple disc diffusion testing to screen for ESBL
enterobactericae. Cefpodoxime and ceftazidime
show the highest sensitivity for ESBL detection
followed by CTX & CAZ so we can rely upon
them in detecting ESBL confirmed by
Microscan in our routine daily work. Especially
for Egypt where many Labs have restricted
resources with no available automated system to
complement lab detection of ESBL we can rely
upon this simple manual method in detecting
ESBL.
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‫‪127,pp344-346.‬‬
‫‪24. Daniel Maina, Gunturu Revathi, Samuel‬‬
‫‪Kariuki, Hastings Ozwara. (2012):‬‬
‫‪Genotypes and cephalosporin susceptibility‬‬
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‫‪6(6):470-477.‬‬
‫)‪25. Bin C, Hui W, Renyuan Z (2006‬‬
‫‪Outcome of cephalosporin treatment of‬‬
‫‪bacteremia due to CTX-M-type extended‬‬‫‪spectrum‬‬
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‫‪Escherichia coli. Diagn Microbiol Infect‬‬
‫‪Dis 56: 351-357.‬‬
‫اﻟﺒﻜﺘﻴﺮﻳﺎ اﻟﻤﻌﻮﻳﺔ اﻟﻤﻮﺳﻌﺔ اﻟﻄﻴﻒ ﺑﻴﺘﺎ ﻻآﺘﺎﻣﻴﺰ آﺸﻒ ﻓﻲ‬
‫وﻓﻘﺎ ﻟﻤﺒﺎدئ ﺗﻮﺟﻴﻬﻴﺔ ﺣﺪﻳﺜﺔ ﻣﻦ ﻣﻌﻬﺪ اﻟﻤﻌﺎﻳﻴﺮ اﻻآﻠﻴﻨﻴﻜﻴﺔ واﻟﻤﺨﺒﺮﻳﺔ‬
‫اﻟﺒﻜﺘﻴﺮﻳﺎ اﻟﻤﻌﻮﻳﺔ اﻟﻤﻮﺳﻌﺔ اﻟﻄﻴﻒ ﺑﻴﺘﺎ ﻻآﺘﺎﻣﻴﺰ ﻳﻤﻜﻦ أن ﺗﺆدي اﻟﻌ ﺪوى ﺑﻬ ﺬﻩ اﻟﺒﻜﺘﻴﺮﻳ ﺎ اﻟ ﻰ اﻟ ﻰ إﺻ ﺎﺑﺎت ﺗﻬ ﺪد اﻟﺤﻴ ﺎة ﻣ ﻊ‬
‫زﻳﺎدة ﻣﻌﺪﻻت اﻻﻋﺘﻼل واﻟﻮﻓﻴﺎت وﺗﻜﺎﻟﻴﻒ اﻟﺮﻋﺎﻳﺔ اﻟﺼﺤﻴﺔ اﻟﻤﺮﺗﺒﻄﺔ ﺑﻬﺎ‪.‬‬
‫اﻟﻬﺪف ﻣﻦ هﺬا اﻟﺒﺤﺚ هﻮ اﻟﺘﺤﻘﻖ ﻣ ﻦ ﻣﺨﺘﻠ ﻒ اﻟﻄ ﺮق اﻟﺒ ﺴﻴﻄﺔ اﻟﻤﺘﺎﺣ ﺔ ﻟﻠﻜ ﺸﻒ ﻋ ﻦ ه ﺬﻩ اﻟﺒﻜﺘﻴﺮﻳ ﺎ و ﻣﻘﺎرﻧﺘﻬ ﺎ ﺑ ﺎﻟﻄﺮق‬
‫اﻟﺤﺪﻳﺜﺔ اﻟﻤﻤﻴﻜﻨﺔ اﻟﻤﺴﺘﺨﺪم ﺑﻬﺎ ﺟﻬﺎز ال ‪ microScan‬وﺗﺤﺪﻳﺪ اﻟﻄ ﺮق اﻻﻧ ﺴﺐ واﻻﻗ ﻞ ﺗﻜﻠﻔ ﺔ ﻟﺘ ﺸﺨﻴﺺ ه ﺬﻩ اﻟﺒﻜﺘﻴﺮﻳ ﺎ‪.‬وﺟ ﺮى‬
‫ﺗﻘﻴﻴﻢ اﻟﺜﻼﺛﺔ ﻃﺮق اﻟﻤﺘﺎﺣﺔ ﺗﺠﺎرﻳﺎ ﻟﺘﺤﺪﻳﺪ اﻷﺣﻴﺎء اﻟﺪﻗﻴﻘﺔ وﻧﻈﻢ اﺧﺘﺒﺎر اﻟﺤﺴﺎﺳﻴﺔ و ﺗﺤﺪﻳﺪ ﻗﺪرﺗﻬﺎ ﻋﻠﻰ اﻟﻜﺸﻒ ﻋﻦ و اﻟﺘﺄآﻴ ﺪ ﻋﻠ ﻰ‬
‫إﻧﺘﺎج اﻟﻤﻮﺳﻌﺔ اﻟﻄﻴﻒ ﺑﻴﺘﺎ ﻻآﺘﺎﻣﻴﺰ ﻓﻲ اﻟﺒﻜﺘﻴﺮﻳﺎ اﻟﻤﻌﻮي‪.‬‬
‫اﻟﻤ ﻮاد واﻷﺳ ﺎﻟﻴﺐ اﻟﻤ ﺴﺘﺨﺪﻣﺔ ﻓ ﻲ ه ﺬا اﻟﺒﺤ ﺚ ه ﻲ وﺳ ﺎﺋﻞ ﺑﺤ ﺚ ﻳﺪوﻳ ﺔ ﺑ ﺴﻴﻄﻪ ﻣﺜ ﻞ ) ‪disc diffusion screening‬‬
‫‪ (tests‬و هﻮاﺧﺘﻴ ﺎر اﻗ ﺮاص ﺑﻬ ﺎ ﻣ ﻀﺎدات ﺣﻴﻮﻳ ﺔ ﻣﻌﻴﻨ ﻪ ﻓ ﻰ ه ﺬﻩ اﻟﻄﺮﻳﻘ ﺔ وﻃﺮﻳﻘ ﺔ اﺧ ﺮى وه ﻲ ) ‪( combined disk test‬‬
‫اﺧﺘﺒﺎر اﻻﻗﺮاص اﻟﻤﺠﻤﻌﺔ ﺗﺄآﻴﺪا ﻋﻠﻰ ذﻟﻚ ﺟﺮى ﺗﻘﻴﻴﻢ اﻟﻄﺮﻳﻘﺘﻴﻦ ﺿﺪ ﻧﺘﻴﺠﺔ ا ل ‪microScan‬‬
‫و ﻗﺪ آﺎﻧﺖ ﻧﺘ ﺎﺋﺞ ه ﺬا اﻟﺒﺤ ﺚ آﺎﻟﺘ ﺎﻟﻲ ‪ :‬ﻋ ﺪد اﻟﺤ ﺎﻻت اﻟﻤ ﺴﺘﺨﺪﻣﺔ ﻓ ﻲ ه ﺬا اﻟﺒﺤ ﺚ ‪ ١١٣‬ﺣﺎﻟ ﺔ ‪ ،‬ﺗ ﻢ ﺗﺎآﻴ ﺪ وﺟ ﻮد اﻟﺒﻜﺘﻴﺮﻳ ﺎ‬
‫اﻟﻤﻌﻮﻳ ﺔ اﻟﻤﻮﺳ ﻌﺔ اﻟﻄﻴ ﻒ ﺑﻴﺘ ﺎ ﻻآﺘ ﺎﻣﻴﺰ ﻓ ﻲ ﻣﺨﺘﻠ ﻒ اﻟﻄ ﺮق آﻼﺗ ﻲ‪ ٦١ :‬ﻋﺰﻟ ﺔ ﻣﺘﻮﻗﻌ ﺔ ﻋ ﻦ ﻃﺮﻳ ﻖ ) ‪disc diffusion‬‬
‫‪ ٧٠، (screening tests‬ﻋﺰﻟﺔ ﺑﺎﻟﻄﺮﻳﻘﺔ اﻟﺜﺎﻧﻴﺔ ) ‪ ( combined disk test‬اﺧﺘﺒﺎر اﻻﻗﺮاص اﻟﻤﺠﻤﻌ ﺔ ‪ ٧٩،‬ﻋﺰﻟ ﺔ ﻣﺆآ ﺪة ﺑ ﺎل‬
‫‪ microScan‬وﺗ ﻢ اﻟﻜ ﺸﻒ ﻋ ﻦ اﻧ ﻪ آ ﻞ اﻟﻌ ﺰﻻت اﻟﻤﺆآ ﺪ اﻧﻬ ﺎ اﻟﻤﻮﺳ ﻌﺔ اﻟﻄﻴ ﻒ ﺑﻴﺘ ﺎ ﻻآﺘ ﺎﻣﻴﺰ ﺑﻄﺮﻳﻘ ﺔ ال ‪ microScan‬ﻓﻬ ﻰ‬
‫ﻣﻘﺎوﻣﺔ اﻳ ﻀﺎ ﻟﻠﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﺔ ‪ ( CPO ) Cefpodoxime‬و ‪.( CAZ ) ceftazidime‬و ﻗ ﺪ ﺗ ﻢ ﺗﻮاﻓ ﻖ اﻟﺜﻼﺛ ﺔ ﻃ ﺮق ﻓ ﻲ‬
‫‪ ٥٨‬ﻣﻦ اﻟﻌﺰﻻت‪.‬‬
‫اهﻢ اﻻﺳﺘﻨﺘﺎﺟﺎت ﻣﻦ هﺬﻩ اﻟﺪراﺳﺔ هﻰ ان ﻧﺘﻴﺠﺔ اﻟﺤﺴﺎﺳﻴﺔ ﻻﻗﺮاص اﻟﺴﻴﻔﻮﺑﻮدوآﺴﻴﻢ ) ‪ ، (CPO‬واﻟ ﺴﻴﻔﺘﺎزﻳﺪﻳﻢ ) ‪( CAZ‬‬
‫ﺗﻈﻬ ﺮ اﻋﻠ ﻰ درﺟ ﺔ ﻣ ﻦ اﻟﺘﻮاﻓ ﻖ ﻣ ﻊ اﻟﻨﺘ ﺎﺋﺞ اﻟﻤﺆآ ﺪة ﻣ ﻦ ال ‪ microScan‬ﻳﻠﻴﻬ ﺎ اﻟﺤ ﺴﺎﺳﻴﻪ ﻻﻗ ﺮاص ال ﺳﻴﻔﻮﺗﺎآ ﺴﻴﻢ ) ‪( CTX‬‬
‫واﻟﺴﻴﻔﺘﺮﻳﺎآﺴﻮن ) ‪ ( CRO‬و ﻟﺬﻟﻚ ﻳﻤﻜﻦ اﻻﻋﺘﻤ ﺎد ﻋﻠ ﻰ ه ﺬﻩ اﻟﻄ ﺮق اﻟﺒ ﺴﻴﻄﻪ اﺛﻨ ﺎء ﻋﻤﻠﻨ ﺎ اﻟﻴ ﻮﻣﻲ ﻓ ﻲ ﺗ ﺸﺨﻴﺺ ه ﺬا اﻟﻨ ﻮع ﻣ ﻦ‬
‫اﻟﺒﻜﺘﻴﺮﻳﺎ اﻟﺨﻄﻴﺮة و ﺧﺎﺻﺔ ﻓﻲ اﻟﺒﻼد اﻟﻤﺤﺪودة اﻟﻤﻮارد ‪.‬‬
‫‪56‬‬
Vol. 22, No. 2
Rapid detection of Extended Spectrum β-lactamase (ESBL) Producing
Strains of Escherichia coli in Urinary Tract Infections Patients in
Benha University Hospital
1
1
Enas Sh. Khater and 2Hammouda W. Sherif
Department of Microbiology and Immunology, Faculty of Medicine, Benha University
2
Department of Urology, Faculty of Medicine, Benha University
ABSTRACT
The aim of this study was done to detect the prevelance of ESBL, AmpC producing and ESBL&AmpC coproducing strains of Escherichia coli (E. coli) in urinary tract infections patients in Benha University
Hospital and to evaluate the performance of CHROMagar™ ESBL media for rapid screening of ESBL
producing E. coli. All patients under study were subjected to: Full history taking and clinical
examination. Bacteriological study included; urine sample collection from each patient and subjected to
urine analysis, urine culture on cysteine lactose electrolyte deficient agar (CLED) agar, CHROMagar™
ESBL media and MacConkey agar supplemented with 2 mg/liter ceftazidime (MCKC). ESBL detection in
E. coli isolated on CLED agar by phenotypic screening by clinical and laboratory standards institute
(CLSI) method then phenotypic confirmation by E. test. The presence AmpC beta-lactamase ESBL was
detected by AmpC disc test and detection of AmpC beta-lactamase & ESBL co-producers by cefepime &
Cefepime+ Clavulanate E test. In this study out of 45 E. coli strains 24 (5 ٣ .3%) ESBL producers were
detected by E. test (golden method for confirmation of ESBL according to CLSI) and 21(46.7%) strains
were non ESBL producers. Out of the 24 isolated ESBL producing E.coli strains 9 (37.5%) were detected
in community acquired UTI patients while 15 (62.5%) were detected in health care associated UTI
patients. The sensitivity of the MCKC and CHROMagar™ ESBL media were 100%. While specificity
were 80.8% and 87.5% respectively. In our study out of 45 isolated E.coli strains 14 (31.1%) were AmpC
producers by AmpC test, 4 (8.9%) were AmpC &ESBL co-producers by cefepime/ cefepime clavulanic
E.test. To conclude it is important to know the prevalence of ESBL, AmpC producing and ESBL&AmpC
co-producing organisms so that judicious use of antibiotics could be done and increase awareness about
the need for routine detection of AmpC &ESBL in clinical isolates. CHROMagar™ ESBL media detect
ESBL producers from clinical specimen and give rapid presumptive identification by means of colony
colour at 24h with good sensitivity and specificity.
cephamycins (cefoxitin and cefotetan), but are
susceptible to β- lactamase inhibitors
(clavulanic acid) [3].
Genotypic methods based on enzyme
assays, PCR and others are not suitable for
routine clinical testing in
most clinical
diagnostic laboratories. The phenotypic
confirmatory tests are highly sensitive and
specific compared to genotypic confirmatory
tests. [4]
Phenotypic tests, which require a screening
step followed by confirmation used to detect
ESBL in most microbiology laboratories. The
screening test is based on testing the organism
for resistance to an indicator cephalosporin.
Cefpodoxime is commonly used as it is
hydrolysed by TEM, SHV, and CTX-M types,
but other cephalosporins such as cefotaxime,
ceftriaxone, and ceftazidime are also used. To
confirm the presence of an ESBL, synergy
between the indicator cephalosporin and
clavulanic acid needs to be demonstrated
INTRODUCTION
Microorganisms responsible for urinary tract
infection (UTI) such as E.coli and Klebsiella spp.
have the ability to produce ESBL in large
quantities. These enzymes are plasmid borne and
confer multiple drug resistance, making urinary
tract infection difficult to treat.[1]
Enterobacteriaceae, especially E. coli and
Klebsiella spp.-producing ESBLs such as SHV
and TEM types, have been established since the
1980s as a major cause of hospital-acquired
infections. However, during the late 1990s,
several community-acquired pathogens that
commonly cause urinary tract infections and
diarrhea have also been found to be ESBL
producers. These include E. coli, Salmonella,
Shigella and Vibrio Cholera.[2]
ESBLs are enzymes capable of conferring
bacterial resistance to the penicillins, first,
second- and third-generation cephalosporins,
and aztreonam. They are not active against the
57
(ESBLs are inhibited by clavulanic acid).[5] E
test is a convenient method for detection of
ESBL by MIC reduction. Two different E test
gradient formats have been in use based on
reduction of ceftazidime or cefotaxime MICs by
3 two-fold dilutions in the presence of
clavulanic acid and have been used successfully
for ESBL detection[6,7].
Various selective media have been
proposed in order to assess the presence of
ESBL producers in stool and urine samples.
Examples of such media include Drigalski agar
supplemented with cefotaxime, MacConkey
agar supplemented with ceftazidime or nutrient
agar
supplemented
with
ceftazidime,
vancomycin and amphotericin B. Chromogenic
media were developed for isolation of ESBL
from clinical specimens. CHROMagar™ ESBL
media allows the detection of ESBL-producing
bacteria while inhibiting the growth of other
bacteria. Identification of ESBL producers
depend on colony colour after 24h incubation[8].
The ESBL phenotypes have become more
complex due to the production of multiple
enzymes which include the inhibitor-resistant
ESBL variants and plasmid-borne AmpC.
AmpC is normally produced in low levels by
many organisms and is not associated with
resistance, but it can be produced at high levels
and cause resistance to all beta lactams, except
carbapenems and 4th generation cephalosporins.
These enzymes have spread worldwide and their
prevalence varies by the geographical area.
Many clinical laboratories currently test
Escherichia coli and Klebsiella spp. for
production of ESBLs but do not attempt to
detect plasmid mediated AmpC beta lactamases.
These enzymes are typically associated with
multiple antibiotic resistances, leaving a few
therapeutic options[9].
The present study was done to detect the
prevelance of ESBL, AmpC producing and
ESBL&AmpC co-producing strains of E. coli
in urinary tract infections patients in Benha
University Hospital and to evaluate the
performance of CHROMagar™ ESBL media
for rapid screening of ESBL producing E. coli.
Vol. 22, No. 2
were diagnosed as UTIs patients in Benha
University hospital either from Urology
Department or attending Urology Benha
University out-patient clinic. Their ages ranged
from 20 to 68 years. The selection of patients
was done according to clinical data suspecting
UTI. The diagnosis of UTIs in urine samples
was based on the presence of 105 CFU o f
microorganisms per ml in urine culture on
CLED[10]. Also, presence of more than 5 pus
cells per high power field in an unspun urine in
male, and more than 10 pus cells in a female.
High number of pus cells in urine, or pyuria
usually indicates infection[11].
All patients under study were subjected to:
Full history taking and clinical examination
with specific stress on fever,urgency, frequency,
dysuria,
suprapubic
tenderness
and
costovertebral angle pain and tenderness.
Bacteriological study include:
Samples: urine sample was collected from each
patient in sterile and screw capped urine
container and subjected to the following:
I- Urine analysis
II- Urine culture
• Urine culture on CLED agar,
CHROMagar™ ESBL and MacConkey
agar
supplemented with 2 mg/liter
ceftazidime (MCKC)
• Identification of bacterial isolates by
colony colour & morphology, gram stain
and biochemical reactions
III- ESBL detection in E. coli isolates by:
1- Phenotypic screening CLSI method,
CHROMagar™ ESBL and MCKC
2- Phenotypic confirmation by E test
IV- AmpC beta-lactamase isolates
were
screened by cefoxitin resistance (zone <
19mm) then confirmed by AmpC disc test
V- AmpC beta-lactamase ESBL co-producers
detection by cefepime & cefepime+
clavulanate E test.
Methods:
A- Urine sample collection:
Early morning and mid-stream urine is
collected in a sterile container for
bacteriological and microscopic examination
after cleaning the periurethral area (meatus,
vulva labial folds). If collected from indwelling
catheter: the wall of the catheter at the juncture
with the drainage tube should be disinfected and
sterile syringe should be used to obtain a urine
specimen under aseptic conditions. Immediate
processing of the samples after collection was
done to avoid samples contamination.
Viable bacterial count was performed for
urine samples using the pour plate method.
MATERIALS & METHODS
This study was done in Microbiology and
Immunology Department of Benha Faculty of
Medicine from October 2012 to March 2013.
Out of the 135 urine samples collected from
patients suspected to have UTI, a total of 100
were culture positive on CLED, collected from
100 patients (56 males and 44 females) who
58
Microscopic examination of centrifuged urine
was performed for pus cell (WBC) count [10]
B- Urine culture
1. The urine samples were centrifuged; cultures
were done from the deposit on CLED agar,
all plates were incubated at 37°C for 48 hrs.
2. Culture
on
CHROMagar™
ESBL
(CHROMagar, France) which was freshly
prepared on the day of the study according to
the manufacturer’s instructions and poured
into 90-mm-diameter petri dishes. Urine was
directly inoculated onto a CHROMagar plate
and streaked for colony isolation. The plates
were incubated overnight at 35°C in ambient
air and then examined for any growth.[12]
• Culture
on
MacConkey
agar
supplemented with 2 mg/liter ceftazidime
(MCKC). The plates were incubated
overnight at 35°C in ambient air and then
examined for any growth.
• Identification of bacterial isolates by
colony morphology, gram stain and
biochemical reactions [13]
Interpretation of CHROMagar™ ESBL
according to colony colour:
E.coli: dark pink to reddish colonies
Klebsiella,
Enterobacter,Citrobacter
spp.:Metallic blue colonies
Proteus: brown halo colonies
Non Resistant Other Gram(-) strains and
gram(+) strains inhibited
C- Testing for the ESBL Production:
1. Phenotypic screening CLSI method for
ESBL Production:
• E. coli isolates were screened for ESBL
production by the disc diffusion method
according to the CLSI guidelines [7]. The
following antibiotics were used; cefotaxime
(30μg), cefpodoxime (30μg),ceftrioxone
(30μg), ceftazidime (30μg), cefepime (30μg),
Aztereonam (30μg). (Oxoid, UK). This
screening method based on measuring the
specific zone diameters for the antibiotic
discs
• All the strains which showed a zone diameter
of ≤ 17 mm for cefpodoxime, ≤ 22 mm for
ceftazidime, ≤ 27 mm for aztreonam, ≤ 25
mm for ceftriaxone and ≤ 27 mm for
cefotaxime were selected for checking the
ESBL production as was recommended by
CLSI M100-S21 (2010)[7]. Every isolate that
showed resistance to at least one of the
screening agents was tested for ESBL
production. The use of more than one of
these agents for screening improves the
sensitivity of detection.
Vol. 22, No. 2
2.
Phenotypic confirmatory test by ESBLE-test:
The ESBL-E-Test strips were obtained
from (AB biodisc, Solna, Sweden) ceftazidime/
ceftazidime + clavulanic acid (TZ/TZL) and
cefotaxime and cefotaxime + clavulanic acid
(CT/CTL)
in
accordance
with
the
manufacturer's instructions. One end of each
strip contains a gradient concentration of either
ceftazidime (TZ) (MIC range 0.5 to 32 μg/ml)
or cefotaxime (CT) (MIC range of 0.25 μg to 16
μg). The other end of the strip with a gradient of
ceftazidime plus a constant concentration of
clavulanate TZ/TZL (0.064-4 μg/ml plus 4
μg/ml of clavulanic acid) or with a gradient of
cefotaxime plus a constant concentration of
clavulanate CT/CTL (0.25 μg- 16 μg plus 4 μg
of clavulanic acid)[14]. After overnight growth,
the organism was emulsified in saline solution
to a turbidity of 0.5 McFarland standard. The
suspension was spread on a Muller Hinton agar
plate with a cotton swab. After the plates were
dried for 15 min, the E-Test strips were placed
on them, after incubation at 35°C for 18 hrs.
The MIC was interpreted as the point of
intersection of the inhibition ellipse with the
edge of the test strip. After overnight growth,
the organism was emulsified in saline solution
to a turbidity of 0.5 McFarland standard. The
suspension was spread on a Muller Hinton agar
plate with a cotton swab. After the plates were
dried for 15min, the E-Test strips were placed
on them,after incubation at 35°C for 18 hrs. The
MIC was interpreted as the point of intersection
of the inhibition ellipse with the edge of the test:
1) ESBL positive:
• If CT ≥ 0.5 and CT/CTL ≥ 8 or TZ ≥ 1
and TZ/TZL ≥ 8.
• Presence of a phantom zone or ellipse
deformation
2) ESBL Negative: If CT < 0.5 or CT/CTL < 8
and TZ < 1 or TZ/TZL < 8.
3) Non-determinable (ND): CT > 16 and CTL
> 1 and TZ >32 and TZL > 4. Strains
showing non-determinable (ND) results
with CT/CTL and TZ/TZL strips should be
further tested using PM/PML strips for
detection of AmpC beta-lactamase ESBL
co-producers.
D- AmpC beta-lactamase detection by AmpC
disc test [16]
The test is based on use of Tris-EDTA to
permeabilize a bacterial cell and release βlactamases into the external environment.
Cefoxitin (30μg). (Oxoid, UK) resistant E.coli
isolates tested by AmpC disc test as following:
1. A lawn culture of a 0.5 McFarland’s
suspension
of
ATCC E.coli 25922
59
(bioMérieux) was prepared on a MuellerHinton agar plate. A 30 μg cefoxitin (fox)
disc was placed on the inoculated surface of
the agar. A sterile AmpC discs (i.e., filter
paper discs containing Tris-EDTA) obtained
from (Becton Dickinson, Sparks, MD) which
was inoculated with several colonies of the
test organism was placed beside the cefoxitin
disc, almost touching it.
2. After an overnight incubation at 37°C, the
plates were examined for either an
indentation or a flattening of the zone of
inhibition, which indicated the enzyme
inactivation of cefoxitin (positive result), or
an absence of distortion, which indicated no
significant inactivation of cefoxitin (negative
result).
E- AmpC beta-lactamase ESBL co-producers
detection:
a) Cefepime/cefepime + clavulanic acid
(PM/PML) strips [15]
All non-determinable ESBLs by TZ/TZL
and CT/CTL E test were further tested using
PM/PML strips for detection of AmpC betalactamase ESBL co-producers.
One end of each strip contains a gradient
concentration of Cefepime (PM) (MIC range
0.25-16 µg/mL). The other end of the strip with
a gradient of Cefepime plus a constant
concentration of clavulanate PM/PML (0.064-4
µg/mL plus 4 μg/ml of clavulanic acid). The
results were interpreted as positive:
• If the MIC ratio for PM/PMLwas ≥8.
• Presence of a phantom zone, deformation
or ellipse
Statistical analysis:
Data were entered into a database using
SPSS 13 for Windows (SPSS Inc., Chicago, IL).
Sensitivity: the ability of the test to detect true
positive cases and specificity: the ability of the
test to detect true negative cases [17]
Vol. 22, No. 2
pneumoniae 22 (21%) and the least was
Staphylococcus aureus 2 (1.9%).
Out of 45 E. coli strains 24 (53.3%) ESBL
producers were detected by CT/CTL and/ or
TZ/TZL E. test (golden method for confirmation
of ESBL according to CLSI) and 21 (46.7%)
strains were non ESBL producers as shown in
figure (1)
Table (1) shows that out of the 24 isolated
ESBL producing E.coli strains 9 (37.5%) were
detected in community acquired UTI either outpatients or in-patients admitted<48 h patients
while 15 (62.5%) were detected in health care
associated UTI patients who were admited >48
h.
Table (2) shows that out of 45 E. coli
isolates 16 (35.6%) yielded no growth on any
selective media and 29 (64.4%) yielded
growth on MCKC, while 27 (60%) yielded
growth on CHROMagar™ ESBL media. In
comparison with the E. test, sensitivity of the
MCKC and CHROMagar™ ESBL media were
100%. While specificity were 80.8% and 87.5%
respectively.
Preliminary screening test by CLSI method
showed that resistance of E coli strains to
Ceftazidime,
Ceftriaxone,
Aztereonam,
Cefpodoxime and Cefepime were 35 (77.7%)
,33(73.3%),31(68.9%), 26 (57.8%), 26 (57.8%)
respectively, so 35 strains (77.8%) of isolates
were considered as preliminary producers of
ESBLs. While preliminary producers of ESBLs
from CHROMagar™ ESBL agar were 27 (60%)
as shown in Table (3). 24 (100) confirmed
ESBLs were detected by both CLSI screening
method and CHROMagar™ ESBL agar. 11
were positive ESBLs by CLSI screening method
and 3 positive ESBLs by CHROMagar™
ESBL agar, while all these strains were
confirmed negative. Sensitivity of both CLSI
screening method and CHROMagar™ ESBL
agar were 100%. While high specificity showed
by CHROMagar™ ESBL agar 100%, low
specificity showed by CLSI screening method
70%.
Among the screen positive isolates
cefoxitin resistance was noted in 41 (91.1%)
isolates. All the ESBL producers (24), 14 AmpC
producers and 4 AmpC &ESBL co-producers
were cefoxitin resistant.
Table (4) shows that out of 45 isolated
E.coli strains 14 (31.1%) were AmpC producers
by AmpC test, 4 (8.9%) were AmpC &ESBL
co-producers by cefepime/ cefepime clavulanic
E.test, 24 (53.3%) were pure ESBL by TZ/TZL
or CT/CTL E.test.
RESULTS
The ages of studied patients ranged from 20
to 68 years. As regards the sex distribution
among the studied patients out of 100 patients
56 were males and 44 were females. 45 were
out-patients and 55 in-patients admitted in
Urology department. Out of 55 in-patients 30
patients were catheterized and 25 were non
catheterized.105 isolates on CLED agar were
obtained from the studied 100 patients urine
samples, while most samples yielded only single
isolate, five samples yielded two isolates. The
most common isolated microorganisms were
E.coli 45 (42.9%) followed by Klebsiella
60
Vol. 22, No. 2
Table (1) Distribution of ESBL producing E. coli among community acquired and health care
associated UTI infections:
Community acquired UTI
Health care associated UTI
NO
%
NO
%
ESBL producing
9
37.5%
15
62.5%
E. coli (n=24)
Table (2) Identification of ESBL producer and non ESBL producer E. coli growth on
CHROMagar™ ESBL agar and MCKC agar
CHROMagar™ ESBL agar
MCKC
Results of growth
NO
%
NO
%
Growth
27
(60%)
29
64.4%
No growth
18
(40%)
16
35.6%
TOTAL
45
45
45
45
Specificity
87.5 %
80.8 %
Sensitivity
100%
100%
PPV
88. 9 %
82.8 %
NPV
100%
100.00 %
Table (3) Comparison between CHROMagar™ ESBL agar and CLSI screening method to screen
ESBL producing E. coli:
CHROMagar™ ESBL agar
CLSI screening method
NO
%
NO
%
Preliminary ESBL producers
27
60%
35
77.8%
Non ESBL producers
18
40%
10
22.2%
Total
45
100
45
100%
Table (4) Distribution of AmpC and AmpC &ESBL co-produers among E. coli isolates:
E. coli isolates n=45
Amp C producers
Amp C &ESBL coPure ESBL producers Non Amp C &ESBL producers
producers
or co-producers
NO
%
NO
%
NO
%
NO
%
14
31.1%
4
8.9%
24
53.3%
3
6.7%
61
Vol. 22, No. 2
Figure (2): Dark pink to reddish E coli colonies isolated on CHROMagar™ ESBL media
Figure (3): Positive AmpC test (indentation of the zone of inhibition around cefoxitin disc)
a community-based ESBL prevalence to be
5.7% in London. The cause of the upsurge in
community-acquired infections with ESBLproducing organisms is not yet clear, but
associations
with
foodstuffs,
animal
consumption of antibiotics, and frequent patient
contact with health care facilities need to be
explored[22].
Methods to detect ESBL-producing
organisms from clinical specimens should have
high sensitivity and high specificity combined
with a short time to the reporting of results. In
order to identify ESBL-producing gramnegative bacilli from clinical samples more
easily and reliably, selective media should
ideally achieve the identification of the
organisms and detection of ESBL in one step.
At the least, it should decrease the workload and
reduce
the
need
of
unnecessary
confirmations.[23]
In the present study out of 45 E. coli
isolates 16 (35.6%) yielded no growth on any
selective media and 29 (64.4%) yielded growth
on MCKC, while 27 (60%) yielded growth on
CHROMagar™ ESBL media. In comparison
with the E. test, sensitivity of the MCKC and
CHROMagar™ ESBL media were 100%. While
specificity
were
80.8%
and
87.5%
DISCUSSION
Extended-spectrum β-lactamases (ESBLs)
producing Gram-negative bacteria are large,
rapidly evolving group of plasmid-mediated
enzymes emerging pathogens. Clinicians,
microbiologists, infection control practitioners
and hospital epidemiologists are concerned
about ESBL-producing bacteria because of the
increasing incidence of such infections [18]
In the present study out of 45 E. coli strains
24 (53.3%) ESBL producers were detected by
E. test (golden method for confirmation of
ESBL according to CLSI[7]).This is in
agreement with Hasan et., al[19] who reported
(57.4%) ESBL rate in uropathogenic E.coli.
Thabit et.,al[20] also reported 53% ESBL
producing E.coli by E. test.
In our study out of the 24 isolated ESBL
producing E.coli strains 9 (37.5%) were
detected in community acquired UTI patients
while 15 (62.5%) were detected in health care
associated UTI patients. This was in agreement
with Thabit et.,al[20]Who reported that ESBL
producing E. coli among community isolates
was (39.47%) while among nosocomial isolates
the rate was (70%).Our results are comparable
to those obtained by Bean et al.[21].who reported
62
respectively.Glupczynski et. al[24] reported
sensitivity and specificity of MCKC 84% and
91% respectively. Regarding sensitivity and
specificity of CHROMagar™ ESBL media
lagace- Wiens et al[25] reported high sensitivity
(99,2%) and Specificity (89%). Also Sito et
al[26] reported sensitivity and specificity of
CHROMagar™ ESBL media 100% and 93%
respectively.
In our study preliminary screening test by
CLSI screening method showed that 35 strains
(77.8%) of
isolates were considered as
preliminary producers of
ESBLs. While
preliminary producers of
ESBLs
from
CHROMagar™ ESBL agar were 27 (60%) as
shown in Table (3). 24 (100) confirmed ESBLs
were detected by both CLSI screening method
and CHROMagar™ ESBL agar. 11 were
positive ESBLs by CLSI screening method and
3 positive ESBLs by CHROMagar™ ESBL
agar, while all these strains were confirmed
negative. Sensitivity of both CLSI screening
method and CHROMagar™ ESBL agar were
100%. While high specificity showed by
CHROMagar™ ESBL agar 100%, low
specificity showed by CLSI screening method
70%. Also Manhas et.,al [27] reported sensitivity
and specificity of CLSI screening method were
99.4% and 66.1% respectively.
Thabit
et.,al[20]reported 76.5% potential producers of
ESBLs by CLSI screening method while
confirmed ESBLs 53% .
With the spread of AmpC and ESBL
producing strains all over the world, it is
necessary to know the prevalence of these
strains in hospitals. Use of cefepime is more
reliable to detect these strains because high
AmpC production has little effect on cefepime
activity. In this study out of 45 isolated E.coli
strains 14 (31.1%) were AmpC producers by
AmpC test, 3 (6.7%) were AmpC &ESBL coproducers by cefepime/ cefepime clavulanic
E.test, 24 (53.3%) were pure ESBL by TZ/TZL
or CT/CTL E.test. This was in agreement with
Singhal et.,al [28] who reported AmpC enzyme
production in 36% of E.coli isolates by AmpC
test,also Sinha et.,al[29] reported AmpC
production in 24% and co-production of ESBL
and AmpC enzymes in 8%. Stürenburg et
al[30]evaluated the performance of the cefepime
clavulanate ESBL E test to detect AmpC
&ESBL co-produers in an Enterobactriaceae
strain collection. The ESBL E test was 98%
sensitive with cefepime-clavulanate.
Vol. 22, No. 2
CONCLUSION
1-
It is important to know the prevalence of
ESBL,
AmpC
producing
and
ESBL&AmpC co-producing organisms so
that judicious use of antibiotics could be
done.
2- CHROMagar™ ESBL media detect ESBL
producers from clinical specimen and give
rapid presumptive identification by means
of colony colour at 24h with good
sensitivity and specificity.
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28. Singhal, S., Mathur, T., Khan, S. and
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for AmpC beta lactamases in tertiary care
65
‫‪Vol. 22, No. 2‬‬
‫اﻟﺘﻌﺮف اﻟﺴﺮﻳﻊ ﻋﻠﻲ اﻧﺰﻳﻢ اﻟﺒﻴﺘﺎﻻآﺘﺎﻣﻴﺰ ذات اﻟﻄﻴﻒ اﻟﻤﻤﺘﺪ اﻟﻤﻘﺎوم ﻟﻠﻤﻀﺎدات اﻟﺤﻴﻮﻳﻪ ﻓﻲ ﺑﻜﺘﻴﺮﻳﺎ‬
‫اﻻﻳﺸﺮﻳﺸﻴﺎ آﻮﻻي ﻓﻲ ﻣﺮﺿﻲ اﻟﻤﺴﺎﻟﻚ اﻟﺒﻮﻟﻴﻪ ﺑﻤﺴﺘﺸﻔﻲ ﺑﻨﻬﺎ اﻟﺠﺎﻣﻌﻲ‬
‫د اﻳﻨﺎس ﺷﻌﺒﺎن ﺧﺎﻃﺮ ﻗﺴﻢ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻲ واﻟﻤﻨﺎﻋﻪ‪ -‬آﻠﻴﻪ ﻃﺐ ﺑﻨﻬﺎ – ﺟﺎﻣﻌﻪ ﺑﻨﻬﺎ‬
‫د ﺣﻤﻮدﻩ وهﻴﺐ ﺷﺮﻳﻒ ﻗﺴﻢ اﻟﻤﺴﺎﻟﻚ اﻟﺒﻮﻟﻴﻪ‪ -‬آﻠﻴﻪ ﻃﺐ ﺑﻨﻬﺎ – ﺟﺎﻣﻌﻪ ﺑﻨﻬﺎ‬
‫ﺗﻬ ﺪف ه ﺬﻩ اﻟﺪراﺳ ﻪ اﻟ ﻲ ﺗﺤﺪﻳ ﺪ ﻣ ﺪي اﻧﺘ ﺸﺎر اﻧ ﺰﻳﻢ اﻟﺒﻴﺘﺎﻻآﺘ ﺎﻣﻴﺰ ذات اﻟﻄﻴ ﻒ اﻟﻤﻤﺘ ﺪ اﻟﻤﻘ ﺎوم ﻟﻠﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﻪ و اﻧ ﺰﻳﻢ‬
‫اﻻﻣﺐ ﺳﻲ واﻳﻀﺎ وﺟﻮد اﻻﻧﺰﻳﻤﻴﻦ ﻣﻌﺎ ﻓﻲ ﺑﻜﺘﻴﺮﻳ ﺎ اﻻﻳﺸﺮﻳ ﺸﻴﺎ آ ﻮﻻي اﻟﻤﻌﺰوﻟ ﻪ ﻣ ﻦ ﻋﻴﻨ ﺎت اﻟﺒ ﻮل اﻟﺨﺎﺻ ﻪ ﺑﻤﺮﺿ ﻲ اﻟﻤ ﺴﺎﻟﻚ‬
‫اﻟﺒﻮﻟﻴﻪ ﻓﻲ ﻣﺴﺘﺸﻔﻲ ﺑﻨﻬﺎ اﻟﺠﺎﻣﻌﻲ وآﻤﺎ ﺗﻬﺪف اﻟﺪراﺳﻪ اﻳﻀﺎ ﺗﻘﻴﻴﻢ اﺳﺘﺨﺪام ﻣﺴﺘﻨﺒﺖ اﻟﻜﺮوم اﺟﺎر ﻓﻲ اﻟﻜ ﺸﻒ اﻟ ﺴﺮﻳﻊ ﻋ ﻦ اﻧ ﺰﻳﻢ‬
‫اﻟﺒﻴﺘﺎﻻآﺘﺎﻣﻴﺰ ذات اﻟﻄﻴﻒ اﻟﻤﻤﺘﺪ اﻟﻤﻘﺎوم ﻟﻠﻤﻀﺎدات اﻟﺤﻴﻮﻳﻪ‪ .‬هﺬا وﻗﺪ ﺧﻀﻊ آﻞ ﻣﺮﺿﻲ اﻟﺪراﺳﻪ اﻟﻲ ﻣﻌﺮﻓﻪ اﻟﺘﺎرﻳﺦ اﻟﻤﺮﺿﻲ و‬
‫اﻟﻔﺤﺺ اﻻآﻠﻴﻨﻴﻜﻲ واﻳﻀﺎ اﻻﺧﺘﺒﺎرات اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻴﻪ اﻟﺘﺎﻟﻴﻪ‪:‬‬
‫ﺗﺠﻤﻴﻊ ﻋﻴﻨﻪ ﺑﻮل ﻣﻦ آﻞ ﻣﺮﻳﺾ‬
‫زرع ﻋﻴﻨﻪ اﻟﺒﻮل ﻋﻠﻲ ﻣﺴﺘﻨﺒﺖ اﻟﻜﻠﻴﺪ واﻟﻜﺮوم اﺟﺎر اﻟﺨﺎص ب اﻟﻜﺸﻒ ﻋﻦ اﻧﺰﻳﻢ اﻟﺒﻴﺘﺎﻻآﺘﺎﻣﻴﺰ ذات اﻟﻄﻴﻒ اﻟﻤﻤﺘ ﺪ اﻟﻤﻘ ﺎوم‬
‫ﻟﻠﻤﻀﺎدات اﻟﺤﻴﻮﻳﻪ واﺟﺎر اﻟﻤﺎآﻮﻧﻜﻲ اﻟﻤﻀﺎف اﻟﻴﻪ ‪٢‬ﻣﻠﻴﺠﺮام‪ /‬ﻟﺘﺮ ﻣﻦ ﻋﻘﺎر اﻟﺴﻴﻔﺘﺎزﻳﺪﻳﻢ‬
‫اﻟﺘﻌ ﺮف ﻋﻠ ﻲ اﻧ ﺰﻳﻢ اﻟﺒﻴﺘﺎﻻآﺘ ﺎﻣﻴﺰ ذات اﻟﻄﻴ ﻒ اﻟﻤﻤﺘ ﺪ اﻟﻤﻘ ﺎوم ﻟﻠﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﻪ ﺑﻮاﺳ ﻄﻪ اﻟﻜ ﺸﻒ اﻟﻤﺒ ﺪﺋﻲ ﺛ ﻢ اﻟﻜ ﺸﻒ‬
‫اﻟﺘﺎآﻴﺪي ﺑﻮاﺳﻄﻪ اﺧﺘﺒﺎر اي ﺗﺴﺖ‬
‫اﻟﺘﻌﺮف ﻋﻠﻲ اﻧﺰﻳﻢ واﻻﻣﺐ ﺳﻲ ﺑﻮاﺳﻄﻪ اﺧﺘﺒﺎر اﻻﻣﺐ ﺳﻲ‬
‫اﻟﺘﻌﺮف ﻋﻠﻲ اﻧﺰﻳﻤﻲ اﻟﺒﻴﺘﺎﻻآﺘﺎﻣﻴﺰ ذات اﻟﻄﻴﻒ اﻟﻤﻤﺘﺪ اﻟﻤﻘﺎوم ﻟﻠﻤﻀﺎدات اﻟﺤﻴﻮﻳﻪ واﻻﻣﺐ ﺳﻲ ﺑﻮاﺳ ﻄﻪ اﺧﺘﺒ ﺎر ﺳ ﻴﻔﻴﺒﻴﻢ اي‬
‫ﺗﺴﺖ‬
‫وﻣﻦ ﻧﺘﺎﺋﺞ اﻟﺒﺤﺚ وﺟﺪ اﻧ ﻪ ﻣ ﻦ ﺑ ﻴﻦ ال‪ ٤٥‬اﻳﺸﺮﻳ ﺸﻴﺎ آ ﻮﻻي ‪ (%٥٣.٣)٢٤‬ﺗﺤﺘ ﻮي ﻋﻠ ﻲ اﻧ ﺰﻳﻢ اﻟﺒﻴﺘﺎﻻآﺘ ﺎﻣﻴﺰ ذات اﻟﻄﻴ ﻒ‬
‫اﻟﻤﻤﺘ ﺪ اﻟﻤﻘ ﺎوم ﻟﻠﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﻪ )‪ 26 (45.7%‬ﻻ ﺗﺤﺘ ﻮي ﻋﻠ ﻲ ﻧﻔ ﺲ اﻻﻧ ﺰﻳﻢ‪ .‬آﻤ ﺎ وﺟ ﺪ اﻧ ﻪ ﻣ ﻦ ﺑ ﻴﻦ ال ‪ ٢٤‬ﺳ ﻼﻟﻪ ﺑﻜﺘﻴﺮﻳ ﺎ‬
‫اﻻﻳﺸﺮﻳ ﺸﻴﺎ آ ﻮﻻي اﻟﺘ ﻲ ﺗﺤﺘ ﻮي ﻋﻠ ﻲ اﻧﺰﻳﻤ ﻲ اﻟﺒﻴﺘﺎﻻآﺘ ﺎﻣﻴﺰ ذات اﻟﻄﻴ ﻒ اﻟﻤﻤﺘ ﺪ اﻟﻤﻘ ﺎوم ﻟﻠﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﻪ ‪(%٣٧.٥)٩‬و‬
‫‪ (%٦٢.٥)١٥‬ﻋﺰﻟﺖ ﻣﻦ ﻣﺮﺿﻲ ﻋﺪوي اﻟﻤﺴﺎﻟﻚ اﻟﺒﻮﻟﻴﻪ اﻟﻤﻜﺘﺴﺒﻪ ﻣﻦ اﻟﻤﺠﺘﻤﻊ وﻣﻦ اﻟﻤﺴﺘﺸﻔﻲ ﻋﻠﻲ اﻟﺘﻮاﻟﻲ‪.‬‬
‫وﻓﻲ هﺬﻩ اﻟﺪراﺳﻪ وﺟﺪ ان اﺧﺘﺒﺎر اﻟﺤﺴﺎﺳﻴﻪ ﻟﻠﺰرع ﻋﻠﻲ ﻣﺴﺘﻨﺒﺖ اﻟﻜﺮوم اﺟﺎر اﻟﺨﺎص ب اﻟﻜ ﺸﻒ ﻋ ﻦ اﻧ ﺰﻳﻢ اﻟﺒﻴﺘﺎﻻآﺘ ﺎﻣﻴﺰ‬
‫ذات اﻟﻄﻴﻒ اﻟﻤﻤﺘﺪ اﻟﻤﻘﺎوم ﻟﻠﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﻪ واﺟ ﺎر اﻟﻤ ﺎآﻮﻧﻜﻲ اﻟﻤ ﻀﺎف اﻟﻴ ﻪ ‪٢‬ﻣ ﺞ\ﻟﺘ ﺮ ﻣ ﻦ ﻋﻘ ﺎر اﻟ ﺴﻴﻔﺘﺎزﻳﺪﻳﻢ آ ﺎن ‪%١٠٠‬‬
‫ﺑﻴﻨﻤ ﺎ آﺎﻧ ﺖ اﻟﺨ ﺼﻮﺻﻴﻪ ‪ 80.8%‬و‪ %٨٧.٥‬ﻋﻠ ﻲ اﻟﺘ ﻮاﻟﻲ‪ .‬وﺟ ﺪ اﻳ ﻀﺎ اﻧ ﻪ ﺗ ﻢ ﻋ ﺰل ‪ (%٣١.١)١٤‬ﺳ ﻼﻟﻪ اﻳﺸﺮﻳ ﺸﻴﺎ آ ﻮﻻي‬
‫ﺗﺤﺘﻮي ﻋﻠﻲ اﻧﺰﻳﻢ اﻣﺐ ﺳﻲ ﺑﻴﻨﻤﺎ ‪ (%٨.٩) ٤‬ﺗﺤﺘﻮي ﻋﻠﻲ اﻧﺰﻳﻤﻲ اﻟﺒﻴﺘﺎﻻآﺘﺎﻣﻴﺰ ذات اﻟﻄﻴﻒ اﻟﻤﻤﺘﺪ اﻟﻤﻘﺎوم ﻟﻠﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﻪ‬
‫واﻻﻣﺐ ﺳﻲ‪.‬‬
‫وﻳ ﺴﺘﺨﻠﺺ ﻣ ﻦ ه ﺬﻩ اﻟﺪراﺳ ﻪ ان ﻣ ﻦ اﻟ ﻀﺮوري ﻣﻌﺮﻓ ﻪ ﻣ ﺪي اﻧﺘ ﺸﺎرﺑﻜﺘﻴﺮﻳﺎ اﻳﺸﺮﻳ ﺸﻴﺎ آ ﻮﻻي اﻟﺘ ﻲ ﺗﺤﺘ ﻮي ﻋﻠ ﻲ اﻧ ﺰﻳﻢ‬
‫اﻟﺒﻴﺘﺎﻻآﺘﺎﻣﻴﺰ ذات اﻟﻄﻴﻒ اﻟﻤﻤﺘﺪ اﻟﻤﻘﺎوم ﻟﻠﻤﻀﺎدات اﻟﺤﻴﻮﻳ ﻪ واﻧ ﺰﻳﻢ اﻻﻣ ﺐ ﺳ ﻲ او ﺗﻮاﺟ ﺪهﻤﺎ ﻣﻌ ﺎ وذﻟ ﻚ ﻟﻴ ﺘﻢ اﻻﺳ ﺘﺨﺪام اﻻﻣﺜ ﻞ‬
‫ﻟﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﻪ ﻣ ﻦ ﻗﺒ ﻞ اﻻﻃﺒ ﺎء اﻟﻤﻌ ﺎﻟﺠﻴﻦ‪ .‬وﻳ ﺴﺘﺨﻠﺺ اﻳ ﻀﺎ ان ﻣ ﺴﺘﻨﺒﺖ اﻟﻜ ﺮوم اﺟ ﺎر اﻟﺨ ﺎص ب اﻟﻜ ﺸﻒ ﻋ ﻦ اﻧ ﺰﻳﻢ‬
‫اﻟﺒﻴﺘﺎﻻآﺘﺎﻣﻴﺰ ذات اﻟﻄﻴﻒ اﻟﻤﻤﺘﺪ اﻟﻤﻘﺎوم ﻟﻠﻤﻀﺎدات اﻟﺤﻴﻮﻳﻪ ﻳﻌﺪ ﻣﻦ اﻟﻄﺮق ﺟﻴﺪﻩ اﻟﺤﺴﺎﺳﻴﻪ واﻟﺨ ﺼﻮﺻﻴﻪ ﻟﻠﻜ ﺸﻒ اﻟ ﺴﺮﻳﻊ ﻋ ﻦ‬
‫اﻟﺒﻜﺘﻴﺮﻳﺎ اﻟﺘﻲ ﺗﺤﺘﻮي ﻋﻠ ﻲ اﻧ ﺰﻳﻢ اﻟﺒﻴﺘﺎﻻآﺘ ﺎﻣﻴﺰ ذات اﻟﻄﻴ ﻒ اﻟﻤﻤﺘ ﺪ اﻟﻤﻘ ﺎوم ﻟﻠﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﻪ ﺑﻌ ﺪ ‪ ٢٤‬ﺳ ﺎﻋﻪ ﻣ ﻦ اﻟ ﺰرع ﻋ ﻦ‬
‫ﻃﺮق ﻟﻮن اﻟﺒﻜﺘﻴﺮﻳﺎ اﻟﻤﺰروﻋﻪ‬
‫‪66‬‬
Vol. 22, No. 2
Expression of MMTV-like env gene in Egyptian
Breast Cancer Patients
Mohamed M. Hafez1, Zeinab K. Hassan1 Mahmoud M. Kamel2,
Mahmoud N. ElRouby1 and Abdel Rahman N.Zekri1
1
Virology and Immunology Unit, Cancer Biology Department, 2Clinical Pathology
Department, National Cancer Institute, Cairo University, Cairo, Egypt
ABSTRACT
Mouse mammary tumor virus (MMTV) causes breast cancer in mice. DNA sequences related to MMTVlike env gene is detected in human breast cancer (BC) tissue suggesting its etiology in human BC. The
objective of our study was to assess the significance of MMTV-like env gene in Egyptian BC women. One
hundred and fifty archival formalin-fixed paraffin embedded breast tissues were used and divided into 2
groups; group one included 100 malignant, group two included50 benign tissues. To amplify the MMTVlike env gene, semi-nested PCR was used and to confirm the homology with the MMTV genome direct
sequencing was used. MMTV-like env was efficiently detected in36%ofmalignantand 4% of benign breast
tissues. Sequence analysis was evident revealed 96% homology with the MMTV genome, but no other
significant similarities with the human genome. The presence of the viral sequences was associated
significantly with estrogen and progesteron positive cases and insignificantly the other pathological
parameters studied. The molecular analysis of breast cancer tissue confirmed the presence of MMTV-like
env sequences with significant high percentagesin cancerous tissues than in benign one. These data
raising the possibility that MMTV viral infection may be involved in the neoplastic process.
(HMTV) and its relation to the biological
characteristic of human breast tumor has been
supposed for years (11). HMTV was cloned from
two MMTV env-like positive human breast
cancer patients. HMTV genome was integrated
in the breast cancer cell line positive for MMTV
env-like, but not in normal human breast
cells(12). Several studies showed a significant
correlation between the presence of MMTV–
like env and the expression of lamina in
receptor, a marker for human BC invasiveness
and poor prognosis(13-15). Gene sequences
homologous to MMTV envelope gene have
been reported in 32–74% of human BC samples
in United States, Italy, Australia and Tunisia
contained, compared to less than 2% in normal
breast tissues(15-18).Seventy percent of the
complete MMTV-like virus genome identified
in the breast cancer tissues has 91-99% a
sequence homology to MMTV from mouse
mammary tumors (12, 19).MMTV env gene-like
sequence is present more in breast cancers with
a family history of breast cancer or from certain
geographical locations(19).The purpose of this
study is to investigate the presence and
correlation of MMTV to the Egyptian breast
cancers.
INTRODUCTION
Breast Cancer is a worldwide cancer(1,2) with
approximately 1.38 million new diagnosed
cases in year 2008(3).Incidence rates were much
higher in more developed countries compared to
less developed countries(3). In Egypt, breast
cancer is the most common cancer among
women, representing 18.9% of total cancer
cases (35.1% in women and 2.2% in men)
according to National Cancer Institute cancer
registry(4). Environmental and epidemiological
factors that contribute to breast cancer are
poorly understood, and the identified etiological
factor is the hereditary transmission of some
predisposing genes, such as BRCA genes(5).
Mouse mammary tumor virus (MMTV) is a
retrovirus of9 kb in length, with envelope
proteins (env) used for its entry via binding with
cell surface molecules(6). Mouse mammary
tumor virus is transmitted horizontally through
the milk (exogenous or milk-borne virus) to
susceptible offspring or vertically through the
germ line (endogenous provirus). The
endogenous or exogenous origins of MMTV
sequences were investigated by analyzing
cancer and normal breast tissues from the same
patients(7) MMTV is the suspected cause for
breast cancer as its strong association with
mammary cancer in mice(8,9). MMTV env-like
fragment was found in tumor but not in normal
breast tissues (10).Human mammary tumor virus
MATERIAL & METHODS
One hundred and fifty archival paraffinembedded breast tissues, Egyptian females,
67
were collected from pathology department,
National Cancer Institute, Cairo University,
Egypt. Samples were classified into two groups,
100 malignant and 50 benign. This study was
performed according to Helsinki Declaration
principles. Primary tumor size and axillary
nodal status were obtained from pathological
records. H&E-stained slides were used for
assessment of the histological type. From
pathology report, the mean age for the BC
patients was 55 years (SE 1.12; range 35 to 85
years). A total of 23cases were less than 40
years, 32 cases were 41-50 years and 45 cases
were above 50 years. All BC cases had invasive
ductal carcinoma. Forty five cases had grade III,
35 had grade II and 20 had grade I. Fifty cases
were positive for ER+/PR+ and HER-2-neu+, 20
cases were ER-/PR- negative and HER-2-neu+
positive, 15 cases were ER+/PR+ positive and
HER-2/neu- negative and 15 cases were ER+
and HER-2-neu+ positive and PR- negative.
Detection of MMTV envGene-like Sequences:
The presence of MTV env gene was
detected by semi-nested PCR. For DNA
extraction, two 8µm tissue sections were
examined under microscope and the selected
tumor areas were removed from slides using
scalpel and placed directly into sterile 2ml
Eppendorf tubes. DNA was extracted from
tumor tissue using Recover All nucleic acid
extraction kit (Ambion, CA, USA),and DNA
quality was assessed by measuring the
concentration and ratio using NanoDrop 8000
(Thermo Scientific, USA).Also the DNA quality
was tested by amplifying a 407-base paired (bp)
sequence of the β-globin gene (13).
For detection of MTV envgene, 300 ng of
DNA were used in PCR with outer primers 1X
5’-TGCGCCTTCCCTGACCAGGGG-3’ and
2NR:5’-GTAACACAGGCAGATGTAGG-3’to
amplify a 356-bp fragment. Two microlitre of
the first-PCR product were used in the second
round with the primers pair MMTV 5F: 5’GTATGAAGCAGGATGGGTAGA-3’
and
2NRto amplify a 190-bp fragment. All PCR
amplifications were performed in 25 µl of
master mix (KAPA Taq ready Mix DNA
polymerase) containing 1.5 mM MgCl2, 400
mM of each dNTP, 0.5 U of Taq DNA
polymerase and 300 nM of each sense and
antisense primers. Thermo cycling was
performed in thermal cycler (Life Technology,
Applied Biosystems, USA) starting by
denaturation at 95ºC for 10 min, then 35 cycles
of denaturation at 95 ºC for 30 s, annealing at 58
ºC for 30 s, extension at 72 ºC for 1 min, finally
an extension step at 72 ºC for 10 min. PCR
master mix with primers was used as negative
Vol. 22, No. 2
controls without DNA template. A 2.7 kb envLTR fragment, a positive control for MMTVlike sequences, was used and kindly provided as
a gift by Dr Beatriz G.-T. Pogo (Department of
Microbiology, Mount Sinai School of Medicine,
New York, USA).PCR products were
electrophoresed into 2% agarose gels containing
ethidium bromide, and visualized with UV-light
Fig 1.
DNA sequencing
To validate the MMTV env-like
sequence,all positive PCR samples were
analyzed by direct DNA sequencing. The PCR
products were purified from agarose gels using
QIA quick Purification Kit according to
manufacturer’s instructions (QIAGEN, Hilden,
Germany). Purified PCR products were labeled
with fluorescent dyes using BigDye Terminator
v3.1 Cycle Sequencing Kit (Applied
Biosystems) using 2NR sequence primer,
followed by purification using BigDyeR X
Terminator™ Purification Kiton ABI PRISM
3300(Life Technology, Applied Biosystems,
USA). A consensus sequence was obtained from
each sample by aligning the nucleotide
sequence data obtained from three independent
amplification reactions of the positive samples.
Chromatograms with sharp peaks and quality
values≥20 with little or no background noise
was subjected to Basic Local Alignment Search
(BLAST) as shown in figure 2.
Statistical analysis
The presence of MMTV-like sequences in
tumors were tested for possible association with
clinicopathological data (age, tumor grade,
tumor size, and lymph node status) and
immunohistochemical parameters (ER, PR and
HER2) using Chi-square test, P values of <0.05
were considered statistically significant. All
analyses were carried out with the SPSS 17.0
(for windows) statistical software package.
RESULTS
The MMTV-like env gene was investigated
in 100 cancer and 50 benign formalin fixed
paraffin embedded sections from breast tissues
Egyptianfemale. MMTV-like env sequences
were detected in 36 out of 100(36%) BC and in
4% (2/50)of benign breast tissues.There was an
association betweenclinicopathological data and
the MMTV-like env positive cases. Overall,
correlation was found between the presence of
MMTV-like env sequences and age groups in
which MMTV-like sequences was found in 34.6
% in age group <40 years, 18.7 % in age group
from 41-50 years and 48.9% among age group
>50 years in cancer group. Whereas in benign
68
Vol. 22, No. 2
The sequences of the PCR products of
malignant and benign positive MMTV samples
were identified and aligned with the
corresponding region of the prototype MMTV
and MMTV-like sequences in the GeneBank
Accession Nos. AY152721 and AF243039,
respectively. The sequencing data analysis with
multiple nucleotide alignment showed 95–97%
homology to both MMTV and MMTV-like env
sequences. Furthermore, no similarity was
found when we compared our sequences with
the canine, feline (JN831356 and GQ996603,
respectively) and human genome sequences
available in the GenBank database using
BLAST software from the NCBI website,
indicating that these amplified products were
not of canine, feline, human genomic, or
endogenous retrovirus origin.
tissues was (1/2) 50% in age group from 41-50
years and (1/2) 50% in age group >50 years.
In relation to tumor grade, MMTV-like env
gene was detected in 35% of grade Iandin
34.3% of grade II and in 37.8% of grade III. In
lymph node positive cases 41.7% were positive
for MMTV while 30.8% was found in lymph
node negative cancer tissues. There was a
significant association between the MMTV
infection and the tumor size in which 40% and
3.8% were positive in tumor size <2 cm and >2
cm respectively. There was no significant
difference in MMTV infection and the
hormonal status and HER2 neu in which 26.7
%of ER/PR positive, 40%of HER2/ER positive,
30%of HER2 positive and 40%of HER2/ER/PR
positive cases were positive for MMTV
infection.
Figure 1: PCR products were electrophoresed into 2% agarose gels containing ethidium bromide, and
visualized with UV-light. Line 1 represents PCR marker, line 2 and 3 represent negative samples, lines
from 4 to 6 are positive samples and line 7 and 8 are positive and negative controls respectively.
MMTV AY152721AAATTCTCCTAAGGATCCCAATGATAGAGATTTTACTGCTCTAGTTCCCCATACAGAATTG
60
HMTV AF243039
AAATTCTCCTAAGGATCCCAATGATAGAGATTTTACTGCTCTAGTTCCCCATACAGAATTG 60
BC-1 AATTACTCCTAAGGATCCCAA-GATAGAGATTTTACTGCTCTAGTTCCCCATACAGAATTG 60
BC-2 AATTTCTCCTAAGGATCCCAATGATAGAGATTTTACTGCTCTAGTTCCCCATACAGAATTG 60
BC-3 AATATCTCCTAAGGATCCCAA-GATAGAGATTTTACTGCTCTAGTTCCCCATACAGAATTG 60
BC-4 AATAACTCCTAAGGATCCCAATGATAGAGATTTTACTGCTCTAGTTCCCCATACAGAATTG 60
BC-5 AATTTCTCCTAAGGATCCCAATGATAGAGATTTTACTGCTCTAGTTCCCCATACAGAATTG 60
BC-6 AATTACT-CTAAGGATCCCAA-GATAGAGATTTTACTGCTCTAGTTCCCCATACAGAATTG 60
BS-1 AATTTCTCCTAAGGATCCCAATGATAGAGATTTTACTGCTCTAGTTCCCCATACAGAATTG 60
BS-2 AATATCTCCTAAGGATCCCAA-GATAGAGATTTTACTGCTCTAGTTCCCCATACAGAATTG 60
BS-3 AATTACTCCTAAGGATCCCAATGATAGAGATTTTACTGCTCTAGTTCCCCATACAGAATTG 60
BS-4 AATATCTCCTAAGGATCCCAA-GATAGAGATTTTACTGCTCTAGTTCCCCATACAGAATTG 60
** **************** *************************************
MMTV AY152721 TCGCTTAGTTGCAGCCTCAAGATATCTTATTCTCAAAAGGCCAGGATTTCAAGAACATGA 120
HMTV AF243039 TCGCTTAGTTGCAGCCTCAAGATATCTTATTCTCAAAAAGCCAGGATTTCAAGAACATGA 120
BC-1
TCGCTTAGTT-CAGCCTCAAGATATCTTATTCTCAAA--GCCAGGATTTCAAGAACATGA 120
BC-2
BC-3
BC-4
BC-5
BC-6
BC-7
BC-8
BS-1
BS-2
BS-3
BS-4
********** ************************* ********************
69
Vol. 22, No. 2
MMTV AY152721 CATGATTCCTACATCTGCCTGTGTTACTTACCCTTATGCCATATTATTAGGATTACCTCA180
HMTV AF243039 CATGATTCCTACATCTGCCTGTGTTACTTACCCTTATGCCATATTATTAGGATTACCTCA 180
BC-1
CATGATTCCTACATCTGCCTGTGTTACTTACCCTTATG-CATATTATTAGGATTACCTCA 180
BC-2
BC-3
BC-4
CATCATTCCTACATCTGCCTGTGTTACTTACCCTTATG-CATATTATTAGGATTACCTCA 180
BC-5
BC-6
BC-7
BC-8
BS-1
BS-2
BS-3
BS-4
*** ******************************** ********************
Figure 2: Nucleotide alignment of MMTV-like env sequences from eight human breast carcinomas and 4
benign with mouse mammary tumor virus (MMTV) and human mammary tumor like virus (HMTV)
sequences retrieved from GenBank database (Accession No.
patients(12). Different MMTV variants can infect
human cells, however other unknown
endogenous retroviral sequences present in
breast carcinomas cannot be lined out(24).
In this study, there was a higher
percentages of MMTV infection (40%) in small
tumor size <2 cm than in large tumor size >2 cm
(3.8%). Concerning the lymph node status and
tumor grade no significant correlation were
found between clinical parameters and the
presence of MMTV-like env similar as stated in
previous studies(15,22,25). Epidermal growth
factor 2 is proto-oncogene is expressed in 30-50
% of human breast cancer and its expression
play role in the cancer development, poor
prognosis and metastasis(26,27).In the current
study, there wasa correlation between MMTV
infection and the hormonal status and HER2 neu
in which 26.7 %of ER/PR positive, 40%of
HER2/ER positive, 30%of HER2 positiveand
40%of HER2/ER/PR positive cases were
positive for MMTV infection. Some studies
failed to detect a correlation between MMTV
infection and the hormonal status(15,28)others
found a significant correlation between the
presence of MMTV-like sequence and ER/PR
status(21,29). A significant correlation was
observed between MMTV-like sequences and
PR in skin cancer but no association found
between MMTV-like env and ER/ PR status in
ovarian and endometrial cancers(30). Studies
found insignificant correlation betweenHER-2
status
and
presence
of
MMTV-like
sequence(15,28). The increase in prevalence of
MMTV-like virus env in breast cancer supports
a possible causal role in breast cancers.
In conclusion this study confirmed the
presence of high prevalence of MMTV-like
sequences in malignant compared to low in
benign breast tissues and suggests that MMTV
infection might be a contributing factor in the
development of breast cancer.
DISCUSSION
Breast Cancer is a worldwide cancer in
women(1,2,20). Etiological factors associated with
BCare genetic alteration and long-term
treatment with estrogens. Mouse mammary
tumor virus env sequence is detected in human
BC tissues and may be a causative agent for the
breast cancer poor prognosis(15,16,21,22).
This study investigated the association of
MMTV with breast cancer in Egyptian women.
The current study reported high percentages of
MMTV env gene in archival BC (36%) samples
than in benign (4%) breast tissues. Similar
studies on BC identified the MMTV-like
sequence in BC cases with 30-40%(10,16,23). Also
studies from Argentina, Italy, and Australia
showed that the MMTV-like sequences are
ranged from31.7% to42.2% in malignant and
less than 2% in benign breast tissues(7,15,22).
In Tunisia, higher levels of MMTV
(74%)was reported in BC(17) showing a
correlation between MMTV-like env and
development of aggressive inflammatory BC.
Low percentage or absence of MMTV-like
envgene was reported in breast cancer
tissues(16,21,22). The contradictions in the
MMTV-like env gene incidences in breast
cancer may be contributed to the detection
methods used or to the specificity of PCR or to
geographic distribution.
The origin and biological significance of
the MMTV envgene-like sequences in humans
are unclear. Detection of virus sequence entirely
in malignant and not in normal breast tissues
suggested its exogenous origin. In the current
study, MMTV-like gene was detected in
malignant breast tissues with 97% homologous
to MMTV and HMMTV but not to any other
known endogenous retroviral sequence.
Similarly data from America and Italy support
the presence of MMTV in 38% of BC
70
Vol. 22, No. 2
11. Callahan,
R.,1996.
MMTV-induced
mutations in mouse mammary tumors: their
potential relevance to human breast cancer.
Breast Cancer Res Treat. 39(1). 33-44.
12. Liu, B., Y. Wang, S.M. Melana, I.
Pelisson, V. Najfeld, J.F. Holland, and
B.G. Pogo,2001. Identification of a
proviral structure in human breast cancer.
Cancer Res. 61(4). 1754-9.
13. Fukuoka, H., M. Moriuchi, H. Yano, T.
Nagayasu, and H. Moriuchi,2008. No
association of mouse mammary tumor
virus-related retrovirus with Japanese
cases of breast cancer. J Med Virol. 80(8).
1447-51.
14. Zammarchi, F., M. Pistello, A. Piersigilli,
R. Murr, C. Di Cristofano, A.G.
Naccarato, and G. Bevilacqua,2006.
MMTV-like sequences in human breast
cancer:
a
fluorescent
PCR/laser
microdissection approach. J Pathol. 209(4).
436-44.
15. Pogo, B.G., S.M. Melana, J.F. Holland,
J.F. Mandeli, S. Pilotti, P. Casalini, and
S. Menard,1999. Sequences homologous to
the mouse mammary tumor virus env gene
in human breast carcinoma correlate with
overexpression of laminin receptor. Clin
Cancer Res. 5(8). 2108-11.
16. Wang, Y., J.F. Holland, I.J. Bleiweiss, S.
Melana, X. Liu, I. Pelisson, A.
Cantarella, K. Stellrecht, S. Mani, and
B.G. Pogo,1995. Detection of mammary
tumor virus env gene-like sequences in
human breast cancer. Cancer Res. 55(22).
5173-9.
17. Levine, P.H., B.G. Pogo, A. Klouj, S.
Coronel, K. Woodson, S.M. Melana, N.
Mourali,
and
J.F.
Holland,2004.
Increasing evidence for a human breast
carcinoma
virus
with
geographic
differences. Cancer. 101(4). 721-6.
18. Ford, C., M. Faedo, W. Delprado, and
W. Rawlinson,2004. Correspondence re:
C. Ford, et al. Mouse mammary tumor
virus-like gene sequences in breast tumors
of Australian and Vietnamese women. Clin.
Cancer Res., 9: 1118-1120, 2003. Clin
Cancer Res. 10(2). 802.
19. Holland, J.F. and B.G. Pogo,2004. Mouse
mammary tumor virus-like viral infection
and human breast cancer. Clin Cancer Res.
10(17). 5647-9.
20. Haya S. Al-Eid, S.B., Ali Al-Zahrani,
2003. Kingdom of Saudi Arabia Ministry of
Health National Cancer Registry Cancer.
21. Hachana, M., M. Trimeche, S. Ziadi, K.
Amara, N. Gaddas, M. Mokni, and S.
Korbi,2008.
Prevalence
and
characteristics
of
the
MMTV-like
Acknowledgements
The authors thank Dr Beatriz G.-T. Pogo
from the Department of Microbiology, Mount
Sinai School of Medicine, New York, USA for
her kind gift of MMTV-like envpositive control.
Conflict of interest: All authors declare that
there is no conflict of interest.
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( MMTV ) ‫اﻟﺘﻌﺒﻴﺮ اﻟﺠﻴﻨﻲ ﻟﺠﻴﻦ ﺷﺒﻴﻪ ﻓﻴﺮوس ﺳﺮﻃﺎن اﻟﺜﺪى ﻟﻠﻔﺌﺮان‬
‫ﻓﻰ ﺣﺎﻻت ﻣﺮﺿﻰ ﺳﺮﻃﺎن اﻟﺜﺪي اﻟﻤﺼﺮﻳﻴﻦ‬
، ١ ‫ ﻣﺤﻤﻮد ﻧﻮر اﻟﺪﻳﻦ اﻟﺮوﺑﻰ‬، ٢ ‫ﻣﺤﻤﻮد ﻣﺤﻤﺪ آﺎﻣﻞ‬، ١ ‫زﻳﻨﺐ ﻗﺮﻧﻲ ﺣﺴﻦ‬، ١ ‫ﻣﺤﻤﺪ ﻣﺤﻤﻮد ﺣﺎﻓﻆ‬
١
‫ﻋﺒﺪ اﻟﺮﺣﻤﻦ ﻧﺒﻮي زآﺮى‬
.‫ اﻟﻤﻌﻬﺪ اﻟﻘﻮﻣﻲ ﻟﻼورام‬، ‫ وﺣﺪة اﻟﻔﻴﺮوﺳﺎت واﻟﻤﻨﺎﻋﺔ ﻗﺴﻢ ﺑﻴﻮﻟﻮﺟﻴﺎ اﻟﺴﺮﻃﺎن‬١
‫ ﻣﺼﺮ‬، ‫ ﺟﺎﻣﻌﺔ اﻟﻘﺎهﺮة‬، ‫ اﻟﻤﻌﻬﺪ اﻟﻘﻮﻣﻲ ﻟﻼورام‬، ‫ ﻗﺴﻢ اﻟﺒﺎﺛﻴﻮﻟﻮﺟﻴﺎ اﻻآﻠﻴﻨﻴﻜﻴﺔ واﻟﻜﻴﻤﻴﺎﺋﻴﺔ‬٢
‫ ﺗ ﻢ اﻟﻜ ﺸﻒ ﻋ ﻦ ﺗﺴﻠ ﺴﻞ ﻟﻠﺤﻤ ﺾ‬.‫ ( ﺳ ﺮﻃﺎن اﻟﺜ ﺪي ﻓ ﻲ اﻟﻔﺌ ﺮان‬MMTV ) ‫ ﻳﺴﺒﺐ ﻓﻴﺮوس ﺳﺮﻃﺎن اﻟﺜﺪى ﻟﻠﻔﺌ ﺮان‬:‫اﻟﺨﻠﻔﻴﺔ و هﺪف اﻟﺪراﺳﺔ‬
‫اﻟﻨﻮوي اﻟﺪﻧﺎ اﻟﻤﺮﺗﺒﻄﺔ ﺑﺸﺒﻴﺔ ﺟﻴﻦ اﻟﻐﻼف ﻟﻔﻴﺮوس اﻟﺜﺪى ﻟﻠﻔﺌﺮان ﻓﻰ أﻧﺴﺠﺔ ﺳﺮﻃﺎن اﻟﺜﺪى ﻋﻨﺪ اﻟﺒﺸﺮﻣﻤﺎ ﻳﺸﻴﺮ اﻧﻪ ﻗﺪ ﻳﻜﻮن اﺣﺪ اﻟﻤ ﺴﺒﺒﺎت‬
‫ ﺗﻬ ﺪف ه ﺬﻩ اﻟﺪراﺳ ﻪ اﻟ ﻰ ﻗﻴ ﺎس ﻣ ﺪى اهﻤﻴ ﻪ وﺟ ﻮد ﺷ ﺒﻴﺔ ﺟ ﻴﻦ اﻟﻐ ﻼف ﻟﻔﻴ ﺮوس اﻟﺜ ﺪى ﻟﻠﻔﺌ ﺮان‬.‫ﻓ ﻲ ﺣ ﺪوث ﺳ ﺮﻃﺎن اﻟﺜ ﺪى ﻋﻨ ﺪ اﻹﻧ ﺴﺎن‬
.‫ ( ﻓﻰ ﺣﺎﻻت ﺳﺮﻃﺎن اﻟﺜﺪى ﺑﺎﻟﺴﻴﺪات اﻟﻤﺼﺮﻳﺎت‬MMTV )
‫ ﺣﺎﻟﻪ ﻣﻦ ﺣﺎﻻت ﺳﺮﻃﺎن‬١٠٠ ‫ ﻣﺠﻤﻮﻋﻪ اﻻوﻟﻰ وﺗﺸﻤﻞ‬:‫ ﺣﺎﻟﻪ وﻗﺴﻤﺖ آﺎﻻﺗﻰ‬١٥٠ ‫ﺗﺤﺘﻮى هﺬﻩ اﻟﺪراﺳﺔ ﻋﻠﻰ ﻋﻴﻨﺎت ﻣﻦ‬:‫اﻟﻤﺮﺿﻰ واﻟﻄﺮق‬
‫ وﺗﻢ اﺳﺘﺨﺪام اﻻﺧﺘﺒﺎرات اﻟﺠﺰﻳﺌﻴﺔ ﻋﻦ ﻃﺮﻳﻖ ﻋﻤﻞ ﺗﻔﺎﻋﻞ اﻧﺰﻳﻢ اﻟﺒﻠﻤﺮﻩ اﻟﺘﺴﻠﺴﻠﻲ اﻟﻨﺼﻒ ﻣﺘﻌ ﺸﺶ‬.‫ ﺣﺎﻟﻪ ﻣﻦ اورام ﺣﻤﻴﺪﻩ ﺑﺎﻟﺜﺪى‬٥٠‫اﻟﺜﺪى و‬
( direct sequencing ) ‫و ﻟﺘﺎآﻴﺪ وﺟﻮد هﺬا اﻟﺠﻴﻦ ﻳﺘﻢ ﻋﻤﻞ اﻟﺘﺴﻠﺴﻞ اﻟﻤﺒﺎﺷﺮ‬. (PCR )
‫ ﻓﻘﻂ ﻣﻦ ﺣ ﺎﻻت اﻻورام اﻟﺤﻤﻴ ﺪﻩ وآ ﺎن ذﻟ ﻚ اﻟﻔ ﺮق‬%٤‫ ﻣﻦ ﺣﺎﻻت ﺳﺮﻃﺎن اﻟﺜﺪى وﻓﻰ‬%٣٦ ‫ ﻓﻰ‬MMTV-like env ‫ ﺗﺒﻴﻦ وﺟﻮد‬:‫اﻟﻨﺘﺎﺋﺞ‬
‫وﻟﻜ ﻦ ﻻ ﺗﻮﺟ ﺪ ﺗ ﺸﺎﺑﻬﺎت هﺎﻣ ًﺔ‬، MMTV ‫ ﻣ ﻊ ﺟﻴﻨ ﻮم‬%٩٦ ‫ وآ ﺎن ﺗﺤﻠﻴ ﻞ اﻟﺘﺴﻠ ﺴﻞ واﺿ ﺢ وآ ﺸﻒ ﻋ ﻦ ﺗﻨ ﺎﻇﺮ ﺑﻨ ﺴﺒﺔ‬.‫ذو دﻻﻟ ﺔ إﺣ ﺼﺎﺋﻴﺔ‬
‫و اآﺘﺸﻔﻨﺎ وﺟﻮد ﻋﻼﻗﻪ ﺗﻮاﻓﻘﻴﻪ ﺑﻴﻦ وﺟﻮد اﻟﻔﻴﺮوس ﻣﻊ ﻣﺴﺘﻘﺒﻼت اﻟﻬﺮﻣﻮﻧ ﺎت اﻟﺨﺎﺻ ﻪ ﺑﻬﺮﻣ ﻮﻧﻰ اﻻﺳ ﺘﺮوﺟﻴﻦ‬.‫اﻷﺧﺮى ﻣﻊ ا ﻟﺠﻴﻨﻮم اﻟﺒﺸﺮي‬
.‫واﻟﺒﺮوﺟﺴﺘﻴﺮون ﻓﻘﻂ‬
‫ ( ﻣﻮﺟﻮد ﺑﻨﺴﺒﻪ آﺒﻴﺮﻩ ﻓﻰ ﺣﺎﻻت اورام اﻟﺜﺪى اﻟﺒ ﺸﺮي ووﺟ ﻮد‬MMTV ) ‫اآﺪت هﺬﻩ اﻟﺪراﺳﺔ ان ﻓﻴﺮوس ﺳﺮﻃﺎن اﻟﺜﺪى ﻟﻠﻔﺌﺮان‬: ‫اﻟﺨﻼﺻﺔ‬
‫ ( وه ﺬا‬PCR ) ‫اﻟﺠﻴﻨﺎت اﻟﺨﺎﺻﻪ ﺑﻬﺬا اﻟﻔﻴﺮوس ﺑﻨﺴﺒﻪ اآﺒﺮ ﻓﻰ ﺣﺎﻻت اﻻورام اﻟﺨﺒﻴﺜﻪ ﻋﻦ اﻟﺤﻤﻴﺪﻩ ﻋﻦ ﻃﺮﻳﻖ ﻋﻤﻞ ﺗﻔﺎﻋﻞ اﻟﺒﻠﻤ ﺮﻩ اﻟﻤﺘﺴﻠ ﺴﻞ‬
.‫ﻗﺪ ﻳﻜﺸﻒ اﻟﺴﺘﺎر ﻋﻦ اﺣﺘﻤﺎﻟﻴﻪ ان ﻳﻜﻮن هﺬا اﻟﻔﻴﺮوس ﻣﻦ اﻟﻤﺴﺒﺒﺎت ﻟﺤﺪوث ﺳﺮﻃﺎن اﻟﺜﺪى ﻋﻨﺪ اﻟﺒﺸﺮ‬
72
Vol. 22, No. 2
A retrospective study of Systemic Lupus Erythematosus (SLE) in
Jazan: Clinical and immunological overview
Maggie Reda Mesbah1, Essam Atwa2, Mousa Meshi3
Assistant Professor of Microbiology and Immunology, Faculty of Medicine, Mansoura
University1, Professor of Rheumatology, Faculty of Medicine, Zagazig University2,
Laboratory and Blood Bank Director; King Fahd Central Hospital (KFCH)-JazanKingdom Saudi Arabia (KSA) 3
ABSTRACT
Introduction: Systemic Lupus Erythematosus (SLE) is a multisystem autoimmune disease of unknown
etiology characterized by the production of non-organ specific autoantibodies directed to nuclear,
cytoplasmic and cell surface antigens It predominantly affects young females has a worldwide
distribution. There are some studies on the characteristics, incidence of SLE from different regions of
Saudi Arabia but studies from the Jazan region of KSA are lacking. Aim of the work: The aim of this
study was to analyze retrospectively the main clinical, and immunologic manifestations of SLE patients
attending the inpatient and outpatient clinics of KFCH in Jazan and to determine the specific
characteristics of organ/system involvement in those SLE patients. Also, detection of incidence and
prevalence of SLE in Jazan. Patients and Methods: This study was done retrospectively and included 101
SLE patients who attended Rheumatology and Nephrology clinics in KFCH between January 2010 and
December 2011. All patients fulfilled the 1997 revised American College of Rheumatology (ACR)
classification criteria for SLE. Files of the all patients were systematically reviewed using a pre-designed
standardized patient sheet. The sheet included: personal data :age,sex…etc, clinical data: arthritis, skin
and hematological manifestations…etc and immunological tests: Antinuclear antibodies (ANA), antidouble strand antibodies (dsDNA), complement 3(C3), complement 4(C4),anticardiolipin antibodies (Acl)
IgM,IgG and β2 glycoprotein (GP) ) IgM, IgG. Results: A total of 101 Saudi patients with SLE were
studied. A female to male ratio was 33: 1. The crude overall point prevalence for SLE on 31 December
2011 was 8.50/100 000 (95% CI = 0.03-0.14). The overall incidence rate (IR) was 1.47/ 100 000 .The IR
was 1.40/100 000 for female (CI 95%= 0.13 – 0.29) and 0.07/100 000 for male (CI 95%= 0.009 –
0.0295). The most common clinical manifestations were arthritis (69%), hematological manifestations
(65%), muco-cutaneous manifestation (40%), fever (32%) , hair loss (24%) and photosensitivity (20%).
The most common associated complications were lupus nephritis (62%), infection (41%), serositis
occurred in (21%) of cases, and secondary antiphospholipid syndrome represents (4%) of cases. ANA
was positive in 100% of cases. The most common form was rim pattern 58(58%), homogenous 27(27%)
and speckeled 15(15%) dsDNA was positive in 99% of casas. Hypocomplementenemia was detected in
(54%) for C3 and (64%) for C4.Anticardiolipin antibodies (Acl) IgM, IgG was detected in (36%) and
(21%) respectively. Beta 2glycoprotein IgM, IgG was detected in (18%) and (13%) of cases respectively.
Conclusion: The prevalence and incidence of SLE were less than other saudi studies. Articular and
hematological manifestations were the main clinical features like other Saudi studies while the cutaneous
manifestations were less detectable than other Saudi and many other studies. The renal involvement,
remained the major cause of morbidity and mortality among our study group. Higher detection of dsDNA
and hybo-complementenemia may reflect more severe disease. This study may be a base for more studies
in the future. In the future considerable effort must be spent on lupus education and medical care in
Jazan aiming to improve the quality of care of patients with SLE aiming to decrease the morbidity and
complications.
females and manifests with a wide spectrum of
clinical and immunological abnormalities(2).The
inflammation
results from production of
autoantibodies that attack cells of host organs
including the skin, muscles, joints, blood,
kidneys, brain and other tissues(3).
There is a wide variation in the natural
history of SLE among different ethnic and
geographical groups(4).
INTRODUCTION
Systemic Lupus Erythematosus
is a
multisystem autoimmune disease of unknown
etiology characterized by the production of nonorgan specific autoantibodies directed to
nuclear, cytoplasmic and cell surface antigens
that may lead to a wide range of tissue
It predominantly affects young
injuries.(1)
73
Vol. 22, No. 2
test were used. The hospital laboratory used
commercially available routine assays for
immunological and other laboratory tests. The
latter file data were then assessed for the
development of complications (e.g., renal
failure, thrombosis, infections). Diagnoses were
performed on clinical grounds and verified by
the appropriate laboratory techniques.
Patient characteristics
The following features were recorded (1)
age, (2) locality, (3) gender (we calculate the
female to male ratio) and (4) age at the time of
diagnosis which is defined as the time at which
a patient fulfilled at least four ACR criteria (12).
(5) disease duration defined as the time interval
between date of diagnosis and end of the study
or death of patient.
Clinical features
The ever presence or absence of different
clinical
manifestations
including
mucocutaneous, musclo-skeletal, serositis,
fever, renal, neurological and hematological
manifestations were meticulously checked from
the patients medical records.
The clinical manifestations involving
different organs and systems were defined in
accordance with ACR criteria (12).Diagnosis of
antiphospholipid syndrome had to meet
diagnostic criteria.
Serologic studies
Antinuclear antibodies (ANA): Sera were
screened on a mouth kidney/ stomach liver
substrate by indirect immunofluorescence
(IMMCO-Diagnostics, Italy) in order to
determine the positivity and pattern of
autoantibodies.
Anti ds-DNA: An Enzyme Linked Immune
Sorbent Assay (ELISA) for dsDNA (Diesse,
Italy).
Plasma C3 and C4: Complement 3 and 4
levels
were
measured
by
radial
immunodiffusion method (Dade Behring and
Siemens, Germany).
Anticardiolipin antibodies (Acl) and β2
glycoprotein (GP) :They were measured by
ELISA assays for IgG and IgM (Diesse,Italy).
Both Acl and β2- glycoprotein (GP) were
present at least on two occasions, at a minimum
of three months apart. Interpretation of all
serological test results was considered according
to the manufacturer, s instructions.
Statistics
All data were stored on a main frame
computer and all analyses were performed using
SPSS (Statistical Package for the Social
Sciences) software.(13,14)
Systemic Lupus Erythematosus has a
worldwide distribution. Reports of the incidence
and prevalence of SLE show a considerable
variation between countries and sometimes
within one country (5).
There are some studies on the
characteristics, incidence of SLE from different
regions of Saudi Arabia (3,6-10) but studies from
the Jazan region of KSA are lacking. The
understanding of the distinctive manifestations,
the course and outcome of SLE among different
ethnic groups could lead to a better
understanding of the factors that contribute to
these differences and to the delivery of better
medical care to those populations. Furthermore,
clinical features of SLE have been described
from different geographical regions in the
world, with some clinical differences among
different racial groups. (11)
The present study had attempted to identify
the demographic, immunologic and clinical
features of SLE patients in Jazan.
The aim of this study was to analyze
retrospectively the main clinical, and
immunologic manifestations of SLE patients
attending the inpatient and outpatient clinics of
KFCH in Jazan and to determine the specific
characteristics of organ/system involvement in
those SLE patients. Also, detection of incidence
and prevalence of SLE in Jazan.
PATIENTS & METHODS
Study design (Data collection)
This study was done retrospectively and
included 101 SLE patients who attended
inpatient and outpatient clinics in KFCH-Jazan
KSA between January 2010 and December
2011.The KFCH is a 500-bed referral hospital
in Jazan, KSA. It provides free health service to
all the patients from the southern area of KSA,
and is, thus, the major treating hospital for the
economically weaker segments of the Saudi
population. All studied patients fulfilled the
1997
revised
American
College
of
Rheumatology (ACR) classification criteria for
SLE. The time at which a patient fulfilled at
least four ACR criteria was chosen as the time
of diagnosis. (12) Files of all patients were
systematically reviewed using a pre-designed
standardized patient sheet. The symptoms of
clinical manifestations were extracted from the
first visit or admission to the hospital.
Whenever multiple results existed, the
immunological and laboratory data of the first
74
Vol. 22, No. 2
27-31 95% CI). The median of age at disease
onset was 22 y with (20.61 – 23.23
95%
CI).The median of disease duration was 7y with
(5 – 9 95% CI).
2-Prevalance and incidence:
The crude overall point prevalence for SLE
on 31 December 2011 was 8.50/100 000 (95%
CI = 0.03-0.14). The prevalence among female
was 8.25/ 100,000 (95% CI = 0.94- 1.0). The
prevalence among male 0.25/100,000 (95% CI
=0.17-0.34). (Table 1).
RESULTS
1-Demographic data:
A total of 101 Saudi patients with SLE
residing in the study area were identified. All
patients were fulfilled at least four of the eleven
revised ACR classification criteria.Of the
patients 98 were female (97%) and 3 were male
(3 %) giving a female to male ratio of 33: 1.
Most of patients were from Jazan(33.7%) and
Sabia (24.8%) then Abo-arish and Samta (12%)
each. The median age of patients was 29y (with
Table (1) Prevalence of SLE in Jazan area.
Gender
n
%
Female
98
(97)
Male
3
(3)
Overall
101
(100)
Prevalence/100 000
8.25
0.25
8.50
The incidence was calculated by identifying
the appearance of new SLE patients in the study
area during the last two years (from 1st. Jan.
2010 to 31th. Dec. 2011). The overall incidence
rate (IR) was 1.47/ 100 000 .The IR was
1.40/100 000 for females (CI 95%= 0.13 – 0.29)
and 0.07/100 000 for males (CI 95%= 0.009 –
0.0295).
95% CI
0.94- 1.0
0.17-0.34
0.03-0.14
3-Clinical manifestation
Table (2) summarizes most relevant clinical
manifestations of the study population. The
most common clinical manifestations were
arthritis (69%), hematological manifestations
(65%), muco-cutaneous manifestation (40%),
fever
(32%),
hair
loss
(24%)
and
photosensitivity
(20%).
Less
common
manifestations included oral ulcers (9%), malar
rash (8%).The least common presentations was
lymphadenopathy (2%).
Table (2): The clinical manifestations of the studied group.
Manifestations
No of Patients(101)
%
1-Arthritis/Arthralgia
69
69
2-Mucocutaneous
40
40
Malar rash
8
8
Photosensitivity
20
20
Discoid Lupus
3
3
Mouth ulcers
9
9
65
65
3-Hematological
Anemia
50
50
Leucopenia
21
21
Thrombocytopenia
39
39
32
32
4-Fever
24
24
5-Hair loss
62
62
6-Lupus nephritis
41
41
7-Infections
2
2
Neuropsychatric 621
21
7-Serositis
4
4
8-2ry APL syndrome
2
2
9- Lymphadenopathy
5
5
10-Others
75
95% CI
0.59-0.77
0.30 – 0.49
0.03 – 0.13
0.12 – 0.28
0.003– 0.063
0.03 – 0.14
0.55-0.74
0.40-0.59
0.13-0.29
0.29-0.48
0.23 -0.41
0.15 – 0.32
0.52-0.71
0.31-0.50
0.01 – 0.05
0.13-0.29
0.002-0.077
0.007– 0.047
0.007 – 0.091
The most common associated complications
were lupus nephritis (62%), infections (41%),
serositis occurred in (21%) of cases, secondary
antiphosphlipid syndrome represents (4%) and
neuropsychatric complications (2%) of cases.
Vol. 22, No. 2
4-Immunological tests:
Table (3) summarizes the results of the
immunological tests of the study group.
Table (3): Immunological profile of SLE patients.
Immunological test
Positive
No= 101
101
1-ANA
100
2-dsDNA
54
3-Low C3
64
4-Low C4
36
5-Acl IgM
21
6-Acl IgG
18
2 gly IgMβ713
2 gly IgGβ8-
95%CI
%
(100)
(99)
(54)
(64)
(36)
(21)
(18)
(13)
1.00-1.00
0.97-1.00
0.44-0.63
0.54-0.73
0.26-0.45
0.13-0.29
0.10-0.25
0.06-0.19
ANA was positive in 100% of cases. The
most common form was rim pattern (Fig.1)
58(58%), homogenous 27(27%) (Fig.2) and
speckeled 15(15%).dsDNA was positive in 99%
of cases. Hypocomplementenemia was detected
in (54%) for C3 and (64%) for
C4.Anticardiolipin antibodies IgM, IgG was
detected in (36%) and (21%) respectively. Beta
2glycoprotein IgM, IgG was detected in (18%)
and (13%) of cases respectively.
Fig.(2) ANA by Indirect Immunoflouresence
(Homogenous pattern).
Comparison of the results of our study with
different results on SLE in KSA and other
studies are shown in table 4.
Fig.(1) ANA by Indirect Immunoflouresence
(Rim pattern).
76
Vol. 22, No. 2
Table (4): Comparison of clinical and laboratory features of SLE patients from different ethnicities worldwide.
Feature
Mesbah
Alballa
Al-Rayes
Heller
Arfaj and
Houman
Uthman
et al
et. al
et. al.
et. al.
Khalil
et. al.
et. al
(6)
(3)
(9)
(10)
(15)
(16)
Year
2013
1995
2007
2007
2009
2004
1999
Country
KSAKSAKSAKSAKSATunisia
Lebanon
Ryad
Jedda
Ryad
Jazan
Ryad
101
87
199
93
624
100
100
Sample size
22
25.3
35
24
25.3
32
26
Onset age
% Female
Arthritis
Malar rash
Photosensitivity
Discoid Lupus
Mucus ulcers
Hematological
Anemia
Leucopenia
Thrombocytopenia
Fever
ANA
dsDNA
Low C3
Low C4
aCL
aCL IgM
G aCL Ig
Lupus nephritis
Infection
Neuro-pscychatric
97
69
8
20
3
9
65
50
21
39
32
100
99
54
64
36
21
62
41
2
89.6
91
72
26
18
16
78
33
20.7
98
40
62
26
81.4
91
27.6
21.6
19.09
22.61
16
53.26
65
56
53.7
58.79
36.18
90.3
68
37
22
7
17
85
24
16
58
95
90
62
61
19
90.7
80.4
47.9
30.6
17.6
39.1
82.7
30.1
10.9
30.6
99.7
80.1
45.4
42.2
33.5
49.7
47.9
48
27.6
92
78
63
53
18
12
21
100
59.9
60.8
66
40.6
63.5
43
25
86
95
52
16
19
40
47
10
17
33
87
50
50
19
Secondry APL
Serositis
4
21
-
29.1
-
27
11
27.4
11
45
40
77
Al-Attia
et. al
(17)
1996
UAE
Rabbani
et. al..
(18)
2004
Pakistan
Laustrup
et.. al
(19)
2010
Danish
Lopez and
Mozo
(1)
2003
Spanish
33
26
196
31
94
32.5
94
91
36
42
3
27
45
9
30
21
89.5
97
-
91
79
63
62
13
26
72
10
99
92
-
54
39
87.8
38
29
6
14
19.7
22
26
53
86
74
35
33
26
367
35(Female)
51(Male)
88.28
95.6
73.8
-
41
12
9.5
22.9
-
33
22
45
-
Vol. 22, No. 2
the mean age at diagnosis was reported as midtwenties to early thirties(3,6,9 15,16,18,19).
It has been suggested that age at onset
modifies the expression of the disease in terms
of clinical presentation, pattern of organ
involvement and serological finding.
Although the articular manifestations were
the most common clinical presentation in our
patients (69%), but it is relatively lower than
reported by many other studies from
Arab
KSA(91%,91%,80.4%)(3,6,10)
(78%,95%)(15,16), and Europe ( 79%) (19) and
higher than detected in Asian study (38%)(18),
while it is similar to Heller et al. who detected
arthritis among 68% of his SLE patients.(9)
Mucocutaneous
manifestations
was
presented in 40% of our patients which is the
same as detected in Tunisia (41%)(15) and
Pakistan(46%) (18 )while it is lower than that was
detected in KSA(64.3%) by Arfaj and Khalil
,2009 (10).
Photosensitivity was the commonest
mucocutaneous presentation (20%), followed by
mucosal ulcers (9%) malar rash (8%) and lastly
discoid lupus (3%). A Photosensitivity detection
was similar to that reported by many Saudi
studies (21.6%, 26%, 22%)(3,6,9) but it is less
than that reported in Tunisia (53%)(15) UAE
(42%)(17) and Europe (62%). (19)
Malar rash seems to be less prevalent in our
patients (8%) and less than other Saudi studies
(27.6%,
72%,37%,47.9%)(3,6,9,10)Tunisia
(15)
(63%) ,Lebanon (52%)(16),Pakistan(29%) (18)
and Europe (63%). (19)
Weather this difference is attributable to
different sun exposure pattern due to climate or
cultural dress code of women or due to racial
differences remains unclear.
Discoid lupus was detected in (3%) of our
patients. The incidence of discoid lupus differed
from area to another. It ranged from as low as
(3%0 in UAE study (17), (10%) in Kuwait (21)and
(7 to 18%) in Saudi studies(6,9,10), (14%) in
Asia(18) and (13%) in Europe.(19)
Mucous ulcers was detected among (9%) of
our patients which is less than many studies
either from Saudi Arabia (16%) (6),(19.09%)
(3)
,(39.1%) (10), Lebanon (40%) (16), UAE ( 27%)
(17)
, Pakistan (19.7%)(18) and Europe( 26%) (19).
Hematological
manifestations
were
observed in (65%) of our patients, which is less
than that was detected in Saudi Arabia by
Alballa (78%) (6) and Arfaj and Khalil (82.7%)
(10)
but it is higher than that was reported in
Lebanon(47%) (16), Kuwait (53%) (21) and UAE
(45%). (17) The most common hematological
manifestation
was
anemia
which
is
corresponding to the previous reports from
DISCUSSION
In this present study we analyzed
retrospectively the main clinical and
immunologic manifestations of 101 SLE Saudi
patients attending the inpatient and outpatient
clinics of KFCH in Jazan –KSA and we
determined the specific characteristics of
organ/system involvement in those SLE
patients. Also, we detected the incidence and
prevalence of SLE in Jazan.
This is the first study on SLE that has
compared data from Jazan with other studies in
KSA and other areas of the world.
Although the etiology of SLE is unknown,
there exists much evidence to suggest that
genetic and environmental factors are involved.
Published studies on the incidence and
prevalence rates of the disease support this
view as dramatic difference were found
depending on the ethnic group and the region of
the world analyzed.(1)
In this study, the first performed in Jazan
we found the overall incidence rate (IR) was
1.47/ 100 000 .It is coordinate to data from all
over the world in which IR ranged from 110/100,000.(20) The IR was 1.40/100 000 for
female( 0.13 – 0.29 CI 95%) and 0.07/100 000
for male ( 0.009 – 0.0295 CI 95%).
The crude overall point prevalence for SLE
on 31 December 2011 was 8.50/100 000
inhabitants (95% CI = 0.03-0.14).The
international reported prevalence rates are all
much higher as they range from 20-70/100,000
this is may be due to under reporting in this
area.(20)In KSA Arfaj and Khalil detected also a
high prevalence of 19/100,000. Other data about
incidence and prevalence from Saudi Arabia
and Arab countries are deficient.(10)
Our lower results may be explained by the
lack of recording epidemiological data from the
peripheral areas. Also, we cannot discard that
certain bias in the recruitment of patients may
influence the results among different studies.
This indicates that ethnicity can be superseded
by local environmental factors as a major
determinant for the expression of SLE.
In our patients, registry only 3% of patients
are males while females represents 97% of cases
giving a female to male ratio of 33: 1,
confirming the well- known fact of a female
predominance of SLE(9,10,15,17,19). The high
frequency of SLE among females has been
attributed to differences in the metabolism of
sex hormones and/or gonadotropin releasing
hormone (9).
The median age of disease onset was 22y it
is somewhat earlier than most reports in which
78
Saudi Arabia by Heller (9). Low detection rate of
anemia were reported in Lebanon (10%) (16) and
UAE (9%) (17).
Leucopenia was detected in 21% of cases
which is coordinate with previous reports from
KSA (22.61%, 24%) (3,9) and Pakistan (22%)
18)
.Thrombocytopenia was detected among 39%
of SLE cases which was higher than all other
reports from KSA (16%, 20.7%, 16%,
10.9%)(3,6,9,10),Tunisia (12%) (15), Lebanon
(33%) (16), UAE (21%) (17) Pakistan (26%)(18)
and Europe (10%) (19).
The most notable clinical complications in
SLE patients in our study were renal impairment
(62%), infections (41%), serositis (21%),
secondary antiphospholipid syndrome (4%) and
neuropsychatric manifestations (2%). Our
finding regarding the higher incidence of renal
involvement among our study group is higher
than many other studies in Kuwait (37%) (22),
Pakistan (33%) (18) and Europe (27.9%) (22), but
it is in agreement with previous reports in Saudi
Arabia by Alballa (62%)(6), El- Rayes (53.7%)(3)
and Heller (61%)(9).
Inspite of differences in occurrence of renal
complications in SLE patients from different
geographical locations, renal impairment
remains one of the major causes of morbidity
and mortality in SLE patients globally. Even
with therapeutic advances, lupus nephritis
remains the major complication of SLE and
those patients with renal involvement are at a
higher risk of dying from this disease.(18)
The risk of infection looms like the sword
of Damocles over SLE patients in our study
infections had reported in (41%) of our study
group.
Neuropsychatric
complications
was
detected among (2%) of our patients which is
less than was reported from KSA (19%,26%,
27.6% 36.18% respectively)(9,6,10,3), Tunisia
(25%)(15), Lebanon (19%)(16), UAE (39%)(17)
Pakistan (26%) (18) and Europe (12%) (19).
As regards to the immunological features of
our SLE patients our results showed that ANA
remains the most sensitive and specific test for
SLE diagnosis as it was detected in
(100%)(95% CI= 1.0) of our patients which is
similar to majority of studies in which ANA
approached 100%(6,10,15,19)but it is higher than
that was reported in Lebanon (87%) (16), UAE
(89.5%) (17) and Pakistan (86%) (19). For dsDNA
higher level of detection (99%) than many
studies in KSA (80.1% and , 90%) (10,9), Tunisia
( 59.9%)(15), Pakistan(18) (74%) and Europe
(73.8%)(1) which may be a reflection of
relatively severe form of the disease in our
study.
Vol. 22, No. 2
Hypo-complementenemia was detected in
(54%) and (64%) for C3 and C4 respectively
which may be a reflection of the disease
activity.
Secondary antiphospholipid syndrome was
manifested in 4% of our SLE patients which is
much lower than previously reported from KSA
by El-Rayes et al. (29.1%)(3) and Arfaj and
Khalil (11%)(10). Interestingly, all those patients
had clinical manifestations suggestive of
antiphospholipid syndrome, either they had
history of recurrent abortion or had a history of
deep vein thrombosis..
CONCLUSION
This is the first study in Jazan. The
prevalence and incidence were less than other
studies although data regarding incidence and
prevalence from Saudi studies are lacking.
Additionally, a remarkable gender and age
similarities with other studies in Saudi Arabia
which support the involvement of genetic
factors in the appearance and outcome of the
disease.
Articular
and
hematological
manifestations were the main clinical features
like other Saudi studies although the cutaneous
manifestations were less detectable than other
Saudi, Arab and many other studies. Weather
this difference is attributable to different sun
exposure or racial differences remains unclear.
Renal involvement remains the major cause of
morbidity and mortality among our study group
like many other studies. As regarding the
immunological features ANA detection was
similar to other Saudi and many other studies
and it remains the most sensitive test for SLE
screening. Higher detection of dsDNA and
hybo-complementenemia reflect more severe
form of the disease.
In the future considerable more studies on a
larger scale and more effort must be spent on
lupus investigations and medical care of patients
in Jazan aiming to improve the quality of care of
patients with SLE. More grants and funding are
required for basic and clinical researches on
SLE.
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‫‪Vol. 22, No. 2‬‬
‫دراﺳﺔ اﺳﺘﻌﺎدﻳﺔ ﻟﻤﺮض اﻟﺬﺋﺒﺔ اﻟﺤﻤﺎﻣﻴﺔ اﻟﺠﻬﺎزﻳﺔ ﻓﻲ ﺟﺎزان‪ :‬رؤﻳﺔ إآﻠﻴﻨﻴﻜﻴﺔ و ﻣﻨﺎﻋﻴﺔ‬
‫د‪/‬ﻣﺎﺟﻲ رﺿﺎ ﻣﺼﺒﺎح ‪ & ١‬اد‪/‬ﻋﺼﺎم ﻋﻄﻮة ‪ & ٢‬د‪/‬ﻣﻮﺳﻲ ﻣﻌﺸﻲ‬
‫‪٣‬‬
‫أﺳﺘﺎذ ﻣﺴﺎﻋﺪ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻲ و اﻟﻤﻨﺎﻋﺔ اﻟﻄﺒﻴﺔ آﻠﻴﺔ اﻟﻄﺐ ﺟﺎﻣﻌﺔ اﻟﻤﻨﺼﻮرة ‪ & ١‬أﺳﺘﺎذ اﻷﻣﺮاض اﻟﺮوﻣﺎﺗﻴﺰﻣﻴﺔ‬
‫‪٣‬‬
‫آﻠﻴﺔ اﻟﻄﺐ ﺟﺎﻣﻌﺔ اﻟﺰﻗﺎزﻳﻖ ‪ & ٢‬ﻣﺪﻳﺮ اﻟﻤﺨﺘﺒﺮ وﺑﻨﻚ اﻟﺪم ﺑﻤﺴﺘﺸﻔﻲ اﻟﻤﻠﻚ ﻓﻬﺪ اﻟﻤﺮآﺰي ﺑﺠﺎزان‬
‫اﻟﻤﻘﺪﻣﺔ‪ :‬ان ﻣﺮض اﻟﺬﺋﺒﺔ اﻟﺤﻤﺎﻣﻴﺔ اﻟﺠﻬﺎزﻳﺔ هﻮ ﻣﺮض ﻣﻦ اﻣﺮاض اﻟﻤﻨﺎﻋﺔ اﻟﺬاﺗﻴﺔ و هﻮ ﻳﺼﻴﺐ آﻞ اﺟﻬﺰة اﻟﺠﺴﻢ ﻋﻦ ﻃﺮﻳﻖ‬
‫اﻓﺮاز اﺟﺴﺎم ﻣﻀﺎدة ﺗﻬﺎﺟﻢ آﻞ اﺟﺰاء اﻟﺨﻠﻴﺔ و هﻮ ﻳﺼﻴﺐ اﻹﻧﺎث أآﺜﺮ ﻣﻦ اﻟﺬآﻮرو هﺬﻩ اﻟﺪراﺳ ﺔ ﺗﻤﺜ ﻞ أول دراﺳ ﺔ ﻋﻠ ﻰ ه ﺆﻻء‬
‫اﻟﻤﺮﺿﻲ ﺑﻤﻨﻄﻘﺔ ﺟﺎزان ﺑﺎﻟﻤﻤﻠﻜﺔ اﻟﻌﺮﺑﻴﺔ اﻟﺴﻌﻮدﻳﺔ‪.‬‬
‫هﺪف اﻟﺒﺤﺚ‪ :‬ﺗﻬﺪف هﺬﻩ اﻟﺪراﺳﺔ إﻟﻲ ﻋﻤﻞ ﺗﺤﻠﻴﻞ ﺑﺄﺛﺮ رﺟﻌﻲ ﻋﻠﻲ ﻣﺮﺿﻲ اﻟﺬﺋﺒﺔ اﻟﺤﻤﺎﻣﻴﺔ اﻟﺠﻬﺎزﻳﺔ ﻣﻦ اﻟﻨ ﻮاﺣﻲ اﻹآﻠﻴﻨﻴﻜﻴ ﺔ و‬
‫اﻟﻤﻨﺎﻋﻴﺔ‪.‬‬
‫اﻟﻤﺮﺿﻲ و ﻃﺮق اﻟﺒﺤﺚ‪:‬ﺷﻤﻠﺖ هﺬﻩ اﻟﺪراﺳﺔ ‪ ١٠١‬ﻣﺮﻳﻀﺎ ﺑﺎﻟﺬﺋﺒﺔ اﻟﺤﻤﺎﻣﻴﺔ اﻟﺠﻬﺎزﻳﺔ اﻟﻤﺮاﺟﻌﻴﻦ ﺑﻤﺴﺘﺸﻔﻲ اﻟﻤﻠﻚ ﻓﻬ ﺪ اﻟﻤﺮآ ﺰي‬
‫ﺑﺠ ﺎزان ﺑﺎﻟﻤﻤﻠﻜ ﺔ اﻟﻌﺮﺑﻴ ﺔ اﻟ ﺴﻌﻮدﻳﺔ ﻓ ﻲ اﻟﻔﺘ ﺮة ﻣ ﻦ ﻳﻨ ﺎﻳﺮ ‪ ٢٠١٠‬ﺣﺘ ﻲ دﻳ ﺴﻤﺒﺮ‪ ٢٠١١‬وه ﺆﻻء اﻟﻤﺮﺿ ﻲ ﻳﻨﻄﺒ ﻖ ﻋﻠ ﻴﻬﻢ ﻣﻌ ﺎﻳﻴﺮ‬
‫اﻟﺠﻤﻌﻴ ﺔ اﻷﻣﺮﻳﻜﻴ ﺔ ﻟﻠﺮوﻣ ﺎﺗﻴﺰم اﻟﻤﻌﺪﻟ ﺔ ﻟﻌ ﺎم ‪ ١٩٩٧‬ﻟﺘ ﺸﺨﻴﺺ ﻣﺮﺿ ﻲ اﻟﺬﺋﺒ ﺔ اﻟﺤﻤﺎﻣﻴ ﺔ اﻟﺠﻬﺎزﻳ ﺔ ‪.‬ﺗ ﻢ ﻓﺤ ﺺ ﻣﻠﻔ ﺎت ه ﺆﻻء‬
‫اﻟﻤﺮﺿ ﻲ ﺑ ﺸﻜﻞ ﻣﻨﻬﺠ ﻲ و ﺟﻤ ﻊ اﻟﺒﻴﺎﻧ ﺎت ﻓ ﻲ ورﻗ ﺔ ﻗﻴﺎﺳ ﻴﺔ ﺳ ﺒﻖ ﺗ ﺼﻤﻴﻤﻬﺎ و ﺗ ﺸﻤﻞ اﻟﻤﻌﻠﻮﻣ ﺎت اﻟﺸﺨ ﺼﻴﺔ ﻣﺜ ﻞ اﻟﻌﻤ ﺮ و‬
‫اﻟﺠ ﻨﺲ‪...‬إﻟ ﺦ آﻤ ﺎ ﺗ ﺸﻤﻞ أﻳ ﻀًﺎ اﻷﻋ ﺮاض اﻹآﻠﻴﻨﻴﻴ ﺔ ﻣﺜ ﻞ إﻟﺘﻬ ﺎب اﻟﻤﻔﺎﺻ ﻞ و اﻷﻋ ﺮاض اﻟﺠﻠﺪﻳ ﺔ و اﻟﺪﻣﻮﻳ ﺔ‪ .‬آﻤ ﺎ ﺷ ﻤﻠﺖ أﻳ ﻀﺎ‬
‫اﻹﺧﺘﺒ ﺎرات اﻟﻤﻨﺎﻋﻴ ﺔ اﻟﺘ ﻲ أﺟﺮﻳ ﺖ ﻟﻠﻤﺮﺿ ﻲ و ه ﻲ اﻷﺟ ﺴﺎم اﻟﻤ ﻀﺎدة ﻟﻠﻨ ﻮاة ﺑﺘﺤﻠﻴ ﻞ اﻷﻣﻴﻮﻧﻮﻓﻠﻮرﺳ ﻨﺲ و اﻷﺟ ﺴﺎم اﻟﻤ ﻀﺎدة‬
‫ﻟﻠﺤﻤﺾ اﻟﻨﻮوي اﻟﺪﻳﺆآﺴﻲ رﻳﺒ ﻮزي و اﻟﻜ ﺎردﻳﻮﻟﻴﺒﻴﻦ و ﺑﻴﺘ ﺎ ‪ ٢‬ﺟﻠﻴ ﻮﺑﺮوﺗﻴﻦ ﺑﺘﺤﻠﻴ ﻞ اﻹﻟﻴ ﺰا و اﻟﻌﻮاﻣ ﻞ اﻟﻤﻜﻤﻠ ﺔ ‪ ٤ &٣‬ﺑﺘﺤﻠﻴ ﻞ‬
‫اﻹﻣﻴﻮﻧﻮدﻓﻴﻮﺷﻦ‪.‬‬
‫اﻟﻨﺘﺎﺋﺞ‪ :‬ﻓﻲ ه ﺬﻩ اﻟﺪراﺳ ﺔ اﻟﻠﺘ ﻲ أﺟﺮﻳ ﺖ ﻋﻠ ﻲ ‪ ١٠١‬ﻣﺮﻳ ﻀﺎ ﻣﺜﻠ ﺖ ﻧ ﺴﺒﺔ اﻹﻧ ﺎث‪ :‬اﻟ ﺬآﻮر ‪ ١ : ٣٣‬آﻤ ﺎ آ ﺎن ﻣﻌ ﺪل اﻹﻧﺘ ﺸﺎر ﻓ ﻲ‬
‫دﻳﺴﻤﺒﺮ ‪ ٢٠١١‬هﻮ ‪ ١٠٠٠٠٠ /٨.٥‬ﻣﻦ اﻟﺴﻜﺎن آﻤﺎ ﻣﺜﻠﺖ ﻧﺴﺒﺔ اﻟﺤﺪوث ‪ ١٠٠٠٠٠ :١.٤٧‬ﻣﻨﻬﺎ ‪ ١٠٠٠٠٠ : ١.٤‬ﺑﻴﻦ اﻹﻧﺎث‪.‬‬
‫آﺎن اﻟﻌﺮض اﻷآﺜﺮ ﺷ ﻴﻮﻋًﺎ ه ﻮ إﻟﺘﻬ ﺎب اﻟﻤﻔﺎﺻ ﻞ )‪ ( %٦٩‬ﺛ ﻢ اﻷﻋ ﺮاض اﻟﺪﻣﻮﻳ ﺔ )‪ (%٦٥‬ﻳﻠﻴ ﻪ اﻷﻋ ﺮاض اﻟﺠﻠﺪﻳ ﺔ اﻟﻤﺨﺎﻃﻴ ﺔ‬
‫)‪ (%٤٠‬و ﺳﻘﻮط اﻟﺸﻌﺮ )‪ (%٢٤‬ﺛﻢ اﻟﺤﺴﺎﺳﻴﺔ اﻟﻀﻮﺋﻴﺔ )‪ . (%٢٠‬آﺎﻧﺖ أآﺜﺮ اﻟﻤﻀﺎﻋﻔﺎت ﺷﻴﻮﻋًﺎ ه ﻲ اﻟﺘﻬ ﺎب اﻟﻜﻠ ﻲ اﻟﺤﻤ ﺎﻣﻲ‬
‫)‪ (%٦٢‬و اﻟﻌﺪوى )‪ (%٤١‬اﻹﻟﺘﻬﺎب اﻟﻤﺼﻠﻲ )‪ (%٢١‬ﺛﻢ ﻣﺘﻼزﻣﺔ اﻟﻤﻀﺎدة اﻟﻔﻮﺳﻔﻮرﻳﺔ اﻟﺜﺎﻧﻮﻳﺔ )‪. (%٤‬‬
‫ﺑﺎﻟﻨﺴﺒﺔ ﻹﺧﺘﺒﺎر اﻷﺟ ﺴﺎم اﻟﻤ ﻀﺎدة ﻟﻠﻨ ﻮاة ﻓﻘ ﺪ ﺗ ﻢ رﺻ ﺪ وﺟﻮده ﺎ ﻓ ﻲ )‪ ( %١٠٠‬ﻣ ﻦ اﻟﺤ ﺎﻻت و آ ﺎن اﻟ ﺸﻜﻞ اﻷآﺜ ﺮ ﺷ ﻴﻮﻋًﺎ ه ﻮ‬
‫اﻟ ﺸﻜﻞ اﻹﻃ ﺎرى )‪ (%٥٨‬و ﻳﻠﻴ ﻪ اﻟ ﺸﻜﻞ اﻟﻤﺘﺠ ﺎﻧﺲ )‪ (%٢٧‬ﺛ ﻢ اﻟ ﺸﻜﻞ اﻟﻤ ﻨﻘﻂ )‪ . (%١٥‬آﻤ ﺎ آﺎﻧ ﺖ ﻧ ﺴﺒﺔ اﻷﺟ ﺴﺎم اﻟﻤ ﻀﺎدة‬
‫ﻟﻠﺤﻤﺾ اﻟﺪﻳﺆآﺴﻲ رﻳﺒﻮﺳﻲ هﻲ )‪ (%٩٩‬آﻤﺎ وﺟﺪ أن ﻧﺴﺒﺔ إﻧﺨﻔﺎض ﻋﻮاﻣ ﻞ اﻟﺘﻜﻤﻠ ﺔ اﻟﺜﺎﻟ ﺚ )‪ (%٥٤‬و اﻟﺮاﺑ ﻊ)‪ (%٦٤‬و آﺎﻧ ﺖ‬
‫ﻧﺴﺒﺔ ﺑﻴﺘﺎ ‪ ٢‬ﺟﻠﻴﻜﻮﺑﺮوﺗﻴﻦ إم )‪ (%١٨‬و ﺟﻲ )‪. (%١٣‬‬
‫اﻟﺨﻼﺻﺔ‪ :‬ﺗﻢ رﺻﺪ ﻧﺴﺒﺔ اﻧﺘﺸﺎر و ﺣﺪوث ﻣﺮض اﻟﺬﺋﺒﺔ اﻟﺤﻤﺎﻣﻴﺔ اﻟﺠﻬﺎزﻳﺔ ﺑﻤﻨﻄﻘﺔ ﺟﺎزان ﺑﻨﺴﺒﺔ أﻗ ﻞ ﻣ ﻦ اﻟﺪراﺳ ﺎت اﻷﺧ ﺮى‪ .‬و‬
‫آﺎﻧﺖ اﻷﻋ ﺮاض اﻟﻤﻔ ﺼﻠﻴﺔ و اﻟﺪﻣﻮﻳ ﺔ ه ﻲ اﻷآﺜ ﺮ ﺷ ﻴﻮﻋًﺎ ﺑ ﻴﻦ ه ﺆﻻء اﻟﻤﺮﺿ ﻲ و آﺎﻧ ﺖ اﻷﻋ ﺮاض اﻟﺠﻠﺪﻳ ﺔ أﻗ ﻞ ﻣ ﻦ اﻟﺪراﺳ ﺎت‬
‫اﻷﺧﺮي داﺧﻞ اﻟﻤﻤﻠﻜ ﺔ و ﺧﺎرﺟﻬ ﺎ‪ .‬و آ ﺎن اﻟﺘﻬ ﺎب اﻟﻜﻠ ﻲ اﻟﻨ ﺎﺗﺞ ﻋ ﻦ ﻣ ﺮض اﻟﺬﺋﺒ ﺔ اﻟﺤﻤﺎﻣﻴ ﺔ اﻟﺠﻬﺎزﻳ ﺔ ه ﻮ أه ﻢ اﻟﻤ ﻀﺎﻋﻔﺎت ‪ .‬و‬
‫ﺑﺎﻟﻨﺴﺒﺔ ﻟﻠﺘﺤﺎﻟﻴﻞ اﻟﻤﻨﺎﻋﻴﺔ ﻓﻘﺪ آﺎن ﺗﺤﻠﻴﻞ اﻷﺟﺴﺎم اﻟﻤﻀﺎدة ﻟﻠﻨ ﻮاة ه ﻮ أه ﻢ اﻟﺘﺤﺎﻟﻴ ﻞ اﻟﻤﺸﺨ ﺼﺔ ﻟﻤ ﺮض اﻟﺬﺋﺒ ﺔ اﻟﺤﻤﺎﻣﻴ ﺔ اﻟﺠﻬﺎزﻳ ﺔ‬
‫آﻤﺎ ان زﻳﺎدة رﺻﺪ اﻷﺟﺴﺎم اﻟﻤﻀﺎدة ﻟﻠﺤﻤ ﺾ اﻟﺪﻳﺆآ ﺴﻲ رﻳﺒﻮﺳ ﻲ و إﻧﺨﻔ ﺎض ﻋﻮاﻣ ﻞ اﻟﺘﻜﻤﻠ ﺔ اﻟﺜﺎﻟ ﺚ و اﻟﺮاﺑ ﻊ ﻣﺆﺷ ﺮًا ﻳﻌﻜ ﺲ‬
‫ﺷﺪة اﻟﻤﺮض ‪ .‬ﻟﺬﻟﻚ ﻳﻮﺻﻲ ﺑﺎﺳﺘﻜﻤﺎل اﻟﺪراﺳﺔﻋﻠﻲ هﺬا اﻟﻤﺮض ﻋﻠﻲ ﻧﻄ ﺎق أوﺳ ﻊ ﺑﻬ ﺪف ﺗﻮﺳ ﻴﻊ ﻗﺎﻋ ﺪة اﻟﺒﻴﺎﻧ ﺎت ﺑﻬ ﺪف ﺗﺤ ﺴﻴﻦ‬
‫ﺟﻮدة اﻟﺨﺪﻣﺔ اﻟﻤﻘﺪﻣﺔ ﻟﻬﺆﻻء اﻟﻤﺮﺿﻲ و ﺗﻘﻠﻴﻞ ﺣﺪوث اﻟﻤﻀﺎﻋﻔﺎت ‪.‬‬
‫‪81‬‬
Vol. 22, No. 2
Application of Pulsed Field Gel Electrophoresis and Ribotyping as
Genotypic Methods Versus Phenotypic Methods for Typing of
Nosocomial Infections Caused by Pseudomonas aeruginosa Isolated
from Surgical Wards In Suez Canal University Hospital
Maii Ahmed Abu-Taleb (1); Abeer Ezzat El-Sayed Mohamed (1);
Hassan Nasr El-Eslam (1); Said Hamed Abbadi (1);
Gehan Saddik El-Hadidy (1) and John J LiPuma (2)
Department of Microbiology & Immunology, Faculty of Medicine, Suez Canal
University, Egypt (1) & Department of Pediatrics & Infectious Diseases, Faculty of
Medicine, Michigan University, Ann Arbor, USA (2).
ABSTRACT
Background: Pseudomonas aeruginosa is an opportunistic pathogen causing nosocomial infections in
many hospitals. The aim of this study was to compare the different epidemiological typing techniques and
their effectiveness in typing and discrimination of nosocomial Pseudomonas aeroginosa isolates.
Methods: Seventy eight confirmed nosocomial Pseudomonus aeroginosa out of 1520 different sample
(nosocomial infections & environmental samples) were collected during a period of one year. Six typing
methods were evaluated, utilizing the confirmed 78 Pseudomonas strains, to assess their usefulness as
tools to study the bacterial diversity. The methods used were antibiogram, pyocin typing, serotyping,
extracellular enzyme typing, automated ribotyping and pulsed field gel electrophoresis (PFGE). Results:
The distinctive capacity of four phenotyping methods was determined and compared to PFGE. Resistance
to the antibiotics tested was in the 37.2% to 98.7% range; Imipenem was the most effective, whereas
augmentin, carbenicillin and ceftazidime were the least effective antibiotics. Antibiogram for 78 isolates
discriminate 13 different patterns. Pulsed field gel electrophoresis yielded 56 distinct types of
P.aeruginosa with 100% distinction capacity (78/78) as all the strains were typable. Compared to PFGE,
the distinctive capacities were 88.5% (69/78) for serotyping, 91% (71/78) for Pyocin typing and 100%
(78/78) for automated ribotyping analysis. The results obtained in PFGE, were the easiest to read and
interpret and most discriminating (0.99), followed by the pyocin typing (0.96), whereas ribotyping had
(0.90) discriminatory power. Conclusion: Our results indicated that P.aeruginosa infections in Suez
Canal University Hospital, mainly affect the hospitalized patients in orthopedic wards, surgical wards
and burn units and played a great role in hospital associated infections. Imipenem was the best antibiotic
as far as bacterial resistance is considered. Although, the lack of major PFGE type confirmed that no
P.aeruginosa outbreak, typing results showed that PFGE was the best used typing method regarding high
typability, sensitivity and discriminatory power. The used typing methods showed that cross transmission
and treatment failure were the two main problems for spread of nosocomial infections inside surgical
wards and should be considered to prevent this bacterial infection in medical units.
Keywords: Pseudomonas aeruginosa, Nosocomial infection, Antibiogram, Pyocin typing, Serotyping,
Extracellular Enzyme typing, Automated Ribotyping and Pulsed Field Gel Electrophoresis.
epidemiology, reservoirs and modes of
Bacterial subtyping is a
transmission(6,7).
necessary step to prove or disprove whether a
single isolate or strain of a given bacterial
species is infecting numerous patients(8-11).
Methods that have been used successfully
included PFGE, ribotyping, amplified fragment
length polymorphism (AFLP) analysis and
random
amplified
polymorphic
DNA
polymerase chain reaction (RAPD-PCR) (12, 13).
Various factors such as method reproducibility,
sensitivity and reliability made it difficult for
laboratories that carry out epidemiological
investigations to choose the proper marker that
INTRODUCTION
Nosocomial Pseudomonas aeruginosa
exhibit high rates of resistance to antibiotics and
had held a nearly unchanged position in the rank
order of pathogens causing hospital-related
infections for more than four decades(1-3).
Predisposing conditions for such infections are:
extensive burn, prolonged antibiotic therapy,
intravenous drug abuse, cystic fibrosis of the
lungs and the presence of indwelling foreign
Evidence-based
prevention
devices(4,5).
strategies targeting specific pathogens should be
based on a thorough knowledge of their
83
suits their facilities and at the same time
answers the question in tracing the source of
outbreak (14). The aim of this study was to
compare the different epidemiological typing
techniques and their effectiveness in typing and
discriminating
nosocomial
Pseudomonas
aeroginosa isolates.
Vol. 22, No. 2
rRNA PCR assay uses the primer set PA-SS-F
GGGGGATCTTCGGACCTCA
(nucleotides
189-206)
and
PA-SS-R
TCCTTAGAGTGCCCACCCG
nucleotides
1124-1144) (22).
- Bacterial lysate preparation:
Ten ml of lysis buffer (250 µl of 10% sodium
dodecylsulfate, 50 µl of 10N NAOH and 9.7 ml
distilled water) were prepared in a 15ml conical
flask and vortexed (Cat. #12-812: Fisher,
Pittsburgh, USA). Twenty µl of lysis buffer in a
1.5ml microfuge tube were used for lysis of
each sample. With a disposable inoculating
needle, only a little bit of bacteria (light touch of
the inoculum) added directly to the lysis buffer,
and vigorously taped against the side and
bottom of the microfuge tube to dislodge all the
bacteria and disperse it evenly in the lysis
buffer. The samples heated at 95oC for 15min in
the heating block and pulse centrifuged to
collect the liquid in the bottom. Filtered double
distilled water (ddH2O, 180µl) were added to
each sample and then centrifuged at 13000
(rpm) for 5min. Bacterial lysates were stored at
4oC until used (23). PCR master mix was first
prepared by calculating the volume of each
reagent in the reaction: 15µl of sterile ddH2O
(Cat. #BP561-1: Fisher), DECP treated
(Diethylpyrocarbonate), 2.5µl of 10X PCR
buffer (Cat. #18038-067: Invitrogen, California,
USA), 2.5µl of 2.5mM GeneMate DNTPs (Cat.
#C-5012-4x25: ISCBioExpress, Utah, USA),
1µl for each forward and reverse primers, 1µl of
50mM MgCl2 (Cat. #18038-067: Invitrogen)
and 0.2µl of Taq polymerase (Cat. #18038-067:
Invitrogen). The reagents were vortexed before
use, except the Taq. After adding the Taq, they
were vortexed again and (23µl) aliquots
dispensed to each reaction tube. Two µl
template lysate and positive control (lysate of
strain PA01) then added to the proper tubes, and
placed in the Rapid-Cycler (Idaho Technology
Inc., Utah, USA) thermo-controller. After an
initial denaturation for 2min at 95oC, 25 cycles
were completed, each consisting of 20 sec at
94oC, 20 sec at the appropriate annealing
temperature 54oC, and 40sec at 72oC. A final
extension of 1min at 72oC was applied (21). PCR
products made to run on 1.5% agarose gel. The
gel was placed in a box filled with 0.5X TBE
running buffer. Two µl of loading dye were
added to each PCR sample. Eight µl of each
sample were loaded, along with 3µl of 100bp
ladder as a size marker on either end of the
samples. The gel made to run at 200 volts for
30min, then placed in ethidium bromide stain
for 15-20min. The gel was visualized with UV
illumination using Gel-Doc2000 (Bio-Rad,
MATERIALS AND METHODS
Pseudomonas aeroginosa isolates were
recovered from different1520 nosocomial
samples isolated from patients admitted in
surgical wards at Suez Canal University
Hospital (Ismailia, Egypt) and environmental
samples during one-year period. Different
specimens from patients, clinically diagnosed as
nosocomial infections according to CDC
criteria, 2006(15), were collected aseptically in
Stuart transport medium (BBL Microbiology
Systems, Maryland, USA) and different
environmental samples transferred to the
bacteriology diagnostic laboratory for primary
isolation of P.aeruginosa(16). Cultures were
done on Columbia agar base supplemented with
5% sterile defibrinated sheep blood and on
MacConkey agar (media prepared according to
manufacture, Difco laboratories, Michigan,
USA). Plates were incubated at 35oC for 1824h(17). Isolates were identified as P.aeruginosa
based on colonial morphology, Gram staining,
positive Oxidase reaction(18), API 20NE
biochemical
test
(BioMerieux,
France)
according to manufacture instructions and
typical PCR was used to confirm the diagnosis.
Isolated colonies inoculated a stock tube culture
containing 1.0% nutrient agar (Difco
laboratories, Michigan, USA) and another
preserved in trypticase soy broth with 13%
glycerol at -70oC until use(19,20). Different
epidemiological typing techniques were applied
in this study, as the work was held in the
bacteriology lab at faculty of medicine, Suez
Canal University and the Infectious Disease
Lab, (MSRB-III, University of Michigan, Ann
Arbor, USA).
Typical PCR: Species-specific 16S rRNA PCR
assays rely on primers that anneal to these
signature sequences(21). Relevant 16S rRNA
sequences were available in the GenBank
database, aligned by using the MegAlign
software package (DNASTAR Inc., Madison,
USA). The Pseudomonas genus specific 16S
rRNA PCR assay uses the primer set PA-GS-F
GACGGGTGAGTAATGCCTA (nucleotides
95-113)
and
PA-GS-R
CACTGGTGTTCCTTCCTATA (nucleotides
693-712). While, P.aeruginosa specific 16S
84
California, USA). PCR fragments of 618bp
indicated positive results (24).
1- Antibiogram Typing:
All P.aeruginosa strains were tested for
antimicrobial
susceptibility
against
22
antimicrobial agents (supplied by BBL) using
the standardized agar disc diffusion method
according to the (CLSI, 2007) (25, 26).
2- Serological Typing:
All P.aeruginosa isolates were serotyped by
utilizing the standard commercially prepared
antisera to 20 somatic-O antigens (Difco
laboratories, Michigan, USA), using the slide
agglutination method. Normal rabbit serum
served as a negative control (27).
3- Pyocin Typing:
Pyocin typing was done by the spotting
method(28).
4- Extracellular Enzyme Profile Typing:
Protease activity was assessed by inoculating
protease assay medium (29); the medium used in
the protease assay was prepared as described
by Skindersoe, et al., 2008 (30). Hemolysin was
assessed according to Leone, et al., 2008 (31).
Gelatinase was assessed according to leone, et
al., 2008 and Molinari, et al., 1993 (31, 32).
Fibrinolytic activity, Coagulase, DNase and
Lipase were assessed according to Murray, et
al., 2007 (33). Elastase was assessed according
to Azghani, et al., 2000 (34).
5- Ribosomal DNA Restriction Profiles
(Ribotyping):
DNA extraction and digestion of DNA with
restriction endonuclease EcoRI were done
according to Maniatis, et al., 2001 (35). Southern
blotting technique was done (36). Restriction of
probe used in ribotyping was done according to
Graves, et al., 1991 and Dubois, et al., 2001 (37,
38)
. Digoxigenin labeling of restricted probe,
Labeling of the standard probe and
Hybridization of the nylon transfer membrane
were done as described by Grimont and
Grimont, 1986 (39) and modified by Graves, et
al., 1991 (37). Immunological detection was done
according to Hunter, 1990, Dawson, et al.,
2002, Sarwari, et al., 2004 and Silbert, et al.,
2004 (40-43). Ribotyping was performed using the
Ribo-Printer Microbial Characterization System
(Qualicon,Delaware, USA) according to the
manufacture instructions.
6- PFGE-:
Genomic fingerprinting of P.aeruginosa isolates
was carried out according to the protocol of
Pflaller, et al., 1999 and Speijer et al., (1999) (44,
45)
. Isolates were grown overnight at 37oC on
Muller Hinton (MH, Difco) plates. Using the
side of a sterile disposable inoculating needle,
approximately 25mg of each bacterium was
Vol. 22, No. 2
suspended in 1ml aliquots of EET (100mM
EDTA, 100mM EGTA, 10 mM Tris, pH 8.0)
buffer. Bacteria were centrifuged at 8000rpm
for 2-3min to obtain a pellet. The liquid was
decanted and cells were re-suspended in fresh
1ml EET buffer. The process was repeated
twice more and then bacteria were resuspended
in 0.5 ml TE buffer( 10 mM Tris –HCL, 1mM
EDTA, pH 8.0). The Optical Density was
adjusted (OD) of 0.4-0.47 at wavelength 620nm
(46)
. Ten ml of 2% low melting point (LMP)
agarose were made and allowed to cool to 45oC
for 15min. Equal amounts of LMP agar and
bacterial suspension were added and mixed
until the suspension was even without airbubbles.one hundred µl were dispensed into the
slot of a molding tray and allowed to solidify on
ice for 10-15min. Each set of plugs were
digested in 15ml conical tubes containing 10ml
PEN buffer (10 g N-Lauroyl sarcosine sodium
salt,186 g EDTA and 1mg/ml Protease pH 9.5)
(Cat. #P-6911; Sigma) and incubated overnight
with slow rocking at 37oC (45). The plugs were
then washed for 30min at room temperature 3-4
times in TE buffer (pH 8.0). Prior to use, plugs
could be stored at 4oC in TE buffer for up to a
week, or in SaET (Sarkosyl 1%, 0.5M EDTA &
10mMTris-HCL, pH9.5) buffer for up to a year
(47)
.
The plugs were cut straight with uniform width
and depth (2mm) without dents, nicks and
bubbles. Excess moisture were removed with a
Kim wipe, gently placed in the mouth of 1.5ml
tube and pushed gently into the digestion
mixture containing 1µl of restriction
endonuclease SpeI (5`-ACTAG- 3`). The plugs
were incubated overnight at 37oC. The reaction
was then stopped by addition of 1ml TE buffer.
The remaining plugs saved in 4-5ml of 1X TE
buffer for future use (46). Pulsed field certified
agarose (Bio-Rad) was prepared and kept in
45oC water-bath until needed. Electrophoresis
was performed in the CHEF-DR II system
(Contour clamped homogenous electric field)
(Bio-Rad). The electrophoresis buffer (0.5X
TBE) (45 mM tris-borate, 1mM EDTA pH8.0)
was at 14oC. In order to improve the typeability
and overcome the degradation problem, 50µl
thiourea was added to the running buffer (48, 49).
After the restriction mixture was drained off, the
plugs were placed on the comb with the flat side
of the comb up. Lambda phage c1857s7 was the
internal standard marker ranging from 48.5kb to
1Mb in size. The gel was casted around the
comb that held 15 wells and left to cool at room
temperature for 30min before pulling the comb
off. The wells were covered with 1% plug
sealing agarose and left to solidify at room
85
temperature. After about 20min, the loaded gel
was placed in the electrophoresis chamber,
covered with cooled buffer to a height of 2mm
above gel surface and electrophoresed at 165
volts (5 volts/cm), in the form of a ramp of 1-25
second, and a running time of 25h (45). The gel
was stained with 0.5µg/ml ethidium bromide in
0.5X TBE buffer for 25-30min. The gel was destained with 0.5X TBE for 50min when needed.
The gel was then visualized with UV
illumination. Gel images were digitized using a
Gel Doc2000 gel analyzer (Bio-Rad) and stored
as TIFF files (41). Digitized image was converted
(track resolution, 250) and then normalized with
the reference lanes (resolution 200, smoothing 5
points) and analyzed by using Molecular
Analyst Fingerprinting Plus software (version
4.1; Applied Maths, Bio-Rad)(50). The rolling
disk background subtraction method was
applied and similarity matrices of densitometric
curves of the gel tracks (the first 16% and last
7.5% of data points) were excluded from the
analysis. The criterion for related clones was
taken as profiles with 85% or more similar
bands (51). Banding patterns were also visually
analyzed according to criteria developed by
Tenover et al., (1995, 1997) (52, 53).
Statistical Analysis:
Data were processed and analyzed using the
Statistical Package of Social Science (SPSS,
version 13.0). Data were analyzed in tables
Vol. 22, No. 2
using percentage. The mean and standard
deviation (SD) were used for numerical data for
description (54).
RESULTS
A total of 1520 different specimens were
collected from cases of nosocomial infections
and environmental samples diagnosed according
to CDC criteria in surgical wards at Suez Canal
University Hospital, Ismailia, Egypt, during
one-year period. Based on colonial morphology,
biochemical reactions and positive Oxidase
reaction, 131 Pseudomonas species isolates
were discovered as a cause of infection, with
isolation rate (8.6%). For more diagnostic
conformation, typical PCR was applied. Using
Genus-specific PCR, 78 Pseudomonades out of
preliminary 131 pseudomonas strains were
confirmed (Figure 1). Overall, wound
infections and burn together were the source of
more than half of the total isolates (55.1%), with
the next most frequent source of isolation
obtained from the environmental samples
(17.9%), as shown in (Table 1). Among the 78
Pseudomonas isolates (51.3%) showed typical
morphology, while (29.5%) were mucoid and
(19.2%) were rough. The epidemiological
distribution of the confirmed Pseudomonades is
illustrated in (Table 2).
Figure 1: PCR analysis of P.aeruginosa. (Right) PCR using genus-specific primers. (Left) PCR using
species-specific primers. Lanes L, 100bp ladder; lane P, positive control, and lane N, negative control.
86
Vol. 22, No. 2
Table 1: Sources of Pseudomonas aeroginosa Isolates Based on PCR Results.
Source of Isolation
No. of Isolates
% of Isolates
34
43.6
Septic surgical wounds
9
11.5
Septic skin burns
11
14.1
Urinary tract infections
5
6.4
Respiratory tract infections
14
17.9
Environmental samples
1
1.5
Septic bed sores
3
3.8
Blood samples
1
3.1
Chronic Osteomyelitis
Total
78
100.0
11
2
11
3
2
5
26
18
78
0
0
3
0
0
0
0
0
3
1
0
2
0
0
5
4
2
14
9
2
0
0
2
0
21
11
45
0
0
5
0
0
0
0
0
5
Urine
11
2
14
4
2
9
31
21
94
Sputum
17
4
18
7
5
9
42
29
131
Morphology
Pus
Burn Unit
Gynecology
ICU
Nephrology
Neurosurgery
Operative
Orthopedics
Surgery
Total
Environ
Blood
Table 2: Epidemiological Distribution of Pseudomonas Strains.
Sample Type
No. of
Oxidase
P.
Ward
Samples
+ve
Isolates
1
0
1
3
0
0
1
5
11
T
8
8
4
2
2
3
12
8
40
M
2
2
3
1
0
1
8
8
23
antibiotics) comprising the remaining 11 groups
with predominant of the AT-3 (11.5%). Mucoid
strains showed high resistance rates to
antibiotics (78.3%) of their total number,
comprising (31%) of AT-1, (44.4%) of AT-3,
and (66.7%) of AT-5, while (100%) of the
sputum samples were AT-1. Antibiotic
resistence and sensitivity are shown in (Table
3). Resistance was most frequently observed to
augmentin, ceftazidime and carbenicillin
(98.7%). On the other hand, sensitivity is higher
to imipenem (62.8%), followed by norfloxacin
(60.3%) and amikacin (57.7%).
Phenotyping Results:
1. Antibiogram Typing:
Susceptibility patterns to 22 anti-microbial
agents differentiated 78 Pseudomonas isolates
into 13 anti-biotypes (AT), with (100%
typeability and 80% reproducibility) and the
discriminatory index was 0.83. Pseudomonas
strains expressed high level of resistance, with
only one strain (AT-2) being sensitive to all
antibiotics. Most of the resistant strains were
pan-resistant to all antimicrobials tested (AT-1,
37.2%). The rest (61.5%) were multidrug
resistant strains (i.e. resistant to three or more
Table 3: Distribution of Antibiotic Sensitivity and Resistance.
Antibiotic
Resistance % Sensitivity % Antibiotic
Amikacin
42.3
57.7
Gentamicin
Augmentin
98.7
1.3
Imipenem
Aztreonam
83.3
16.7
Levofloxacin
Carbenicillin
98.7
1.3
Norfloxacin
Cefepime
89.7
10.3
Ofloxacin
Cefoperazone
87.2
12.8
Pefloxacin
Cefotaxime
73.1
26.9
Piperacillin
Ceftazidime
98.7
1.3
Sulperazone
Ceftriaxone
94.9
5.1
Ticarcillin
Cefuroxime
87.2
12.8
Tobramycin
Ciprofloxacin
59.0
41.0
Unasyn
87
Resistance %
56.4
37.2
64.1
39.7
70.5
78.2
89.7
89.7
89.7
82.1
94.9
Sensitivity %
43.6
62.8
35.9
60.3
29.5
21.8
10.3
10.3
10.3
17.9
5.1
R
1
1
4
0
0
1
6
2
15
Vol. 22, No. 2
The minor pyocin types (a, d and c) occur at
frequencies of (12.8%, 10.3%, and 9%
respectively). Only seven isolates (9%) were untypeable, while another two isolates showed no
activity with the set A-E only. S-pyocin activity
against one or more of the indicator strains were
produced by (75.6%) of the Pseudomonas
isolates. All indicators showed some sensitivity
to S-pyocins, twenty different S-type patterns
were distinguished, the most common of these
were S5 (12.8%), S5, B (10.3%), S7 (9%) and
S7, B (7.7%). For easier interpretation, the
combination of both major and minor types was
used to assign the pyocin type, giving rise to 29
different patterns of inhibition with (91%
typeability, and Discriminatory index 0.96). The
variation between types-1 and 4, and types 2, 4
and 5 were significant, because these types
differed by the reaction difference of only one
indicator strain. The most common pyocin types
were PT-1, PT-3, and PT-2, with frequencies
(10.3%, 9% and 7.7% respectively). Pyocin
profiles PT-5 to PT-9 contained three isolates
each, and the remaining 21 profiles contained
only one or two isolates each. Ten pyocin types,
mostly PTs-1, 2, and 15 represented
environmental samples; eight of them (80%)
showed cross-relation with clinical samples.
This may indicate some degree of crossing
between both types of samples (Table 4).
2. Serotyping:
Sixty-nine isolates out of 78 were
successfully typed (88.5%) into 16 serogroups.
None of the isolates was in O-types 7, 8, 13, 15,
17, 18, or 20. Around (6%) of the isolates were
poly-agglutinable (agglutinated in two or more
antisera). Three combinations (O7 and O8, O9
and O10, O5 and O16) accounted for these
multiple reactions. Only (5.1%) of all isolates
did not agglutinate in any of the antisera when
the live antigen used. With the heated antigen,
only (3.8%) of the isolates remained un-typed
with these antisera. Serotype O6 was the most
prevalent (24.4%) of all serogroups, followed
by O11 (16.7%) and O3 (10.3%) of the total
isolates.
Environmental
isolates
were
represented by eight serotypes; sex of them
(75%) showed cross-relation with clinical
samples. The discriminatory index of the
serotyping was 0.88.
3. Pyocin Typing:
Pyocin typing of the 78 Pseudomonas
strains with the eight indicator strains set 1-8
showed 19 major pyocin types (PT), further
sub-typing with the five A-E set revealed 17
minor pyocin types. Types 1 and 2 isolates were
the most common major types (19.2% each),
followed by type 3 (10.3%). It was interesting
that only one indicator strain (no.6) reaction
separates the two most common pyocin types.
Table 4: Cross-Relating Pyocin Types between Clinical and Environmental Samples.
Cross-relating Pyocin types (%)
Sample Type
PT1
PT2
PT3
PT5
PT9
PT14
PT20
75
66.7
85.7
66.7
66.7
50
50
Clinical
25
33.3
14.3
33.3
33.3
50
50
Environmental
Total
8
6
7
3
3
2
2
PT21
50
50
2
clinical strains. Clinical strains also had higher
percentages of protease and gelatinase activities,
although more than one-half of the
environmental strains also expressed such
activities. Protease activity was present in
(96.9%) of the clinical isolates and in only
(57.1%) of the natural isolates. Out of the 23
extracellular enzyme types, environmental and
clinical samples were represented by 7 and 18
groups respectively.
4. Extracellular Enzyme Profile Typing:
Eight extracellular enzymes and pyocyanin
pigment were used to differentiate Pseudomonas
isolates into 23 (XT) types with (100%
typeability,
98%
reproducibility
and
discriminatory index 0.88). Type-12 was the
most common profile (25.6%), followed by
type-19 contained (20.5%) of the total isolates.
As shown in (Table 5), higher frequencies of
(hemolysin, fibrinolysin, coagulase, lipase,
elastase, and DNase) were found more in the
88
Vol. 22, No. 2
Table 5: Frequency of Extracellular Enzyme Production by Pseudomonas Isolated From Clinical
and Environmental Sources.
Clinical Isolates
Environmental Isolates
Total Isolates
Enzyme
n = 64
n = 14
n = 78
n
%
n
%
n
%
Protease
62
96.9
8
57.1
70
89.7
Elastase
31
48.8
1
7.1
32
41.0
Hemolysin
63
98.4
1
7.1
64
82.1
Gelatinase
59
92.2
10
71.4
69
88.5
Coagulase
48
75.0
0
0.0
48
61.5
Fibrinolysin
53
82.8
1
7.1
54
69.2
Lipase
51
79.7
2
14.3
53
67.9
DNase
20
31.3
0
0.0
20
25.6
Pyocyanin
60
93.8
12
85.7
72
92.3
with extremely reproducible banding patterns
(97.5%). The majority of strains (20.5%)
belonged to RT-9, followed by (14.1%) for RT6, (12.8%) for RT-15, and (11.5% and 10.3%)
for RTs: 18 and 17 respectively. Comparison of
the major ribogroups did not reveal significant
difference
in
their
clinical
origins.
Environmental samples encompassed nine
different ribotypes representing major groups
(IV, V, VIII, and IX). The environmental
isolates except for RTs-5, 21, and 22 were
shared with clinical isolates, with sharing
frequencies correlated to the size of the
ribogroup.
Genotypic method:
1. Ribotyping:
All Pseudomonas isolates included in this
study were typeable by ribotyping (100%
typeability) using EcoRI. The isolates were
clustered into 11 major ribotypes at the 70%
similarity index level. These major groups were
further subdivided into 26 subgroups (RTs; 126) based on the 90% similarity index level.
Percent similarities based on the dice coefficient
and clustering by the UPGMA method are
presented in (Figure 2). The profiles had 5-10
bands ranging in size from 2-50kb, and a
relatively high discriminatory index of 0.90,
Figure 2: Dendogram derived from the UPGMA linkage of correlation coefficients of selected ribotyping
patterns and their wards distribution. Clusters delineated with a 90% similarity cutoff level, and close
relation assigned 11 major groups at the 70% level.
89
Vol. 22, No. 2
represented by a single pulsotype that may form
a close or possible relation with other patterns.
PFGE showed similarity indices ranged between
83.6 and 97.2% (mean ± SD, 90.27% ± 3.88%),
the reproducibility was >96.2%. The
discriminatory Index at 90% similarity cutoff
value was 0.99 based on Hunter’s equation-1
(Hunter, 1990). Isolates with identical fragment
patterns were identified within each set from the
same location in (7.1%) of the cases. The
majority (82.1%) of the genotypes were not
found in more than one hospital ward, most of
these genotypes were composed of a single
isolate. Environmental isolates were represented
by 13 PFGE-types, versus 46 PFGE-types
within the clinical samples. Only three
genotypes (5.4%) showed cross-reaction
between both categories.
2. Pulsed-Field Gel Electrophoresis (PFGE)
Typing:
Figure 3 showed a representative PFGE
typing of 12 selected Pseudomonas strains.
Strains in Lanes 5 and 6, and 11 and 12 show
indistinguishable patterns. PFGE distinguished
unrelated strains and genetically related ones at
clustering level of ≥ 85%. Overall, 56
pulsotypes were identified, showing multiple
degrees of heterogeneity between different
pulsotypes and within the same pulsotype, but
no predominant pulsotype was identified. The
maximum number of strains in a single
pulsotype was 4 and was found in only ET-9
and ET-36. Pulsotypes ET-6, ET-47 and ET-51
each contained three strains. Ten pulsotypes
contained two strains each (Figure 4). Fortyone isolates (52.6%) fell into a unique pattern
Figure 3: A representative PFGE typing of selected 12 P.aeruginosa strains. Lanes 4 & 5, and 10 & 11
included two indistinguishable (cluster) strains S; Lambda phage c1857s7, and M; P.aeruginosa
ATCC27853 strain.
90
Vol. 22, No. 2
Figure 4: Digitized PFGE patterns and dendogram for selected P.aeruginosa isolates cut with SpeI and
their wards distribution. The dendogram was constructed by cluster analysis by UPGMA with GelCompar
3.1 software (Applied Math). Percentages of similarity are shown above the dendogram. Clusters
delineated at 85% similarity cutoff level, close relation assigned at the 70% level, and the possible
relation at 55%.
PFGE was found to be more sensitive than
ribotyping since it was able to differentiate 54
PFGE patterns among the 78 isolates. Using
ribotyping, the 78 strains could be grouped into
only 26 ribotypes. Table (6) shows the
comparison of the different typing methods
regarding their typeability, reproducibility and
discriminatory indices. The results obtained in
PFGE, were the easiest to read and interpret and
most
discriminating
among
molecular
techniques. Pyocin typing had a good
discrimination results, whereas in ribotyping,
band doubling and non-specific bands
represented the main difficulty in reading the
results and could be overcome by repeating the
procedure. Comparison based on sensitivity
(typeability) of the typing methods showed that
PFGE, ribotyping and antibiogram were the
most sensitive method (100%), followed by the
extracellular enzyme typing, while serotyping
was the least sensitive method. Ribotyping and
PFGE typing gave the most reproducible results,
while pyocin and antibiogram typing gave the
least reproducible methods.
Comparison between Different Typing
Methods:
Pulsotype-2 was the only sensitive to all
antibiotics used, while ET-36 was the most
resistant. Isolates with the same antibiotype did
not always have the same genotype as reported
in 10 of the 13 antibiotypes (76.9%). Isolates of
the same PFGE-type exhibited different Oserotypes in (21.4%) of the genotypes. At the
same time, isolates with the same serotype did
not always have the same PFGE-type. The
dominant PT-1 showed seven different
genotypes, while PTs-2 and 3 showed six
different genotypes each. Isolates with the same
pyocin type having more than two PFGE-types
were reported in seven of the 29-pyocin types
(24.1%). So, isolates with different pyocin types
did not always have different PFGE-type.
Comparing the pulsotypes to the
extracellular enzyme types, it was reported that
all the multi-strained extracellular enzyme types
were represented by more than one pulsotype.
For instance, the most dominant extracellular
enzyme types ET-12 and ET-19 were
represented by 18 and 14 pulsotypes (90% and
87.5% respectively).
91
Vol. 22, No. 2
Table 6: Comparative Assessment of Different Typing Methods.
Typeability % Reproducibility % No. of Patterns
Typing Method
100.0
80.0
13
Antibiogram
88.5
89.7
16
Serotyping
91.0
73.1
29
Pyocin
100.0
98
23
Extracellular Enzymes
100.0
97.5
26
Ribotyping
100.0
96.2
56
PFGE
Discriminatory index
0.83
0.88
0.96
0.88
0.90
0.99
Leone et al. (2008) reported that serotyping
failed to identify the (37.8%) of 135 strains,
may be due to the presence of new bacterial
serotypes not identifiable by currently used
commercial antisera or due to the loss of some
antigens during the long-lasting infections (31).
The discriminatory power of pyocin typing
was superior to serotyping 0.96 versus 0.88. The
same results were obtained by El-Sweify
(1993)(8). Although typeability of extracellular
enzymes was (100%), the difficulties in
preparation of testing media and the time
needed to get typing results made it unsuitable
as a rapid preliminary typing method. This
method was highly reproducible, with nearly all
isolates were consistent in their XT profile when
retested (98%), yet the discriminatory power is
low 0.88. In this study, higher frequencies of
(hemolysin, fibrinolysin, coagulase, lipase,
elastase, and DNase) were found, protease
activity was present in (96.9%) of the clinical
isolates and in only (57.1%) of the
environmental isolates. According to Leone et
al. (2008) (31), many strains produced enzymes,
105 (77.7%) and 98 (72.6%) strains were
protease and gelatinase producers, respectively.
According to Tingpej et al. (2007)(63), more than
one-half of the strains produced elastase,
hemolysin and the alginate layer responsible for
the colony mucosity. Most strains produced
large amounts of protease, which is considered a
colonization factor. Although, pyocin typing
had the least reproducibility (73.1%) it has the
highest discriminatory power among other
phenotypic methods (0.96). Antibiogram and
extracellular enzyme typing had the best
typeability (100%), yet antibiogram had the
least discriminatory power. Extracellular typing
and serotyping had the same discriminatory
index of (0.88); still extracellular typing is more
reproducible than other methods. These findings
emphasizes that there is a tremendous difference
in the ability of typing and subtyping methods
to differentiate between strains.
Automated ribotyping produced a less
diverse range of ribotypes (26 RTs) and grouped
isolates into defined clusters. Ribo-printing of
DISCUSSION
Confirmed Pseudomonas strains were
found to be the causative pathogens in (5.1%) of
1520 nosocomial specimens obtained from
patients admitted to different hospital surgical
wards and environmental samples. This result
was in agreement with previous studies (1, 55-57).
Using antimicrobial susceptibility typing,
13 antibiogram patterns were detected with
predominance of (37.2%) AT-1 that was
resistant to all used antimicrobial agents.
Therefore, the presence of almost one-thirds of
the total strains in one antibiotype indicated that
antimicrobial susceptibility typing is not
sensitive enough for use in routine typing of
these isolates. P.aeruginosa expresses natural
resistance to many antibiotics and has the
capacity to acquire many mechanisms of
resistance(58). Susceptibility irrespective of the
isolates’ origin in our study, ranged from (1.3%)
for carbenicillin, augmentin, and ceftazidime to
(62.8%) for imipenem compared to (79%) for
cefepime to (97%) for piperacillin-tazobactam.
Norfloxacin, amikacin and gentamicin proved to
be effective against most P.aeruginosa isolates
(60.3% - 43.6% susceptibility). The rates of
resistance to carbenicillin and ceftazidime were
in agreement with the findings of other
investigators (59-61).
Serological typing of Pseudomonas strains
revealed that 9 strains were not typeable
(11.5%). Serotype O6 was the most frequently
encountered in our study (24.4%), followed by
O11 (16.7%) and serotype O3 (10.3%) these
results were in concordance with the results of
El-Sweify (1993)(8). While in a study by
(Jamasbi and Proudfoot, 2008)(27), serotype O11
was the most frequently encountered.
Reproducibility of serotyping in this study was
lower than other studies (89.7%) and the index
of diversity was higher than other studies
(0.88)(8,31). The differences between our study
and others can be explained as the frequency of
individual O-serotypes of P.aeruginosa in
clinical specimens differs with the country of
investigation and the source of isolates (62).
92
Vol. 22, No. 2
in our hospital. Juan et al. (2005) (69), stated that
in the absence of epidemic clones in a particular
setting, an endogenous source of P.aeruginosa
infections seems to be more likely than
nosocomial spread, as evidenced by the fact that
unique clones infected most patients in the
present study based on PFGE results. Based on
our observations, antimicrobial resistance
clusters were highly variable, even among
strains belonging to the same PFGE pattern. The
dominant AT-1 showed 22 different genotypes,
most of them were ET-36 (13.8%). Isolates with
the same antibiotype did not always have the
same genotype as reported in 10 of the 13
antibiotypes (76.9%). Simultaneously, in four
PFGE-types (7.1%) more than one antibiotype
were associated with the same genotype. Leone
et al. (2008)(31), obtained similar finding. Strains
belonging to the same PFGE pattern exhibited
different O-serotypes in (21.4%) of the
genotypes. At the same time, isolates with the
same serotype did not always have the same
PFGE-type. Strains of the dominant serotype O6
showed 17 different genotypes. Bergmans et al.
(1997)(70), observed similar findings as within a
group of isolates with the same serotype, PFGE
analysis showed < a 3band difference in 85 of
91 isolates (93%). In six of 91 isolates (7%), > 7
band differences were present according to
PFGE analysis. The study concluded that,
isolates with a different serotype also had a
different PFGE type. Isolates with the same
serotype did not always have the same PFGE
type.
Simultaneously, the dominant PT-1 showed
seven different genotypes, while PTs-2 and 3
showed six different genotypes each. Isolates
with the same pyocin type having more than
two PFGE-types were reported in seven of the
29-pyocin types (24.1%). Moreover, when
comparing the pulsotypes to the extracellular
enzyme types, it was reported that all the multistrained extracellular enzyme types were
represented by more than one pulsotype. At the
same time, a single extracellular enzyme type
each represented only two of the 15 multistrained pulsotypes; namely, ET-51 was
represented by XT-12 and ET-23 was
represented by XT-19. Therefore, any
combination of these methods did not improve
distinctive capacity.
In here, that may go with the indication of
Leone et al. (2008)(31), that no particular
relationship between bacterial genotypes and
phenotypes. They assumed that PFGE is not the
best technique for detecting subtle genotypic
differences and to the fact that the expression of
some genes may be affected by environmental
P.aeruginosa isolates was shown to be sensitive
(100% typeability) and compared to other
epidemiological markers automated ribotyping
was extremely reproducible (97.5%), and
reliable (discriminatory index 0.90), and could
be easily read and interpreted more than the
conventional ribotyping. Pfaller et al. (1996)(64),
identified several distinct profiles among
P.aeruginosa
isolates
using
automated
ribotyping. Moreover, according to the results
of (De Cesare et al. 2001)(65), automated
ribotyping is a useful genotyping techniques for
identifying unique and common subtypes
epidemiological investigations (66).
PFGE discriminated the isolates into 56
patterns and showed a high degree of
heterogeneity. The vast majority of PFGE
patterns differed from each other in more than
six band positions. Similarly, minor changes
were observed in some isolates, such relatively
small differences may occur in genotypically
highly related strains and may reflect relatively
recent structural changes in their genome. Fifty
six strains were found to be clonally related and
37 of these strains were indistinguishable
divided in 15 groups; range of group was 2 to 4
strains. When the epidemiologic data of these
patients were considered, no link was found
among six pulsotypes having clonally related
strains. The remaining nine pulsotypes having
related strains by PFGE had been found
epidemiologically related. Cross transmission
between patients and environmental sources
may occur via the hands of the health care staff
or through contaminated materials and reagents.
The genomic relatedness of 573 P.aeruginosa
strains were typed using PFGE by Römling et
al. (1994)(67) and he concluded that PFGE
analysis of P.aeruginosa strains representing
various bio-and pathovars revealed that almost
all strains had different SpeI fingerprints. This
variability of the overall genomic structure
reflects the well-known versatility of the
species. However, three strains with identical or
very similar chromosomal architecture were
isolated more than once. While Kayabas et al.
(2008)(68), typed 14 P.aeruginosa (12 from
patients and two from environmental samples)
by PFGE, the typing yielded two possibly
related clines, which differed from each other
by four major bands. Ten of the patient isolates
were clonally identical with the strains isolated
from two forceps. According to our results,
there was no dominant pulsotype. Lack of a
major PFGE type confirmed no P.aeruginosa
outbreak and both the clonally and
epidemiologic relationship found, support cross
transmission and problems in infection control
93
conditions. In this study, comparison of the
different genotypic methods resulted in the
detection of similarities and differences in the
relationships among the isolates. For each of the
methods,
the
homogenous
nature
of
P.aeruginosa was apparent. Each technique
used showed a higher degree of consistency in
fingerprint or profile and clustering pattern with
this species than with any other. Therefore, the
results from our study clearly confirmed that the
P.aeruginosa was established as good coherent
species, distinguishable from the other species.
With PFGE, the macrorestriction fragment
patterns reflect the distances between rare
restriction sites around the chromosome;
therefore, the method can detect differences
between isolates resulting from chromosomal
changes that affect these sites or the distances
between them. In this sense, PFGE provides a
global chromosomal overview, scanning more
than 90% of the chromosome but it has only
moderate sensitivity because minor genetic
changes may go undetected (71).
Several criteria have been used in
evaluating bacterial genotyping methods,
including
typeability,
reproducibility,
discriminatory power, and ease of interpretation.
This study had shown that PFGE and ribotyping
had similar typeability and high reproducibility,
yet PFGE discriminate more than ribotyping
0.99 versus 0.90. All the multi-strained
ribotypes were represented by more than one
pulsotype. Ribotype RT-9 showed 11 different
pulsotypes (68.8%), RT-6 and RT-15 each
showed five different pulsotypes (45.5% and
50%) respectively. On the other hand, when
compared to ribotypes, 12 out of the 15 multistrained pulsotypes were represented by a single
ribotype. Four of these belonged to RT-15.
Ribotype-6 and RT-9 had three multi-strained
pulsotypes each. The remaining two pulsotypes
belonged to RT-18. According to Botes et al.
(2003) (72), PFGE and riboprinting discriminated
similarly between ICU isolates demonstrating
endemnicity was more likely than an outbreak.
While according to Silbert et al. (2004) (43),
P.aeruginosa isolates were typed by PFGE,
ribotyping and ERIC-PCR and all three methods
had the same discriminatory power (0.98) for
this species.
The high reproducibility of PFGE is in
agreement with findings of most previous
studies (52, 73).
Overall, the dendograms based on the data
of PFGE and ribotyping is not always
consistent, signifying the genetic diversity and
complexity of the species of Pseudomonas. This
also suggests that these methods are still
Vol. 22, No. 2
problematic for the evaluation of genetic
relatedness in all assayed isolates, especially for
those isolates with distant relationship.
Conversely, the isolates with close genetic
relatedness can be discriminated by these
methods. The close genetic relatedness is a
notable concern for judging epidemiological
relationship of clinical isolates.
All P.aeruginosa included in this study
were typeable by ribotyping and PFGE. While
according to Silbert et al. (2004)(43), all strains
where typeable by PFGE. Only one (1.6%) of
the 64 P.aeruginosa isolates could not be typed
by PFGE. Our results revealed that, it was
possible to obtain identical typing results when
repeated analysis of the same isolate of each
species was performed by either method. We
found that PFGE was reproducible (96.2%).
Although a particular typing method may have a
high discriminatory power and good
reproducibility, its complexity, difficulty
regarding interpretation of the results and costs
may be beyond the capabilities of the
laboratory. Therefore, the choice of a molecular
typing method will depend on the needs, skill
level, and resources of the laboratory.
Ribotyping by means of the Ribo-Printer
has shown high typeability but the level of
discrimination may vary according to the
restriction
enzyme
used.
Chromosomal
digestion
with
EcoRI
may
provide
discriminatory power comparable to that of
PFGE. The automated ribotyping system was
easy to use, was rapid (8 hours), provided 100%
typeability, and was able to obtain identical
results when the same isolate was tested. The
Ribo-Printer also automates the analysis process
and provides excellent information about the
similarity among the samples tested. The main
drawback of this method is the high cost as it is
the most expensive of the molecular typing
techniques analyzed in this study.
The data obtained by PFGE in our study
also showed that PFGE has excellent
discriminatory power for typing P.aeruginosa.
The problem with un-typeable isolates may be
solved by adding thiourea to the gel buffer. In
general, PFGE is an easy method to implement
in a laboratory, but it is slower and more labor
intensive than the other two methods analyzed
in the current study. The PFGE unit is more
expensive than a conventional one but will serve
for hundreds of runs without any additional
costs. Technically, PFGE was much easier and
faster than either ribotyping, with as many as 15
isolates typed at the same time. Nevertheless,
PFGE is not suitable for the average clinical or
hospital laboratory because of the expense, the
94
need for highly trained personnel and the time
involved. Our data showed a high degree of
phenotypic and genotypic diversity among
P.aeruginosa isolates which are in concordance
with the results obtained by Vosahikova et al.
(2007) (46) and Jamasbi and Proudfoot (2008) (27)
in which the majority of strains (>80%) showed
unique genotypes. It also reported that PFGE
provide the highest discriminatory power
between P.aeruginosa isolates and has,
therefore, been advocated as the method of
choice to investigate outbreaks of infections due
to this species.
Based on the previous typing results and
the comparison of the epidemiological markers
used in this study, none of the methods is ideal
for all laboratories and for all epidemiologic
purposes. This is due to wide variations in costs,
time, skills and experience in applying the
different markers and due to marked differences
in their sensitivity and reproducibility.
Furthermore, different types of laboratories
have different needs. Therefore, Cooperation
between hospitals and health care center
laboratories in the nearby localities, in
exchanging epidemiologic data is of great
significance to effectively control P.aeruginosa
and all nosocomial infections. Through such
cooperation, awareness of the P.aeruginosa
profiles that are endemic in the locality will
provide a database for dealing with any future
outbreak; and will provide an epidemiologic
map of the organism in a given region.
In conclusion:
Genotypic markers were generally more
sensitive than phenotypic markers. Both
extracellular enzyme profile typing and pyocin
typing markers were the most sensitive and
reliable phenotypic techniques. Reproducibility
was a problem in serotyping and pyocin typing
markers. Ribotyping was more reproducible
than PFGE but PFGE was the most
discriminatory. Pulsed Field Gel Electrophoresis
was the best typing method and could be added
to one phenotypic marker to trace the sources of
infection.
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‫اﺳﺘﺨﺪام ﻃﺮﻳﻘﺔ اﻟﻔﺼﻞ اﻟﻜﻬﺮﺑﻰ ذى اﻟﻤﺠﺎل اﻟﻨﺎﺑﺾ وﻃﺮﻳﻘﺔ اﻟﺘﺼﻨﻴﻒ اﻟﺮ ي ﺑﻮزوﻣﻰ آﻄﺮق ﺗﻨﻤﻴﻂ‬
‫ﺟﻴﻨﻴﺔ ﻣﻘﺎرﻧﺔ ﺑﻄﺮق اﻟﺘﻨﻤﻴﻂ اﻟﻤﻈﻬﺮﻳﺔ ﻟﺘﺼﻨﻴﻒ اﻟﺰاﺋﻔﺔ اﻟﺰﻧﺠﺎرﻳﺔ اﻟﻤﺴﺒﺒﺔ ﻟﻠﻌﺪوى اﻟﻤﻜﺘﺴﺒﺔ‬
‫ﻟﻠﻤﺴﺘﺸﻔﻴﺎت واﻟﻤﻌﺰوﻟﺔ ﻣﻦ أﻗﺴﺎم اﻟﺠﺮاﺣﺔ ﺑﻤﺴﺘﺸﻔﻰ ﺟﺎﻣﻌﺔ ﻗﻨﺎة اﻟﺴﻮﻳﺲ‬
‫ﻣﻰ أﺣﻤﺪ اﺑﻮ ﻃﺎﻟﺐ )‪ ، (١‬ﻋﺒﻴﺮ ﻋﺰت اﻟﺴﻴﺪ ﻣﺤﻤﺪ )‪ ، (١‬ﺣﺴﻦ ﻧﺼﺮ اﻷﺳﻼم )‪ ، (١‬ﺳﻌﻴﺪ ﺣﺎﻣﺪ ﻋﺒﺎدى )‪، (١‬‬
‫)‪( ٢‬‬
‫ﺟﻴﻬﺎن ﺻﺪﻳﻖ اﻟﺤﺪﻳﺪى )‪ (١‬وﺟﻮن ل ﻟﻴﺒﻮﻣﺎ‬
‫)‪)١‬‬
‫ﻗﺴﻢ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻰ واﻟﻤﻨﺎﻋﺔ‪ ،‬آﻠﻴﺔ اﻟﻄﺐ‪ ،‬ﺟﺎﻣﻌﺔ ﻗﻨﺎة اﻟﺴﻮﻳﺲ‪ ،‬ﻣﺼﺮ‬
‫وﻗﺴﻢ ﺑﺤﻮث اﻷﻃﻔﺎل واﻷﻣﺮاض اﻟﻤﻌﺪﻳﺔ‪ ،‬آﻠﻴﺔ اﻟﻄﺐ‪ ,‬ﺟﺎﻣﻌﺔ ﻣﻴﺸﻴﺠﺎن‪ ,‬اﻟﻮﻻﻳﺎت اﻟﻤﺘﺤﺪة اﻷﻣﺮﻳﻜﻴﺔ‬
‫)‪)٢‬‬
‫اﻟﻤﻘﺪﻣ ﺔ‪ :‬ﻳﻌ ﺪ ﻣﻴﻜ ﺮوب اﻟﺰاﺋﻔ ﺔ اﻟﺰﻧﺠﺎرﻳ ﺔ ﻣ ﻦ ﻣ ﺴﺒﺒﺎت اﻷﻣ ﺮاض اﻻﻧﺘﻬﺎزﻳ ﺔ اﻟﺘ ﻰ ﺗ ﺴﺒﺐ ﻋ ﺪوى اﻟﻤﺴﺘ ﺸﻔﻴﺎت ﻓ ﻲ اﻟﻌﺪﻳ ﺪ ﻣ ﻦ‬
‫اﻟﻤﺴﺘﺸﻔﻴﺎت‪ .‬ﺗﻬﺪف هﺬة اﻟﺪراﺳﺔ اﻟ ﻰ اﻟﻤﻘﺎرﻧ ﺔ ﺑ ﻴﻦ اﻟﻄ ﺮق اﻟﻮﺑﺎﺋﻴ ﺔ اﻟﺨﺘﻠﻔ ﺔ ﻟﺘ ﺼﻨﻴﻒ اﻟﻤﻴﻜ ﺮوب واﻳ ﻀﺎح آﻔﺎﺋﺘﻬ ﺎ ﻓ ﻰ ﺗ ﺸﺨﻴﺺ‬
‫ﺳﻼﻻت اﻟﺰاﺋﻔﺔ اﻟﺰﻧﺠﺎرﻳﺔ اﻟﻤﻌﺰوﻟﺔ ﻣﻦ ﻋﺪوى اﻟﻤﺴﺘﺸﻔﻴﺎت‪.‬‬
‫اﻟﻄﺮق‪ :‬ﻧﺘﺞ ﻋﻦ ﺟﻤﻊ ‪ ١٥٢٠‬ﻋﻴﻨﻪ ﻣﺨﺘﻠﻔﺔ ﻣﻦ اﻟﻤﺮﺿﻰ اﻟﻤﺼﺎﺑﻴﻦ ﺑﺎﻟﻌﺪوى اﻟﻤﻜﺘﺴﺒﺔ ﻣﻦ اﻟﻤﺴﺘﺸﻔﻰ واﻟﻤﻨﻮﻣﻴﻦ ﺑﺎﻗ ﺴﺎم اﻟﺠﺮاﺣ ﺔ‬
‫ﺑﺎﻟﻤﺴﺘﺸﻔﻰ اﻟﺠﺎﻣﻌﻰ ﺑﺎﻻﺳﻤﺎﻋﻴﻠﻴﺔ وﻋﻴﻨﺎت ﺑﻴﺌﻴﺔ ﻣﺨﺘﻠﻔﺔ ﻋﻠﻰ ﻣﺪى ﻋ ﺎم و ﺗ ﻢ ﻋ ﺰل وﺗ ﺸﺨﻴﺺ ‪ ٧٨‬ﺳ ﻼﻟﺔ ﻣ ﻦ ﻣﻴﻜ ﺮوب اﻟﺰاﺋﻔ ﺔ‬
‫اﻟﺰﻧﺠﺎرﻳ ﺔ وﺗﺎآﻴ ﺪ اﻟﺘ ﺸﺨﻴﺺ ﺑﺎﺳ ﺘﺨﺪام ﻃﺮﻳﻘ ﺔ ﺗﻔﺎﻋ ﻞ ﺳﻠ ﺴﻠﺔ اﻟﺒﻠﻤ ﺮة اﻟﺘ ﻀﺎﻋﻔﻰ ‪ .‬ﺛ ﻢ ﺗ ﻼ ذﻟ ﻚ ﺗ ﺼﻨﻴﻒ اﻟ ﺴﻼﻻت اﻟﻤﺆآ ﺪة )‪٧٨‬‬
‫ﻋﻴﻨﺔ( ﺑﻮاﺳﻄﺔ ﺳﺘﺔ ﺗﺼﻨﻴﻔﺎت ﻟﻠﺘﺄآﺪ ﻣﻦ ﺟﺪواهﺎ آﻄﺮق ﻟﺪراﺳﺔ اﻟﺘﻌﺪد اﻟﺠﺮﺛﻮﻣﻰ و ﺗﺼﻨﻴﻒ اﻟﺴﻼﻻت اﻟﻤﻌﺰوﻟﺔ‪ .‬وآﺎﻧ ﺖ اﻟﻄ ﺮق‬
‫اﻟﻤﺴﺘﺨﺪﻣﺔ ﻟﻠﺘﺼﻨﻴﻒ هﻰ اﺧﺘﺒﺎر ﺣﺴﺎﺳﻴﺔ اﻟﺴﻼﻻت ﻟﻠﻤﻀﺎدات اﻟﺤﻴﻮﻳﺔ واﻟﺘﺼﻨﻴﻒ اﻟﺒﻴﻮﺳﻴﻨﻰ واﻟﺴﻴﺮوﻟﻮﺟﻰ ﺑﺎﺳﺘﺨﺪام اﻷﻣ ﺼﺎل‬
‫ﻟﻠﻤﻀﺎدة ﻟﻠﺠﻴﻨﺎت اﻟﺠﺴﺪﻳﺔ وﺗﺼﻨﻴﻒ اﻻﻧﺰﻳﻤﺎت ﺧﺎرج اﻟﺨﻠﻴﺔ واﻟﺘﺼﻨﻴﻒ اﻟﺮﻳﺒﻮزوﻣﻲ وﺗﺼﻨﻴﻒ اﻟﺴﻼﻻت ﺣ ﺴﺐ ﻧ ﻮاﺗﺞ ﺗﻘﻄﻴ ﻊ‬
‫ﺻﺒﻐﻴﺎﺗﻬﺎ اﻟﻮراﺛﻴﺔ ﺑﺎﺳﺘﺨﺪام اﻟﻔﺼﻞ اﻟﻜﻬﺮﺑﻰ ذى اﻟﻤﺠﺎل اﻟﻨﺎﺑﺾ‪.‬‬
‫اﻟﻨﺘﺎﺋﺞ‪ :‬ﺗﻢ ﺗﺤﺪﻳﺪ اﻟﻘﺪرة اﻟﻤﻤﻴﺰة ﻷرﺑﻌﺔ ﻃﺮق ﻟﻠﺘﺼﻨﻴﻒ اﻟﺸﻜﻠﻰ ﻣﻘﺎرﻧﺔ ﺑﻄﺮﻳﻘﺔ ﺗﺼﻨﻴﻒ اﻟﺴﻼﻻت ﺣﺴﺐ ﻧ ﻮاﺗﺞ ﺗﻘﻄﻴ ﻊ اﻟ ﺼﺒﻐﺔ‬
‫اﻟﻮراﺛﻴﺔ ﺑﺎﺳﺘﺨﺪام اﻟﻔﺼﻞ اﻟﻜﻬﺮﺑ ﻰ ذى اﻟﻤﺠ ﺎل اﻟﻨ ﺎﺑﺾ و آﺎﻧ ﺖ ﻧ ﺴﺒﺔ اﻟﻤﻘﺎوﻣ ﺔ ﻟﻠﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﺔ اﻟﺘ ﻰ ﺗ ﻢ اﺧﺘﺒﺎره ﺎ ه ﻲ ﻓ ﻲ‬
‫ﺣ ﺪود ‪ ٪٣٧.٢‬إﻟ ﻰ ‪ ،٪٩٨.٧‬وآ ﺎن ﻋﻘ ﺎر اﻹﻣﻴﺒﻴﻨ ﻴﻢ ه ﻮ اﻷآﺜ ﺮ ﻓﻌﺎﻟﻴ ﺔ‪ ،‬ﻓ ﻲ ﺣ ﻴﻦ ﻋﻘ ﺎرات اﻟﻼوﺟﻴﻤﻨﺘ ﻴﻦ واﻟﻜﺮﺑﻨﻴ ﺴﻴﻠﻴﻦ‬
‫واﻟ ﺴﻴﻔﺘﺎزﻳﺪﻳﻢ ه ﻲ أﻗ ﻞ اﻟﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﺔ ﻓﺎﻋﻠﻴ ﺔ‪ .‬وﺗ ﻢ اﻟﺤ ﺼﻮل ﻋﻠ ﻰ ‪ ١٣‬ﻧﻤﻄ ﺎ‪ ٠‬أﺳ ﻔﺮت ﻧﺘ ﺎﺋﺞ ﺗﻘﻄﻴ ﻊ اﻟ ﺼﺒﻐﺔ اﻟﻮراﺛﻴ ﺔ‬
‫ﺑﺎﺳ ﺘﺨﺪام اﻟﻔ ﺼﻞ اﻟﻜﻬﺮﺑ ﻰ ذى اﻟﻤﺠ ﺎل اﻟﻨ ﺎﺑﺾ ﻋ ﻦ ‪ ٥٦‬ﻧﻮﻋ ﺎ ﻣﺘﻤﻴ ﺰا ﻣ ﻊ ﻗ ﺪرة ﺗﻤﻴﻴ ﺰ ‪ .(٧٨/٧٨) ٪١٠٠‬ﺑﺎﻟﻤﻘﺎرﻧ ﺔ ﻣ ﻊ ﺗﻘﻄﻴ ﻊ‬
‫اﻟﺼﺒﻐﺔ اﻟﻮراﺛﻴﺔ ﺑﺎﻟﺘﺼﻨﻴﻒ اﻟﺮﻳﺒ ﻮزوﻣﻰ ﻟ ﻪ ﻗ ﺪرة ﺗﻤﻴﻴ ﺰ ‪ ،(٧٨/٧٨) ٪١٠٠‬وآﺎﻧ ﺖ ﻗ ﺪرة اﻟﺘﻤﻴﻴ ﺰ ﻟﻠﺘ ﺼﻨﻴﻒ اﻟ ﺴﻴﺮوﻟﻮﺟﻰ ه ﻲ‬
‫‪ ،(٧٨/٦٩) ٪٨٨.٥‬وﻟﻠﺘ ﺼﻨﻴﻒ اﻟﺒﻴﻮﺳ ﻴﻨﻰ ‪ . (٧٨/٧١) ٪٩١‬وآﺎﻧ ﺖ اﻟﻨﺘ ﺎﺋﺞ اﻟﺘ ﻰ ﺗ ﻢ اﻟﺘﻮﺻ ﻞ اﻟﻴﻬ ﺎ ﺑﻄﺮﻳﻘ ﺔ ﺗﻘﻄﻴ ﻊ اﻟ ﺼﺒﻐﺔ‬
‫اﻟﻮراﺛﻴ ﺔ ﺑﺎﺳ ﺘﺨﺪام اﻟﻔ ﺼﻞ اﻟﻜﻬﺮﺑ ﻰ ذى اﻟﻤﺠ ﺎل اﻟﻨ ﺎﺑﺾ اﻷﺳ ﻬﻞ ﻓ ﻰ اﻟﻘ ﺮاءة واﻟﺘﺤﻠﻴ ﻞ واﻷآﺜ ﺮ ﺗﻤﻴﻴ ﺰا )‪ (٠,٩٩‬وﺗﻼه ﺎ ﻃﺮﻳﻘ ﺔ‬
‫ﺗﺼﻨﻴﻒ اﻟﺒﻴﻮﺳﻴﻨﻰ )‪ ،(٠,٩٦‬ﺑﻴﻨﻤﺎ آﺎﻧﺖ ﻗﺪرة ﻃﺮﻳﻘﺔ اﻟﺘﺼﻨﻴﻒ اﻟﺮﻳﺒﻮزوﻣﻰ )‪ (٠,٩٠‬ﻓﻘﻂ‪.‬‬
‫اﻟﺨﻼﺻﺔ‪ :‬ﺗﺸﻴﺮ اﻟﻨﺘﺎﺋﺞ اﻟﻰ أن اﻟﻌﺪوى ﺑﻤﻴﻜﺮوب اﻟﺰاﺋﻔﺔ اﻟﺰﻧﺠﺎرﻳﺔ ﻓﻰ ﻣﺴﺘﺸﻔﻰ ﺟﺎﻣﻌﺔ ﻗﻨﺎة اﻟﺴﻮﻳﺲ ﺗ ﺆﺛﺮ ﺑ ﺸﻜﻞ رﺋﻴ ﺴﻰ ﻋﻠ ﻰ‬
‫اﻟﻤﺮﺿﻰ اﻟﻤﻨﻮﻣﻴﻦ ﻓﻰ أﻗﺴﺎم اﻟﻌﻈﺎم واﻟﺠﺮاﺣﺔ ووﺣﺪات اﻟﺤﺮوق‪ .‬ﻳﻌﺪ ﻋﻘﺎر اﻹﻣﻴﺒﻴﻨﻴﻢ أﻓﻀﻞ اﻟﻤﻀﺎدات اﻟﺤﻴﻮﻳ ﺔ اذا وﺿ ﻌﻨﺎ ﻓ ﻰ‬
‫اﻷﻋﺘﺒﺎر ﻣﻘﺎوﻣﺔ هﺬا اﻟﻤﻴﻜﺮوب ﻟﻠﻤﻀﺎدات اﻟﺤﻴﻮﻳﺔ‪ .‬وﻋﻠﻰ اﻟﺮﻏﻢ ﻣ ﻦ ﻋ ﺪم ﺗﺄآﻴ ﺪ ﺗ ﺼﻨﻴﻒ ﺗﻘﻄﻴ ﻊ اﻟ ﺼﺒﻐﺔ اﻟﻮراﺛﻴ ﺔ ﻟﻮﺟ ﻮد وﺑ ﺎء‬
‫ﺑﻤﻴﻜﺮوب اﻻ أن ﻧﺘﺎﺋﺞ اﻟﺘﺼﻨﻴﻒ أوﺿ ﺤﺖ أن أﻓ ﻀﻞ اﻟﻄ ﺮق اﻟﺘ ﺼﻨﻴﻔﻴﺔ ه ﻰ ﻃﺮﻳﻘ ﺔ ﺗﻘﻄﻴ ﻊ اﻟ ﺼﺒﻐﻴﺔ اﻟﻮراﺛﻴ ﺔ ﺑﺎﺳ ﺘﺨﺪام اﻟﻔ ﺼﻞ‬
‫اﻟﻜﻬﺮﺑﻰ ذى اﻟﻤﺠﺎل اﻟﻨﺎﺑﺾ‪ .‬وﻳﻮﺻﻰ ﺑﺎﺿﺎﻓﺔ ﻧ ﻮع ﻣ ﻦ اﻟﺘﻨﻤ ﻴﻂ اﻟﻤﻈﻬ ﺮى ﻣﺜ ﻞ ﺗ ﺼﻨﻴﻒ اﻟﺒﻴﻮﺳ ﻴﻦ ﻣ ﻊ ﻃﺮﻳﻘ ﺔ ﺗﻘﻄﻴ ﻊ اﻟ ﺼﺒﻐﺔ‬
‫اﻟﻮراﺛﻴﺔ ﺑﺎﺳﺘﺨﺪام اﻟﻔﺼﻞ اﻟﻜﻬﺮﺑﻰ ذى اﻟﻤﺠﺎل اﻟﻨﺎﺑﺾ ﻟﺰﻳﺎدة دﻗﺔ اﻟﺒﺼﻤﺔ اﻟﺠﻴﻨﻴﺔ ﻟﻬﺬا اﻟﻤﻴﻜ ﺮوب ﻓ ﻰ دراﺳ ﺎت ﻣ ﺴﺘﻘﺒﻠﻴﺔ أﺧ ﺮى‬
‫وﻟﺘﺘﺒﻊ ﻣﺼﺎدر اﻟﻌﺪوى ﺑﺎﻟﻤﺴﺘﺸﻔﻰ‪.‬‬
‫‪100‬‬
Vol. 22, No. 2
Detection and Identification of Staphylococcus Aureus Enterotoxins in
Some Milk Products and their Handlers
Michael N. Agban1 and Ahmed S. Ahmed 1
1
Microbiology and Immunology Department,
Faculty of Medicine, Assiut University, Egypt
ABSTRACT
Background: Staphylococus aureus may contain one or more genes that encode staphylococcal
enterotoxins (SE) that cause food poisoning. The previously known toxins were the five major classical
types; however, with the extensive analysis of the S. aureus genome, new genes encoding enterotoxinlike superantigens have been identified. Milk and dairy products are frequently contaminated with
enterotoxigenic S. aureus, which is often involved in staphylococcal food poisoning; these
contaminations are either from animal or human sources. Aim of the work: To detect the presence and
prevalence of coagulase positive S. aureus in milk, kariesh cheese and ice-cream samples and in nasal
swabs and stool samples from milk handlers, and to detect types of S. aureus enterotoxins by sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and to detect the type of enterotoxins
genes by PCR. Material and Methods: 250 samples of Milk, ice-cream, kariesh cheese, nasal swabs and
stool samples from milk handlers were examined for the presence of Coagulase positive Staph aureus,
using Mannitol salt agar, Baird-Parker agar, tube coagulase test, and latex agglutination test for protein A
and capsular polysaccharides. Confirmed S. aureus isolates were examined for the production of SEs
using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the type of SE
genes by polymerase chain reaction (PCR). Results: Coagulase positive S. aureus isolates were detected
in 82% of Staph colonized raw milk, 80% of Staph colonized ice-cream and 82.3% of Staph colonized
kariesh cheese samples and 86.6% of Staph colonized nasal swabs, and 60% of Staph colonized stool
samples; with 70.9% of total samples staph colonization exceeds the Egyptian standards. Collectively,
44.3% of coagulase positive S. aureus isolates were enterotoxigenic and the highest percentages were
detected in raw milk taken directly from animals (75%) and kariesh cheese from street distributors
(66.6%). In all samples, the major classical enterotoxin genotype was SEA which was detected in 44.4%
of toxigenic isolates. SEC was detected in 22.2% of isolates and SED in 18.5% of isolates. SEB could not
be detected. For the newly described genes, SEG was detected in 7.4% of isolates and SEH in 7.4% of
isolates. Conclusions: Raw milk and some dairy products in the markets in Assuit Governorate rural
areas-Egypt, are contaminated with enterotoxigenic S. aureus. The most common type in both milk and
dairy products as well as in nasal swabs and stool samples was SEA which is known to be less common
among strains from animal origin than from human. Nasal and fecal carriage in human milk handlers is
considered a primary source of contamination of milk and dairy products.
Keywords: Staphylococcus aureus enterotoxins, raw milk, dairy products, nasal swabs, SDS-PAGE,
PCR.
There were five major classical SEs types,
named; SEA, SEB, SEC, SED, and SEE. But
now, new genes encoding enterotoxin such as
SEG to SEU are identified. One or more of
these genes are thought to be involved in
staphylococcal food poisoning (1, 4).
S. aureus is the major pathogen of
intramammary infection in bovines, and can
cause contagious clinical/sub-clinical mastitis in
dairy herds5.
The organism may colonize the teat skin
and teat canal, which may predispose to
intramammary infection leading to mastitis. The
bacterium adheres to the internal mucosal
surfaces, producing several virulence factors6.
INTRODUCTION
Staphylococus aureus; one of common
pathogens that causes disease in a lot of hosts.
S. aureus carry many genes that encode a
variety of pyrogenic toxins (PTs), the most
important is staphylococcal enterotoxins (SEs)
and toxic shock syndrome toxin (TSST)1. The
PTs interact with several cellular targets to
produce disease, such as food poisoning and
toxic shock syndrome2.
Staph Food Poisoning (SFP) is a mild
intoxication occurring after the ingestion of
food containing staphylococcal enterotoxins
(SEs) 3.
101
Vol. 22, No. 2
an ice box, left in water bath at 44◦C for < 15
minutes to melt, thoroughly mixed by a sterile
stirrer, 5 ml were transferred to 45 ml of sterile
saline, and then ten fold serial dilutions were
carried out. kariesh cheese samples: Five gram
from each sample were mashed thoroughly in a
sterile morter and 45 ml of 2% sodium citrate
were added to obtain a dilution of 1:10, then ten
fold serial dilutions were carried out. Nasal
swabs and stool samples from milk handlers (1,
12)
.
Isolation, identification and confirmation of
S. aureus:
5 ml or gm of the previously prepared
samples, nasal swabs and stool samples were
inoculated into 5 ml of Staphylococcus broth
(Difco), incubated at 35oC for 20 h (1, 13). Then,
subculture on selective media; Mannitol Salt
agar (Bio Merieux, BBL, 11407)(1,14).
Identification of S. aureus by lecithinase and
lipase activities on Baird-Parker agar (Oxoid,
BO0458J, Basingstoke, England) (1, 14). S.
aureus isolates were confirmed by tube
coagulase test 15, detection of clumping factor,
protein A and capsular polysaccharides by latex
agglutination test using Oxoid Dry Spot
Staphytect Plus (DR 100 M) which uses blue
latex particles coated with both porcine
fibrinogen and rabbit immunoglobulin G (IgG)
including specific antibodies raised against
capsular polysaccharides of S. aureus16.
Viable count of S.aureus in milk and dairy
products:
A quantity of 100 ul from each prepared
dilutions of samples was cultured on BP-A
using surface plating technique. The number of
suspected colonies in countable plates was
enumerated and the S. aureus count per ml or
gm was calculated and recorded17.
Detection of enterotoxigenicity by SDSPAGE (1, 18):
- Samples preparation:
One colony of each strain was washed and
stirred after the addition of 25 µl SDS lysing
buffer, and the proteins were denatured in
boiling water for 5 min. Supernatant was then
centrifuged and collected in an eppendorf tube
for electrophoresis.
-Preparation of Polyacrylamide Gels:
Two layers of discontinuous gels were
prepared; the upper layer (stacking gel) has low
acrylamide concentration (4.5%), low pH (6.8)
and low resolving ability. The lower layer
(resolving gel) has 12.5% acrylamide
concentration, much smaller pores and pH 8.8.
-Sample Application and Electrophoresis:
10µl of the protein extracts of tested strains with
Page Ruler prestained protein ladder (SM0671
Milk and dairy products are frequently
contaminated with enterotoxigenic S. aureus,
which is often involved in staphylococcal food
poisoning; these contaminations are either from
animal or human sources. Recently by the
identification of the new SEs, the perceived
frequency of enterotoxigenic strains has
increased, suggesting that the pathogenic potential
of Staphylococci may be higher than previously
thought7.
As milk is a very suitable medium for the
growth of many pathogens including S. aureus
and because raw milk is subjected to
contamination either directly or indirectly from
different sources including producing animal,
milk producers and handlers, many outbreaks of
SFP traceable to dairy products do still occur
inspite of advanced dairy manufacturing
processes8.
Nasal and stool carriage could be the
primary source of infection of manually handled
milk and milk products. Up to 20% of healthy
humans are persistently colonized by S. aureus,
while as many as 60% can be colonized
intermittently9.
It was demonstrated that S. aureus is
associated with some human diseases as well as
that S. aureus isolated from healthy carriers
share a very high prevalence of enterotoxin gene
cluster10.
The throat and nasal carriers of pathogenic
S. aureus in normal community of healthy
individuals give opportunities for postpasteurization contamination. So, further studies
should be done to clarify the epidemiological
association and bacteriological characteristics of
human and animal S. aureus in food11.
Aim of the work
To detect the presence and prevalence of
coagulase positive S. aureus in milk, kariesh
cheese and ice-cream samples and in nasal
swabs and stool samples from milk handlers,
and to detect types of S. aureus enterotoxins by
sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE), and to detect the
type of enterotoxins genes by PCR.
MATERIAL & METHODS:
This study is a cross section study that was
conducted in Assiut Governorate rural areas on
250 samples (100 milk samples, 50 ice-cream,
50 kariesh cheese and 25 nasal swabs and 25
stool samples from milk handlers).
Samples preparation:
Milk samples: 5 ml added to 45 ml of
sterile saline, then ten fold serial dilutions were
carried out. Ice-cream samples: cooled to 2oC in
102
Vol. 22, No. 2
mixture was added to 9 µl of Master Mix: dd
H2O, 2.5 mM concentration of each
deoxynucleoside triphosphate (dNTP) (Jena
Bioscience, Cat No. NU-1005S), 2U of
BioReady r Taq DNA polymerase (BioFluxCat No. BSA12M1) and 10 x PCR buffer with
15 mM MgCl2 (Jena Bioscience ). The primers
used for detection of SE genes are listed in table
(1). A PERKIN ELMER GeneAmp 9600 PCR
System was used for amplification. The thermal
cycling conditions were as follow: initial cycle
of denaturation (at 95oC for 5 min), followed by
30 cycles of denaturation at (95 oC for 15 s),
annealing (at 50oC for 30 s), and extension (at
72 oC for 30 s), with a final extention step of
72oC for 8 min.
3-Detection of amplified products:
1.5% agarose gel (Molecular Biology
Grade) stained with ethedium bromide
(0.5µl/ml) was loaded with 10 µl of PCR
products for each well, mixed with 2 µl of 6x
loading
dye
(Jena
Bioscience)
and
electrophoresed in 5x TBE buffer (Tris-BoricEDTA) at 100 V for 30 min. PCR products
were sized against a 100 – 1000 bp DNA step
ladder (5 µl of ladder was added in one well)
(Cat No. 239125, Qiagen, Germany) and were
visualized in the gel using U.V. transilluminator
(Hoefer Scientific).
Fermentas Life Sciences) that was used as a
protein marker. The gel was electrophoresed by
using Hoefer Mighty Mini-Vertical Unit with
bromophenol blue dye for 4 hrs. ± 10 min. until
the dye migrates 100mm down the length of
separating gel.
- Protein Visualization:
Protein electrophoresis, then fixation and
staining with 1% Coomassie Blue R-250 with
gentle agitation overnight (16 h.). The stain
solution was replaced with ethanol and acetic
acid. Positive samples of SEs give band from
25-30 kDa and were visualized by Ultraviolet
transilluminator Hoefer Scientific Instrument
(Cat No. 7015668).
Detection of SEs genes by polymerase chain
reaction (PCR) (1, 19):
1-Extraction of S. aureus DNA:
Manual extraction from fresh culture by
heating at 90oC for 17 minutes, then keeping at 20oC until needed. When required, 10 µl of the
supernatant of thawed and centrifuged sample
was used.
2-Amplification of SEs genes:
Each gene was amplified separately, using
specific primer pair for each reaction mixture.
PCR amplification was conducted in final
volume of 25 µl. The PCR mixture: 3µl forward
primer, 3 µl reverse primer (Metabion,
Germany) and 10 µl extracted DNA. Each
Table (1): The Primers used for detection of SEs genes20.
Primer
Primer Sequence
SEA forw.
GCAGGGAACAGCTTTAGGC
SEA rev.
GTTCTGTAGAAGTATGAAACACG
SEB forw
ACATGTAATTTTGATATTCGCACTG
SEB rev.
TGCAGGCATCATGTCATACCA
SEC forw.
CTTGTATGTATGGAGGAATAACAA
SEC rev.
TGCAGGCATATCATACCA
SED forw.
GTGGTGAAATAGATAGGACTGC
SED rev.
ATATGAAGGTGCTCTGTGG
SEG forw.
CGTCTCCACCTGTTGAAGG
SEG rev.
CCAAGTGATTGTCTATTGTCG
SEH forw.
CAACTGCTGATTTAGCTCAG
SEH rev
GTCGAATGAGTAATCTCTAGG
- Statistical analysis:
Was performed using SPSS software
version 16 (SPSS Inc., Chicago, USA), and
expressed as tests of significance (X2 and Fisher
exact tests for categorical variables and Student
t test and Mann Whitney tests for continuous
variables). Statistical significance was assumed
when P < 0.05.
Product Size (bp)
521
667
284
385
328
359
RESULTS
This study was conducted on 250 different
samples which were classified as 100 raw milk
samples, 50 ice-cream samples, 50 kariesh
cheese samples and 25 nasal swabs and 25 stool
samples from milk handlers.
Table 2: shows that 28%, 20%, and 34% of
raw milk, ice cream, and Kariesh cheese
103
samples respectively were colonized by
Staphylococcus species organisms with 57%,
100%, and 76.4% of them respectively were
exceeding Egyptian standards, and 60% of nasal
samples and 20% of stool samples were
colonized by Staphylococcus species. P<0.001.
Figure (1) shows the colony count of
Staphylococcus aureus organisms in different
types of samples.
Vol. 22, No. 2
Table (3) shows that 52.2%, 37.5%, 57.1%,
23.1% and 33.3% of raw milk, ice cream,
Kariesh cheese, nasal samples and stool samples
respectively, were colonized by enterotoxigenic
Coagulase Positive Staphylococci.
Table (4) shows that 44.4%, 22.2%, 18.5%,
7.4% and 7.4% of enterotoxigenic strains were
encoded by SEA, SEC, SED, SEG and SHE
genes respectively. But SEB gene was not
detected in any enterotoxigenic isolated strains.
Table (2): Prevalence of Staph colonization and their count in different samples.
Type of
No of
% of
Count/ml
Exceeding
CPS
CNS
sample
samples positive
(cfu)
E.S
samples
Raw milk samples
From
25
5 (20%)
10-<102 3 (60%)
4 (80%)
1 (20%)
animals
2x105
Dairy farms
25
10 (40%)
10-<102 6 (60%)
8 (80%)
2 (20%)
2.8x105
Dairy shops
25
7 (60%)
10-<102 4 (57%)
6 (85.7%)
1
2x104
(14.3%)
Street
25
6 (52%)
10-<102 3 (50%)
5 (83.3%)
1
distributors
1.7x104
(16.7%)
P- value
P <0.01*
P=0.425 n.s
P=0.346 n.s
P=0.431 n.s P=0.486 n.s
Total
100
28 (28%)
10-<102 16 (57%)
23 (82%)
5
2.8x105
(18%)
Ice – cream samples
Street
25
6 (24%)
10-<102 6 (100%)
5 (83.3%)
1 (16.7%)
vendors
4x103
Small scale
25
4 (28%)
10-<102 4 (100%)
3 (75%)
1 (25%)
producers
2x102
P- value
P=0.537 n.s
P=0.237 n.s
--P=0.378
P=0.254n.s
Total
50
10 (20%)
10-<102 10 (100%)
8 (80%)
2
4x103
(20%)
kariesh cheese samples
8 (80%)
9 (90%)
1 (10%)
Street
25
10 (40%)
10-<102 distributors
5x105
Grocery
25
7 (28%)
10-<102 5 (71%)
5 (71.4%)
2 (28.6%)
shops
2x105
P- value
P<0.03*
P<0.001**
P=0.243n.s
P=0.214 n.s P<0.02*
Total
50
17 (34%)
10-<102 13 (76.4%)
14 (82.3%)
3 (17.7%)
5x105
Milk handlers samples
Nasal swabs
25
15 (60%)
13 (86.6%)
2 (13.4%)
Stool
25
5 (20%)
3 (60%)
2 (40%)
P- value
P<0.001**
P=0.437n.
P<0.001**
Total
250
75 (30%)
39 (70.9%)
61 (81.3%)
14 (18.7%)
P- value for
P<0.001**
total samples
NB.: cfu: colony forming unit, ES: Egyptian standards, CPS: Coagulase Positive Staphylococci, CNS:
Coagulase Negative Staphylococci.
104
10-<100
100-<1000
1000-<10000
Vol. 22, No. 2
10000-<100000
100000-<1000000
100%
80%
60%
40%
20%
ps
sh
o
te
rs
y
ce
r
G
ro
St
re
et
di
st
r
pr
od
ib
u
uc
er
s
s
Sm
al
ls
ca
le
et
ib
u
ve
nd
or
te
rs
ps
sh
o
di
st
r
St
re
D
i re
ct
ly
St
re
et
D
ai
ry
fa
rm
s
D
ai
ry
om
an
im
al
s
0%
fr
% of S. aureus positive milk samples based on
their count/ml
Sources of samples
Figure (1): shows Staphylococcus species count in different samples.
Table (3): Percentage of enterotoxigenic strains of S. aureus among CPS isolated from different
types of samples examined by SDS-PAGE.
Examined
Samples
Raw milk samples
(direct from animal)
Dairy Farm
Dairy shops
Street distributers
Total
P – value
Street vendors
small scale producers
Total
P – value
Kariesh cheese samples
Street distributers
Grocery shops
Total
P – value
Milk Handlers samples
Nasal Swabs
Stool samples
Total
No. of
tested CPS
Isolates
Toxigenic
Isolates
No.
4
8
6
5
23
3
5
2
2
12
P<0.03*
75%
62.5%
33.3%
40%
52.2%
5
3
8
2
1
3
P=0.471n.s
40%
33.3%
37.5%
9
5
14
6
2
8
P=0.375n.s
66.6%
40%
57.1%
13
3
61
3
1
27
23.1%
33.3%
44.3%
105
%
Vol. 22, No. 2
Table (4): Types and prevalence of toxigenic genes in Staphylococcus aureus isolated from
different samples.
Classical superantigen genes
New superantigen genes
Examined
Toxigenic
samples
isolates
SEA
SEB
SEC
SED
SEG
SEH
No. (%)
No.%
No. %
No. %
No. %
No. %
Raw milk samples
From animals
3
1
1
1 (33.3%)
(33.3%)
0
(33.3%)
0
0
Dairy farms
5
2
1
1
1
(40%)
0
(20%)
(20%)
0
(20%)
Dairy shops
2
1
1
(50%)
0
(50%)
0
0
0
Street
2
1
1
distributors
(50%)
0
0
(50%)
0
0
*Total
12
5
3
2
1(8.3%)
1 (8.3%)
(41.6%)
0
(25%)
(16.6%)
P – value
P=0.378
P=0.327
P<0.02*
P<0.01*
P=0.378
n.s
n.s
n.s
Street vendors
2
1 (50%)
Small
scale
producers
Total
1
1 (33.3%)
3
2 (66.6%)
P – value
P=0.483n.
s
0
0
0
-
1 (50%)
0
0
0
-
0
0
0
-
0
0
0
-
1 (16.6%)
1 (16.6%)
1 (16.6%)
1 (16.6%)
0
1 (12.5%)
1 (50%)
2 (25%)
0
1 (12.5%)
0
1 (12.5%)
P=0.354n.
s
P=0.346n.
s
P=0.346n.
s
P=0.367n.
s
0
1 (33.4%)
P=0.379n.
s
kariesh cheese samples
street
distributers
Grocery shops
6
2 (33.3%)
2
1 (50%)
Total
8
3 (37.5%)
P – value
Total Milk samples
23
Milk Handlers samples
Nasal swabs
3
Stool samples
1
Total
27
0
0
0
P=0.256n.
s
10
0
5
4
2
2
1
1
12
(44.4%)
0
0
0
1
0
6 (22.2%)
1
0
5 (18.5%)
0
0
2 (7.4%)
0
0
2 (7.4%)
106
Vol. 22, No. 2
Photo (1): Gel stained with coomassie blue for protein visualization of
S. aureus enterotoxins.
Lane1: SEs detected in a milk sample (directly from animal)
Lane 2: SEs detected in an Ice-cream sample (street vendor)
Lane 3: SEs detected in a Kariesh cheese sample (street distributer)
Lane 4: SEs detected in stool samples.
Lane 5: SEs detected in nasal swabs samples.
Lane 6: Marker (PageRuler prestained protein ladder)
Photo (2): Agarose gel electrophoresis of amplified superantigen genes.
Lane 1: Positive for SEG (328 bp).
Lane 2: Positive for SEH (359 bp).
Lane 3: Negative sample.
Lane 4: Positive sample for SEC (284 bp).
Lane 5: Positive sample for SEA (521 bp).
Lane 6: Marker (100 – 1000 bp).
107
Vol. 22, No. 2
For the kariesh cheese samples:
colonization of street distributers (40%) was
higher than that of grocery shops (28%) with
significant statistical analytical result (P < 0.03),
and the maximum count was higher in street
distributers 5x106 than in grocery shops 2x105.
all of them were exceeding the Egyptian
standards which is against the Egyptian
Standards26 that pointed out that the kariesh
cheese must be free from S. aureus. 82.3% of
cases were Staph aureus. This percentage was
nearly similar to that postulated by Noha et al. 1
Tawfeek et al.,27 Hassan22 and Awida21 who
could isolate S. aureus from 70%, 66%, 72%
and 100% of the examined kariesh cheese
samples respectively.
Nasal and stool carriage is considered as a
primary source of contamination of manually
handled milk and milk products as well as a
source of S. aureus transmission between
human. 60% and 20% of cases respectively
were colonized with Staph species, 80% were
CPS. This is concomitant with Collery et al.23
who found that approximately, 20% of healthy
human were estimated to be persistently
colonized by S. aureus, while as many as 60%
could be colonized intermittently. Noha et al. 1
found that 80% of milk handlers were colonized
by Staph species and 50% of them were CPS.
As a whole there were significant
statistical difference between different types of
samples (P<0.001) concerning colonization,
colony count, percent of CPS and CNS.
It's interesting that; 52.2% of CPS isolated
from milk samples were toxigenic. The highest
percentage of toxigenic CPS isolated from milk
samples was observed in milk taken directly
from animal (75%) and the lowest percentage
was in milk observed from dairy shops (33.3%),
with significant statistical difference (P < 0.03).
As regard ice-cream samples, 37.5% of CPS
was toxigenic and there was no significant
difference between the percentages of
enterotoxigenic S. aureus among CPS isolates.
As
regard
kariesh
cheese
samples,
enterotoxigenic S. aureus was detected in
57.1% of CPS isolated from kariesh cheese
samples. The percentage of enterotoxigenic S.
aureus in kariesh cheese from street distributers
(66.6%) was higher than that in kariesh cheese
from grocery shops (40%). 23.1% and33.3% of
CPS isolates from nasal swabs and stool
samples of food handlers respectively were
enterotoxigenic. Nearly similar result was
observed by Noha et al. 1Bania et al.9 and
Chapaval et al.28 who found out that 49.5%,
54% and 44.54% respectively of the detected
CPS isolates in milk and milk products samples
DISCUSSION
In the present study, we tried to detect the
presence of S. aureus and their ability for
enterotoxin production in milk and some dairy
products and their milk handlers in Assiut City
rural areas, Egypt.
In the present study, Staph species was
isolated from 28% of raw milk samples, 20% of
ice-cream samples, 34% of kariesh cheese
samples, 60% of nasal swabs and 20% of stool
samples, with statistical significant difference
between colonized and non colonized cases (P <
0.001). This is lower than Rall et al.5 who found
that 70.4% of raw milk samples were
contaminated with Staph organism, with
Awida21 who found that 30% of ice-cream
samples were contaminated with Staph
organism, with Hassan22 who found that 72% of
Kariesh cheese samples were contaminated with
Staph organism, and lastly Collery et al.23 who
found that 100% of nasal samples were
contaminated with Staph organism. A recent
study was conducted in Egypt1 and found
slightly higher results: 59.5%, 40%, 70% and
80% for raw milk, ice cream, kariesh cheese and
nasal swabs respectively.
From raw milk samples; S. aureus was
isolated from 80% of animal samples, 80% of
dairy farms samples, 85.7% of dairy shops
samples and 83.3% of street distributors'
samples. All of them were exceeding Egyptian
standards, with no significant statistical
difference in the count of these colonies
between different types of samples (P = 0.431).
So, the highest percentage of samples with
counts exceeding the Egyptian standards was
also observed in milk taken from dairy shops.
The frequency of distribution of the positive
examined milk samples based on their S. aureus
count/ml was nearly similar to that postulated
by Noha et al. 1 and Awida21 where the highest
frequency distribution lies below 102 in the
examined raw milk samples taken from dairy
farms (59.2%), dairy shops (55.5%) and street
distributers (93.3%).
The percentage of Staph species in icecream samples from street vendors (24%) was
near to that of small scale producers (28%) and
the maximum count was higher in street vendors
(4x103) than in small scale producers (2x102)
with no significant statistical analytical result (P
= 537). all of them exceeds the Egyptian
standards. Totally 80 % was Staph Aureus. This
result was lower than the results obtained by
Noha et al. 1 40%, Ali24 44% and Hammad25
52%.
108
produced enterotoxins. Although the selected 61
isolates from milk, milk products, nasal swabs
and stool samples were strongly producing
coagulase,
only
27
(44.3%)
were
enterotoxigenic, this confirms what was stated
by Ryser29 that the attempts to associate
enterotoxin production by S. aureus with
specific biochemical properties generally failed.
Consequently, confirmation of the toxin by
SDS-PAGE or other means provide the only
proof
that
the
particular
strain
is
enterotoxigenic.
The PCR technique was applied to all 27
toxigenic strains of S. aureus isolated from
different samples. In milk and dairy products,
the major classical enterotoxin genotype was
SEA which was detected in 12 isolates (44.4%).
SEC was detected in 6 isolates (22.2%) and
SED was detected in 5 isolates (18.5%). For the
newly described genes, SEG was detected in 2
isolates (7.4%) followed by SEH which was
detected in 2 isolates (7.4%).
The higher percentage of SEA gene
among S. aureus strains isolated from milk and
milk products may be due to the fact that
enterotoxins A are less common among the
strains of animal origin than from of human
origin. These strains of human origin
contaminate milk and dairy products during
different stages of production and processing or
even at consumer outlet. On the other side, the
presence of SEC and SED can be attributed to
the increased incidence of staphylococcal
mastitis, as enterotoxins C and D were found to
be produced by S. aureus strains isolated from
bovine mastitis and were designated as "animal
strains". Out of 4 toxigenic isolates of nasal
swabs and stool samples, the distribution of the
classical enterotoxin genes was SEA in two
isolates (50%), SEC in one isolate (25%) and
SED in one isolate (25%).
3.
Chiang, Y.C.; Liao, W.W.; Fan, C.M.;
Pai, W.Y.; Chiou, C.S. and Tsen, H.Y.:
PCR
detection
of
Staphylococcus
enterotoxins N, O, P,Q,R,U and survey of
SE types in S. aureus isolates from food
poisoning cases in Taiwan. Int J Food
Microbiol. 2008; Jan 15;121(1) : 66-73.
4. Rall, V.L.M.; Vieira, F.P.; Rall, R.;
Vieitis, A.; Fernandes, J.R.; Candeias,
J.M.C.; Cardoso, K.F.G. and Araujo,
j.R.: PCR detection of staphylococcal
enterotoxin genes in S. aureus strains
isolated from raw and pasteurized milk.
Vet. Microbiol. 2008;132: 408-413.
5. Taverna, F.; Negri, A.; Piccinini
R.;Zecconi, A.; Nonnis, S.; Ronchi, S.;
Tedeschi, G. (2007): characterization of
cell wall associated proteins of a S. aureus
isolated from bovine mastitis. Vet.
Microbiol. 119: 240-247.
6. Leloir, Y.; Baron, F. and Gautier, M.
(2003): S. aureus and food poisoning.
Genet. Mol. Res. 2: 63-76.
7. Zschoch, M.; Kloppert, M.B.; Wolter,
W.; Hamann, H.P. and Lammler, C.:
Pattern of enterotoxin genes SEG SE, SEI
and SEJ positive S. aureus from bovine
mastitis. Vet. Microbiol. 2005; 108:243249.
8. Srinivasan, V.;Sawant, A. A.; Gillespie,
B.E.; Headrick, S. J.; Ceasaris, L. and
Oliver, S.P. (2006): Prevalence of
enterotoxin and toxic shock syndrome toxin
genes in S. aureus isolated from milk of
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9. Bania, J.; Dabrowska, A.; Bystron, J.;
Korzekwa, K.; Chrzanowska, J.; and
Molenda, J. (2006): The profiles of
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10. Mempel, M.; Lina, G.; Hojika, M.
Schnopp, C.Seidl, H.P.; Schafer, T.;
Ring, J.; and Vandenesch, F. (2008):
High
prevalence
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associated with the egc locus in S. aureus
isolates from patients with atopic eczema.
Int. J. Clin. Microbiol. Infect. Dis., 22: 306309.
11. Hata, E.; Katsuda, K.; Kobayashi, H.;
Nishimori, K.; Uchida, I.; Higashide, M.;
Ishikawa, E.; Sasaki, T. and Eguchi, M.
(2008): Bacteriological characteristics of
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12. A.P.H.A.: Standard Methods for the
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‫‪Vol. 22, No. 2‬‬
‫إﻳﺠﺎد و ﺗﻮﺻﻴﻒ اﻟﺴﻤﻮم اﻟﻤﻌﻮﻳﺔ ﻟﻠﻤﻴﻜﺮوب اﻟﻌﻨﻘﻮدي اﻟﺬهﺒﻲ ﻓﻲ اﻷﻟﺒﺎن‬
‫وﺑﻌﺾ ﻣﻨﺘﺠﺎﺗﻬﺎ واﻟﻘﺎﺋﻤﻴﻦ ﺑﺘﺪاوﻟﻬﺎ‬
‫ﻣﻴﺨﺎﺋﻴﻞ ﻧﻈﻤﻲ ﻋﺠﺒﺎن )‪ , (1‬أﺣﻤﺪ ﺻﺎدق أﺣﻤﺪ‬
‫)‪(1‬‬
‫ﻗﺴﻢ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻴﺎ اﻟﻄﺒﻴﺔ واﻟﻤﻨﺎﻋﺔ‪ -‬آﻠﻴﺔ اﻟﻄﺐ ‪-‬ﺟﺎﻣﻌﺔ أﺳﻴﻮط)‪ .(1‬ﺟﻤﻬﻮرﻳﺔ ﻣﺼﺮ اﻟﻌﺮﺑﻴﺔ‪.‬‬
‫ﺧﻠﻔﻴ ﺔ اﻟﺒﺤ ﺚ‪ :‬ﻣﻴﻜ ﺮوب اﻟﻤﻜ ﻮر اﻟﻌﻨﻘ ﻮدي اﻟ ﺬهﺒﻲ ﻳﺤﻤ ﻞ اﻟﻌﺪﻳ ﺪ ﻣ ﻦ اﻟﺠﻴﻨ ﺎت اﻟﺘ ﻲ ﻟﻬ ﺎ اﻟﻘ ﺪرة ﻋﻠ ﻰ إﺣ ﺪاث ﺗ ﺴﻤﻢ ﻏ ﺬاﺋﻲ‬
‫وﺗﺆدى اﻟﻰ ﻧﺴﺒﺔ آﺒﻴﺮة ﻣﻦ اﻻوﺑﺌﺔ‪.‬هﺬا و ﻗﺪ آﺎﻧ ﺖ اﻟ ﺴﻤﻮم اﻟﻤﻌﻮﻳ ﺔ اﻟﺨﻤ ﺲ اﻟﻤﻌﺮوﻓ ﺔ ه ﻰ ‪ SEA‬اﻟ ﻰ ‪، SEE‬وﻟﻜ ﻦ هﻨ ﺎك‬
‫اﻧﻮاع ﺟﺪﻳﺪة ﺗﻢ اﻟﺘﻌﺮف ﻋﻠﻴﻬﺎ ﻣﺜﻞ ‪ SEG‬و ‪ .SEH‬و هﺬﻩ آﻠﻬﺎ ﻳﻔﺮزه ﺎ اﻟﻤﻴﻜ ﺮوب ﻓ ﻲ اﻷﻟﺒ ﺎن و ﻣﻨﺘﺠﺎﺗﻬ ﺎ و ﻣ ﺼﺪرهﺎ ه ﻮ‬
‫اﻹﻧﺴﺎن أو اﻟﺤﻴﻮان‪.‬‬
‫اﻟﻬﺪف ﻣﻦ اﻟﺒﺤﺚ‪ :‬ﺗﻘﻴﻴﻢ ﻧﺴﺒﺔ ﺗﻮاﺟﺪ اﻟﻤﻴﻜﺮوب اﻟﻌﻨﻘ ﻮدي اﻟ ﺬهﺒﻲ وﺳ ﻤﻮﻣﻪ ﻓ ﻰ اﻷﻟﺒ ﺎن و اﻷﻳ ﺲ آ ﺮﻳﻢ و اﻟﺠﺒﻨ ﺔ اﻟﻘ ﺮﻳﺶ و‬
‫اﻟﻘﺎﺋﻤﻴﻦ ﺑﺘﺪاوﻟﻬﺎ واﻟﺘﻮﺻﻴﻒ اﻟﺠﻴﻨﻰ ﻟﻠﺴﻤﻮم اﻟﻌﺮوﻓﺔ واﻳﻀﺎ اﻟﺴﻤﻮم اﻟﺘﻰ ﺗﻢ اﻟﺘﻌﺮف ﻋﻠﻴﻬﺎ ﺣﺪﻳﺜﺎ‪.‬‬
‫اﻟﻄﺮق اﻟﻤﺴﺘﺨﺪﻣﺔ‪ :‬ﺗﻢ ﺟﻤﻊ ‪250‬ﻋﻴﻨﺔ ) ‪ 100‬ﻣﻦ اﻟﻠﺒﻦ اﻟﺨﺎم و‪ 50‬ﻣ ﻦ اﻷﻳ ﺲ آ ﺮﻳﻢ و ‪ 50‬ﻣ ﻦ اﻟﺠ ﺒﻦ اﻟﻘ ﺮﻳﺶ( ﺑﺎﻻﺿ ﺎﻓﺔ اﻟ ﻰ ‪25‬‬
‫ﻣﺴﺤﺔ أﻧﻔﻴﺔ و ‪ 25‬ﻋﻴﻨﺔ ﺑﺮازﻋ ﺸﻮاﺋﻴﺎ ﻣ ﻦ اﻟﻤﺘ ﺪاوﻟﻴﻦ ﻟﻬ ﺬﻩ اﻟﻤﻨﺘﺠ ﺎت ﺑﻤﺪﻳﻨ ﺔ أﺳ ﻴﻮط‪ -‬و ﻣﻨﺎﻃﻘﻬ ﺎ اﻟﺮﻳﻔﻴ ﺔ ‪ -‬ﺟﻤﻬﻮرﻳ ﺔ ﻣ ﺼﺮ‬
‫اﻟﻌﺮﺑﻴ ﺔ ‪ ،‬وﻗ ﺪ ﺗ ﻢ ﻓﺤ ﺼﻬﺎ ﺑﻜﺘﺮﻳﻮﻟﻮﺟﻴ ﺎ ﺑﺎﺳ ﺘﺨﺪام ﻣ ﺴﺘﻨﺒﺖ ‪ Baird-Parker agar‬ﻟﻤﻌﺮﻓ ﺔ ﻧ ﺴﺒﺔ ﺗﻮاﺟ ﺪ اﻟﻤﻴﻜ ﺮوب‬
‫اﻟﻌﻨﻘ ﻮدي اﻟ ﺬهﺒﻰ‪،‬وﺗﻢ اﻟﺘﺄآﻴ ﺪ ﺑﺎﺳ ﺘﺨﺪام اﺧﺘﺒ ﺎراﻧﺰﻳﻢ اﻟ ﺘﺠﻠﻂ واﺧﺘﺒ ﺎر اﻟﺘﻠ ﺰن ﻟﻠﻜ ﺸﻒ ﻋ ﻦ ﻋﺎﻣ ﻞ اﻟﺘﺠﻤ ﻊ وﺑ ﺮوﺗﻴﻦ‬
‫‪A‬واﻟﺤﺎﻓﻈﺔ ﻣﺘﻌ ﺪدة اﻟ ﺴﻜﺮﻳﺎت‪ ،‬وﺗ ﻢ اﻟﻔ ﺼﻞ اﻟﻜﻬﺮﺑ ﺎﺋﻲ ﻟﺒﺮوﺗﻴﻨ ﺎت اﻟﻤﻴﻜ ﺮوب ﺑﺎﺳ ﺘﺨﺪام ‪ SDS-PAGE‬ﻟﻠﺘﻌ ﺮف ﻋﻠ ﻰ‬
‫اﻟﻌﺘﺮات اﻟﺴﺎﻣﺔ‪ .‬و ﻗﺪ اﺳﺘﺨﺪم ﺗﻔﺎﻋﻞ اﻧﺰﻳﻢ اﻟﺒﻠﻤﺮة اﻟﻤﺘﺴﻠﺴﻞ ﻟﻠﺘﻌﺮف ﻋﻠﻰ ﻧ ﻮع وﺗﻮزﻳ ﻊ اﻟﺠﻴﻨ ﺎت اﻟﻔﺎﺋﻘ ﺔ اﻟﺨﺎﺻ ﺔ ﺑﺎﻟ ﺴﻤﻮم‬
‫اﻟﻤﻌﻮﻳﺔ ﻟﻠﻤﻴﻜﺮوب اﻟﻌﻨﻘﻮدي اﻟﺬهﺒﻲ )‪.(A,B,C,D,G,H‬‬
‫اﻟﻨﺘﺎﺋﺞ‪ :‬وﻗﺪ ﺗﺒﻴﻦ ﺑﺎﻟﻔﺤﺺ اﻟﺒﻜﺘﺮﻳﻮﻟﻮﺟﻲ أن ﻧﺴﺒﺔ ﺗﻮاﺟﺪ ﻣﻴﻜﺮوب اﻟﻤﻜﻮر اﻟﻌﻨﻘﻮدي اﻟﺬهﺒﻲ اﻟﻤﻮﺟﺐ ﻻﻧﺰﻳﻢ اﻟﺘﺠﻠﻂ آﺎﻧ ﺖ‬
‫‪٪82‬و‪٪80‬و‪٪82.3‬و‪ ٪86.6‬و ‪ %60‬ﻣﻦ ﻋﻴﻨﺎت اﻟﻠﺒﻦ اﻟﺨ ﺎم واﻷﻳ ﺲ اﻟﻜ ﺮﻳﻢ واﻟﺠ ﺒﻦ اﻟﻘ ﺮﻳﺶ وﻣ ﺴﺤﺎت اﻷﻧ ﻒ و ﻋﻴﻨ ﺎت اﻟﺒ ﺮاز‬
‫ﻋﻠ ﻲ اﻟﺘ ﻮاﻟﻰ اﻟﺤﺎﻣﻠ ﺔ ﻟﻤﻴﻜ ﺮوب اﻟﻤﻜ ﻮر اﻟﻌﻨﻘ ﻮدي‪ ،‬آﻤ ﺎ وﺟ ﺪ أن ‪ %70.9‬ﻣ ﻦ اﻟﻌﻴﻨ ﺎت ﺗﺠ ﺎوز ﻋ ﺪد اﻟﻤﻴﻜ ﺮوب ﺑﻬ ﺎ اﻟﻨ ﺴﺒﺔ‬
‫اﻟﻤﺴﻤﻮح ﺑﻬﺎ ﺑﻤﺼﺮ‪ .‬هﺬا وﻗﺪ وﺟﺪ أن ‪ ٪44.3‬ﻣﻦ اﻟﻌﺘﺮات اﻟﻤﻮﺟﺒﺔ ﻻﻧ ﺰﻳﻢ اﻟ ﺘﺠﻠﻂ ﺗﻤﺜ ﻞ ﻋﺘ ﺮات ﺳ ﺎﻣﺔ‪ ،‬وﻗ ﺪ وﺟ ﺪ ان اﻋﻠ ﻰ‬
‫ﻧﺴﺒﺔ آﺎﻧﺖ ﻓﻲ اﻟﻠﺒﻦ اﻟﺨﺎم اﻟﻤﺠﻤﻊ ﻣﻦ اﻟﺤﻴﻮان ﻣﺒﺎﺷﺮة وﻣﻦ اﻟﺠﺒﻦ اﻟﻘﺮﻳﺶ اﻟﻤﺠﻤﻊ ﻣﻦ اﻟﺒﺎﺋﻌﻴﻦ اﻟﺠﺎﺋﻠﻴﻦ ﺑﻨ ﺴﺒﺔ ‪%66.6، ٪75‬‬
‫ﻋﻠﻲ اﻟﺘﻮاﻟﻲ‪ .‬ﺑﺎﻟﻨﺴﺒﺔ ﻟﻼﻟﺒﺎن و ﻣﻨﺘﺠﺎﺗﻬﺎ‪ ،‬ﻓﻘﺪ آﺎن ﺗﻮاﺟﺪ اﻟﺠﻴﻦ اﻟﺨﺎص ﺑﺎﻟﺴﻢ اﻟﻤﻌﻮى ‪ A‬هﻮ اﻷآﺜﺮ ﺗﻮاﺟ ﺪا ﺣﻴ ﺚ وﺟ ﺪ ﻓ ﻲ‬
‫‪ %44.4‬ﻣﻦ اﻟﻌﺰﻻت اﻟﺴﺎﻣﺔ‪ ،‬اﻟﺠﻴﻦ اﻟﺨﺎص ﺑﺎﻟﺴﻢ اﻟﻤﻌﻮى ‪ C‬ﻓﻲ ‪ ،%22.2‬اﻟﺠﻴﻦ اﻟﺨﺎص ﺑﺎﻟﺴﻢ اﻟﻤﻌ ﻮى ‪ D‬ﻓ ﻲ ‪ ،%18.5‬ﺑﻴﻨﻤ ﺎ‬
‫اﻟﺠﻴﻦ اﻟﺨﺎص ﺑﺎﻟﺴﻢ اﻟﻤﻌﻮى ‪ B‬ﻟﻢ ﻳ ﺘﻢ اﻟﺘﻌ ﺮف ﻋﻠﻴ ﻪ ﻓ ﻰ أى ﻣ ﻦ اﻟﻌﻴﻨ ﺎت‪ .‬وآ ﺎن ﻣ ﻦ اﻧ ﻮاع اﻟﺠﻴﻨ ﺎت اﻟﺘ ﻲ اآﺘ ﺸﻔﺖ ﺣ ﺪﻳﺜﺎ‬
‫اﻟﺠﻴﻦ اﻟﺨﺎص ﺑﺎﻟﺴﻢ اﻟﻤﻌﻮى ‪ G‬وﻗﺪ وﺟﺪ ﻓﻲ ‪ %7.4‬و اﻟﺠﻴﻦ اﻟﺨﺎص ﺑﺎﻟﺴﻢ اﻟﻤﻌﻮى ‪ H‬ﻓﻲ ‪.%7.4‬‬
‫اﻻﺳﺘﻨﺘﺎج‪ :‬ﻧﺴﺒﺔ آﺒﻴﺮة ﻣﻦ اﻷﻟﺒﺎن وﺑﻌﺾ ﻣﻨﺘﺠﺎﺗﻬﺎ وﺧﺎﺻﺔ اﻟﺠﺒﻦ اﻟﻘﺮﻳﺶ اﻟﻤﻌﺮوﺿﺔ ﻟﻠﺒﻴﻊ ﻓﻲ ﻣﺤﺎﻓﻈﺔ أﺳﻴﻮط‪-‬ﺟﻤﻬﻮرﻳﺔ‬
‫ﻣﺼﺮ اﻟﻌﺮﺑﻴﺔ‪ ،‬ﻣﻠﻮﺛﺔ ﺑﺎﻟﻤﻴﻜﺮوب اﻟﻌﻨﻘﻮدي اﻟﺬهﺒﻰ وﺳﻤﻮﻣﻪ واآﺜﺮ أﻧﻮاع اﻟﺴﻤﻮم ﺗﻮاﺟﺪا هﻮ اﻟﺴﻢ ‪ A‬وهﻮ ﻣﻌﺮوف اﻧﻪ اﻗﻞ‬
‫ﺗﻮاﺟﺪا ﻓﻲ اﻟﻤﺼﺪر اﻟﺤﻴﻮاﻧﻲ ﻋﻦ ﻧﻈﻴﺮﻩ ﻓﻰ اﻟﻤﺼﺪر اﻟﺒ ﺸﺮى‪ .‬ه ﺬا وﻗ ﺪ ﺗﺒ ﻴﻦ أن اﻟﺒ ﺎﺋﻌﻴﻦ اﻟﺤ ﺎﻣﻠﻴﻦ ﻟﻠﻤﻴﻜ ﺮوب ﻓ ﻲ اﻟﻐ ﺸﺎء‬
‫اﻟﻤﺨﺎﻃﻰ ﻟﻸﻧﻒ و ﻋﻴﻨﺎت اﻟﺒﺮاز ﻳﻌﺘﺒﺮون اﻟﻤﺼﺪر اﻟﺮﺋﻴﺴﻰ ﻟﺘﻠﻮث اﻷﻟﺒﺎن وﻣﻨﺘﺠﺎﺗﻬﺎ‪.‬‬
‫‪111‬‬
Vol. 22, No. 2
Role of SHV Genes in Nosocomially Isolated Extended Spectrum β
Lactamase Producing Klebsiellae Pneumonia from Ventilator
Associated Pneumonia among ICU Patients Using PCR Assay
Abeer Ezzat El Sayed 1, 2; Samia Ragab El Azab3, 4
Department of Microbiology & Immunology, Faculty of Medicine, Suez Canal
University (1, 2) and Department of Anaesthesia & Intensive Care, Faculty of Medicine,
Al-Azhar for Girls University (3, 4)
ABSTRACT
Background: The genus Klebsiellae pneumonia is considered an important pathogen causing ventilatorassociated pneumonia (VAP). Beta lactam antimicrobial agents are the most commonly used treatment of
bacterial infections. Extended spectrum β-lactamases (ESBLs) are enzymes that mediate resistance to
extended-spectrum cephalosporins and monobactams but not cephamycins or carbapenems. ESBLs are
considered an important source of morbidity and mortality in patients receiving mechanical ventilation.
These enzymes are numerous and mutate continuously in response to heavy pressure of antibiotics use
and misuse. This study aimed to study the role of SHV genes as they are one of the most common and
important genes encode these enzymes and prevalence of ESBLs producing Klebsiellae pneumonia
causing VAP in ICU patients and antimicrobial susceptibility pattern to prevent spread of this type of
isolates. Methods: Endotracheal tube aspirates specimens from patients clinically diagnosed as VAP
were collected according to CDC criteria within one year in two hospitals in Saudi Arabia; Buraidah
Central Hospital and King Fahd Specialist Hospital. Klebsiellae pneumonia was identified using
conventional identification method and by automated MicroScan machine. Diagnosis of ESBLs
producing isolates were done using both phenotypic double disk Synergy diffusion method (DDST) and
also was confirmed by the automated MicroScan machine using panel for Gram negative by minimal
inhibitory concentration (MIC) method. Detection of SHV genes were done to ESBLs positive isolates
using polymerase chain reaction (PCR) assay. Results: Twenty one isolates of Klebsiellae pneumonia
were isolated; 18 of them were ESBLs positive by MIC method (85.7%). DDST failed to diagnose one
isolate only as it gave false negative result compared to MIC confirmatory method. The rate of VAP was
5.6 cases/1000 ventilator-days within one year in the two hospitals. The mean of length of stay in ICU of
both hospitals was (mean±SD = 9.7±7.0). The mean of ventilator days was 8.4 days (range 2-35 days).
Prevalence of confirmed ESBLs Klebsiellae pneumonia was 26.5% isolated from all VAP cases (18/68)
causing VAP in both hospitals. Using PCR method 12 isolates out of 18 were positive for the presence of
SHV genes (66.7%). This enumerated the importance of these genes for production of ESBLs. All ESBLs
isolates were resistant to ampicillin, cefazolin, cefuroxime, ceftazidime, cefotaxime, cefepime,
piperacillin, (100%), meanwhile these were sensitive to cefoxitin except two isolates only were resistant
(11.1%). All these isolates were sensitive to tigecycline and colistin (100%). Resistance of ESBLs isolates
to amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin, moxifloxacin, tetracycline and
trimethoprimsulfamethoxazole were 33.3%, 38%, 33.3%, 22%, 22%, 16.7%, 55.6% and 83.3%
respectively. It was found that 55.5% of ESBLs isolates were resistant to amoxicillin/k clavulanate and
piperacillin/tazobactam. The least resistance was to imipenem, meropenem, ertapenem and cefoxitin in 2
isolates only (11.1%) for each. Conclusion: Our findings showed the importance of these ESBLs isolates
in hospital associated infections especially VAP in ICU and antibiotic resistance especially
cephalosporins and penicillin group that could lead to failure of management and restriction for the
suitable choice of chemotherapeutic agents by clinicians. Wise use of antimicrobial agents is
recommended inside ICU as critically ill patients are more susceptible to infection by these strains. It is
recommended to screen for the presence of SHV and other genes by rapid, accurate molecular method in
suspected ESBLs isolates to prevent spread of infection among critically ill patients in ICU. Further
molecular studies like multiplex PCR, genetic sequencing, restriction fragment length polymorphism
(RFLP), isoelectric focusing are needed to study other genes that mediate production of these enzymes.
Keywords: Ventilator Associated pneumonia (VAP) in ICU, Extended Spectrum Beta-Lactamase ESBL,
(sulphydryl variable) SHV genes.
113
Vol. 22, No. 2
Data Collection
Baseline data including age, sex, length of
hospital stay in ICU, Acute Physiology and
Chronic Health Evaluation II (APACHE score
II) 24 hours after admission to ICU (18). Length
of stay in ICU (LOS), number of patient days,
number of ventilator days and total number of
cases of VAP were collected to calculate the
rate. Criteria for VAP were drawn from the
CDC definition (clinical and laboratory in
addition to endotracheal tube aspirates culture
and sensitivity) and were applied to patients
with onset of signs and symptoms after any
period of intubation or within 48 hours after
extubation.
Quantitative culture of endotracheal tube
aspirates (EA) according to Marquette et al.,
(1993) (19):
The endotracheal tube aspirate secretions
(EA) were collected in sterile containers and
immediately were sent to the microbiology
laboratory then were liquefied and homogenized
by adding an equal volume of sterile 1% Nacetyl-L-cysteine (Sigma) solution vortexing for
2 minutes and incubating at room temperature
for 10 minutes. The homogenized respiratory
secretions were serially 10 fold diluted in sterile
saline (0.9%) using 2 dilutions (1/100 and
1/1000). Ten µL from last dilution was
inoculated into blood agar and MacConkey`s
agar plates then incubated aerobically at 35 °C
for 24 hrs. The bacterial isolates were identified
by colonial morphology (mucoid), Gram
staining, oxidase test, spot indole test and
catalase test. The suspected Gram negative
bacteria were further identified in addition to
colonial morphology by using automated
MicroScan walkaway 96, SI according to the
manufacture instructions. Pure bacterial
suspension adjusted turbidity was done using
Combo 42 panel for Gram negative bacilli
(Siemens Healthcare Diagnostics Inc. USA).
Significant bacterial count was considered ≥ 105
CFU/ml for EA secretions counted in the blood
agar plate. Bacterial strains were collected after
identification and preserved in 1.0% trypticase
soy agar incubated at 37 °C for 24 hrs. Another
copy was kept in trypticase soy broth with 13%
glycerol and kept at -70°C until used. Identified
strains were tested for production of ESBL
using the following
1-Phenotypic diffusion method:
Using double disk synergy test (DDST) by
agar diffusion method (CLSI 2011)(21). DDST
was done to determine synergy between a disc
of amoxicillin/ clavulanic acid (20 μg/10 μg)
and 30 mg disc of each 3rd generation
INTRODUCTION
Extended spectrum β- lactamases (ESBls)
are group of enzymes that mediate resistance
and inactivation to extended spectrum
penicillins
and
the
third
generation
cephalosporins and monobactams(1). They are
highly susceptible in vitro to inhibition by βlactamases such as clavulanic acid but are not
active against cephamycins or carbapenems(2,3).
These enzymes are generally plasmid mediated
and detected most commonly in Klebsiellae
pneumonia, E.coli and other members of the
family Enterobacteriaceae(4,5). Although many
types had been observed (SHV, TEM, CTX-M,
PER, OXA, ampC …..); SHV (sulphydryl
variable) type are the most common found in
the
genus
Klebsiellae
pneumonia(2,6-8).
Ventilator-associated pneumonia (VAP) is one
of the most common healthcare-associated
infections and the most lethal, resulting in up to
36,000 deaths per year in the United States (9).
VAP prolongs ventilator days and length of stay
(LOS) in both the ICU and in the hospital itself.
In addition, VAP is the leading cause of death
among hospital-acquired infections, exceeding
the rate of death as the result of central line
infections, severe sepsis and respiratory tract
infections in non-intubated patients(10). VAP
caused by ESBLs producing Klebsiellae
pneumonia is associated with prolongation of
mechanical ventilator days, ICU & hospital stay
and it can lead to treatment failure and increases
in costs(5,10). The incidence of Klebsiellae
pneumonia causing VAP has been increased in
the hospital settings(11-15). Rapid detection and
identification of ESBLs appear essential in
studying the epidemiology of antimicrobial
resistant bacteria(8,16). This study aimed to study
the role of SHV genes as they are one of the
most common and important genes encode these
isolates and prevalence of ESBLs producing
Klebsiellae pneumonia causing VAP in ICU
patients with antimicrobial susceptibility
pattern.
PATIENTS & METHODS
Our study had been performed in Buraidah
Central Hospital (BCH) and King Fahd
Specialized Hospital (KFSH) in the period
between June 2011 and July 2012. All patients
who developed clinical signs of VAP were
included according to CDC criteria(11, 17). Under
aseptic technique, endotracheal tube aspirate
samples were collected for quantitative cultures.
114
cephalosporins
(3GC)
(as
ceftazidime,
cefotaxime and cefopodoxime) antibiotics
(Oxoid, Basingstoke, Hamphshire, England).
Muller Hinton agar plates were prepared and
inoculated with a suspension made from an
overnight blood agar culture of the test strain as
recommended for a standard disc diffusion
susceptibility test with standardized inoculum
(0.5 McFarland tube) to form a pure culture.
Disc (30 μg) of each 3GC antibiotics was placed
on the agar at a distance of 15mm centre to
centre from amoxicillin/ clavulanic acid disc
(AMC) and the plates were incubated for 24
hours at 37°C. ESBL production was interpreted
if the inhibition zone around the test antibiotic
disc increased towards the amoxicillin/
clavulanic acid disc (An increase in zone
diameter of ≥5mm in the presence of clavulanic
acid indicated the existence of ESBL in the test
organism). This increase occurred because the
β-lactamases produced by the isolates were
inactivated by clavulanic acid) according to
(CLSI 2011)(20,21,22).
2-The Confirmatory MIC Test:
It is a ≥ 3 twofold dilution decrease in
MICs of suspected organisms to ceftazidime or
cefotaxime in the presence of a fixed
concentration of clavulanic acid versus its MIC
when tested alone in the used Gram negative
combo 42 panel according to the manufacture
instructions of automated MicroScan walkaway
96 SI (Siemens Healthcare diagnostics Ltd, CA,
USA). Antimicrobial susceptibility pattern to
different chemotherapeutic agents was done to
each isolate by the used automated mentioned
Combo panels based on determination of
minimal inhibitory concentration method
according to Clinical and Laboratory Standards
Institute guideline instructions (CLSI, 2011) (21).
Detection of SHV Genes by PCR Method:
DNA Preparation:
Bacterial DNA was extracted using
commercially available kits (spin column
technology) (E.Z.N.A. bacterial DNA kit,
OMEGA Biotech HiBind). Bacterial cells were
grown to log-phase and harvested. The bacterial
cell wall was removed by lysozyme digestion,
followed by proteinase K digestion. Following
lysis, binding conditions were adjusted and the
sample was applied to a HiBind® DNA spincolumn. Then two rapid wash steps were done
to remove trace salts and protein contaminants
and finally DNA was eluted in 300µl low ionic
strength buffer TE-buffer (10mmTris, 1mm
EDTA pH 8.0)(8). Purified DNA was used later
for PCR amplification. The used primers for
detection of SHV genes were SHVF
5´TCAGCGAAAAACACCTTG 3´and SHVR
Vol. 22, No. 2
primer 5´ TCCCGCAGATAAATCACC 3´
(Sigma, USA) that yield 471bp band after
amplification.
PCR Amplification of SHV Assay:
It was performed using Taq PCR master
mix (Boehringer, Mannheim, Germany) using
the previous mentioned primers. Amplification
was performed according to the manufacture´s
instructions (Amersham Biosciences). Two µl
of sense 5´primer and 2 µl of antisense
3´primer, 3 µl of template DNA and sterile
distilled water to a total volume of 25 µl were
added to each tube containing a PCR bead.
Total reaction volume of 100 µl containing
1xPCR buffer (10mMTris, 50 mM Kcl), 2mM
MgCl2, 0.2mM (each) dNTPs (dATP, dGTP,
dCTP & dTTP), stabilizer and 1.0 U of Taq
polymerase The contents of the tubes were
mixed by gentle vortexing then rapid brief
centrifugation was made to collect the contents
at the bottom of the tube. DNA Thermal Cycler
(Perkin- Elmer, Cetus, USA) was programmed
to perform 30 cycles consisting of: an initial
denaturation step at 94°C for 2 min followed by
30 cycles of denaturation at 95°C for 30
seconds, annealing at 58°C for 60 sec then an
extension step at 72°C for 60 sec and final
extension step at 72°C for 5 minutes after the
last cycle. The amplified DNA was kept at 4 °C
until detection. The amplified products were
analyzed (twenty five µl) on 1.5% agarose gel
(NuSieve GTG agarose; FMC products,
Rockland, Maine) in 1xTAE buffer pH 8.0
(0.04MTris-acetate,
0.001M
EDT
A).
Electrophoresis was done at 90 voltages for 45
minutes using ethidium bromide stain (1mg/L)
and visualized under UV transilluminator
(Fisher, USA). Twenty five µl of amplified
DNA were loaded each with 10µl of gel loading
buffer and loaded in the submerged gel using a
disposable sterile micropipette tips. The DNA
molecular weight marker ladder (5 µl mixed
with 10 µl of gel loading buffer), (100 base pair
sigma 100-1000bp ,USA) was run in parallel for
detection of SHV genes band at 471 bp
according to Lal et al.,2007 (23) and El-Bialy &
abu-zeid 2009(24). Negative control sample
include all previous mixture without extracted
DNA but replaced with sterile deionized water
then was also run for accuracy. Positive control
isolate was used from quality control strain
ESBLs producer ATCC® 700603 (Becton
Dickinson BD, France, S.A.S). The DNA was
extracted with the same procedure mentioned
for accuracy.
Statistical Analysis:
VAP rate was calculated as the number of
cases of VAP divided by the number of
115
ventilator days per 1000 ventilator days. Data
were analysed in tables using percentage. Mean,
median and range for data were calculated.
Comparison of data associated with the
presence of ESBLs production was done by
SPSS statistic program Version 16.0 (25).
Vol. 22, No. 2
from 12 out of 18 ESBLs producing isolates
(66.7%). Non ESBLs producer strains of
K.pneumoniae by phenotypic method were
negative for the presence of SHV genes by
PCR. The overall data revealed that 12 out of 18
positive ESBLs isolates (66.7%) contained SHV
genes. Antimicrobial resistance of ESBLs
producing
K.pneumoniae
to
different
chemotherapeutic agents using MIC method
(Table 2) were shown according to CLSI
(2011)(21). All the strains were sensitive to
tigecycline (TGC) and colistin (CL) (100%). All
the strains were resistant to ampicillin (AMP),
cefazolin (CF), cefuroxime (CXM), cefotaxime
(CTX), ceftazidime (CAZ), cefepime (CPM)
and piperacillin (PRL) (100%); meanwhile these
were sensitive to cefoxitin (FOX) except two
isolates were resistant (11.1%). Resistance of
ESBLs isolates to amikacin (AK), gentamicin
(GM), tobramycin (TOB), ciprofloxacin (CIP),
levofloxacin (LEV), moxifloxacin (MOX),
tetracycline
(TE)
and
trimethoprimsulfamethoxazole( SXT) were 33.3%, 38%,
33.3%, 22%, 22%, 16.7%, 55.6% and 83.3%
respectively. Resistance to amoxicillin/ K.
clavulanate (AMC) and piperacillin/tazobactam
(TZP) was 55.6% for each (10/18). The least
resistance was to imipenem (IMI), meropenem
(MEM) and ertapenem (ETP), was 11.1% (2 out
of 18) as carbapenem group.
RESULTS
Twenty one isolates of Klebsiellae
pneumonia were isolated from both hospitals
during one year study causing VAP in ICU.
Eighteen strains out of 21 (85.7%), were ESBLs
producer by confirmatory MIC method using
combo 42 panel in automated MicroScan
machine. Three isolates were non ESBLs
producer by both DDST and confirmatory MIC
method. The rate of VAP was 5.6 cases/ 1000
ventilator days during this period. ESBLs
producing Klebsiellae pneumonia represented
26.5% (18/68) from both hospitals causing VAP
in ICU as shown in (Table 1). Phenotypic
synergy (DDST) test failed to diagnose one
isolate only as it gave false negative result as
compared to the confirmatory automated MIC
Combo 42 Gram negative panel. The mean of
length of stay in ICU of both hospitals was 9.7
(mean±SD = 9.7±7.0) (Table 1). Ventilator
days ranged from 2-35 days with mean of 8.4
days (Table 1). Using PCR method it detected
SHV genes at 471 bp as shown in (Figure 1)
Table (1): Epidemiological Data and Patient Characteristics.
Patient Characteristics
Age in years [Mean (Range)]
Sex (Male/ Female) (No & %)
APACHI II on admission [median (range)]
LOS in ICU in days (Length of Stay) Mean ± SD
Ventilator days (Mean)(range)
Total VAP Rate/ 1000 Vent Days
ESBLs K.pneumoniae +ve (No & %) causing VAP
Non-ESBLs producer (No & %) causing VAP
SHV positive cases by PCR from positive ESBLs
Total No of Ventilator days (Both hospitals) =
No. of patients with VAP=68
57.2 (45-100)
42/ 26 (61.7%/ 38.3%)
24 (2-45)
9.7 ±7.0
8.4 days (2-35)
5.6
18/68 (26.5%)
3/68 (4.4%)
12/18 (66.7%)
12142 days
Table (2): Antimicrobial Resistance of ESBLs producing K.pneumoniae (18) to different
chemotherapeutic agents using MIC Method.
Antibiotics
Resistant%
Antibiotics
Resistant%
Antibiotics
Resistant%
Ak
33.3
AMC
55.6
AMP
100
CF
100
CPM
100
CTX
100
CAZ
100
FOX*
11.1
CXM
100
CIP
22
CL**
0
ETP*
11.1
GM
38
IMI*
11.1
LEV
22
MEM*
11.1
MOX
16.7
TZP
55.6
PRL
100
TE
55.6
TGC**
0
TOB
33.3
SXT
83.3
----------** means that no resistant antimicrobial agent.
* means the least resistant antimicrobial agent.
116
Vol. 22, No. 2
Figure 1: Agarose Gel Electrophoresis of Amplified Product of SHV Genes In K.pneumonia.
Lane 1: contain positive control band of 417 bp isolated from K.pneumonia ATCC700603. Lanes 2, 3, 7,
8, 9, 10, 11, 12, 13, 14 & 15: Positive bands (471bp) of ESBLs +ve for SHV gene from. K.pneumonia
isolates. Lane 4: Negative control without DNA. Lanes 5, 6: Negative isolates for SHV genes. Lane L:
The molecular size standard of a 100-bp DNA ladder.
confirmatory automated MIC method. This
result may be due to inability of the phenotypic
DDST method to detect all ESBLs due to agar
diffusion problem or presence of high level of
other enzymes like AmpC enzymes that are
inhibitor resistant β-lactamase thus clavulanate
may act as an inducer of high level AmpC
production and increase the resistance of the
isolate to other drugs producing a false negative
result for ESBLs production. This necessitates
the need for further confirmatory molecular
method for diagnosis. This isolate did not
harbour SHV genes by the used PCR method.
In this study isolation rate of ESBLs
producing K. pneumonia causing VAP was
26.5% and this was near to the results of
previous studies(2,13,20,24). All three nonproducers of ESBLs by both DDST and MIC
were negative for SHV genes by PCR (100%
agreement), meanwhile 12 out of 18 positive
producers of ESBLs (66.7%) were positive for
the presence of SHV genes. These results clarify
the important role of these genes for production
of ESBLs and thus increase the percentage of
antimicrobial resistance and were near similar to
some studies (23, 24, 29), who detected these genes
in 66.7% of different origins isolates of
nosocomial K.pneumonia in Suez Canal
University hospital. El-Bialy and Zeid (24) found
that all 15 nosocomially isolated ESBLs K.
pneumonia from neonatal ICU carried SHV-2
gene and 40 % had SHV-1 gene using
restriction fragment length polymorphism
DISCUSSION
The rate of VAP was 5.6 cases/ 1000
ventilator days during this period and this
represented a major concern of device
associated infection in critical area and were
consistent with other studies(9,26). Eighteen
strains out of 21 (85.7%) were ESBLs producer
by confirmatory phenotypic (DDST and MIC
method) (using combo 42 panel). Our results
were consistent with many studies(12,13,27-29),
worldwide with rates exceeding 50% in some
countries and clarified the importance and
concern of ESBLs in hospital associated
infection. Our results were in agreement with
previous studies(9,30,31), that found that ESBLs
causing VAP and other hospital associated
infections cause a major threat to life, difficult,
expensive to treat and can delay discharge of
patients prolongation of ventilator days and
length of stay (LOS) in both the ICU and the
hospital itself. VAP is the leading cause of death
among hospital-acquired infections, exceeding
the rate of death as the result of central line
infections, severe sepsis and respiratory tract
infections in non-intubated patients. So, we
need more infection control measures for
prevention to decrease hospital morbidity and
mortality rate. ESBLs represented 20.9%
(18/68) from both hospitals causing VAP in
ICU. Phenotypic double disk synergy (DDST)
test failed to diagnose one isolate only as it gave
false negative result as compared to the
117
(RFLP). So, we are in agreement with many
studies(5,8,24,29,32,33), that necessitate correct
identification of the genes involved in ESBLmediated resistance for surveillance and
epidemiological studies of their transmission in
hospitals. Also, methods like PCR need to be
used for the characterization and differentiation
of β-lactamase producing isolates at molecular
level.
Our results were consistent with similar
studies from other parts of the world which
showed that SHV-5 gene was common in
K.pneumonia isolates (6, 23) and similar to Chia
et al.,(2005)(8). According to Jemima and
Verghese (2008)(33), ESBL-producing isolates
coded for SHV have been reported from various
countries in Europe, such as Austria, France,
Italy and Greece as well as in the United States
and Australia(5). Among the SHV type of ßlactamases, SHV-5 was found to be responsible
for outbreaks of nosocomial infections in
several countries(34). The negative ESBLs
isolates for SHV genes could be due to presence
of other genes encode for production of ESBLs
such as TEM, CTX-M, PER, GES, OXA,
AmpC….) or due to other mechanisms of
resistance as efflux process.
Antimicrobial susceptibility pattern to
positive ESBLs using automated MIC through
MicroScan machine revealed the marked
resistance to penicillin group, cephalosporines
(100%),
followed
by
trimethoprim
sulfamethoxazole, tetracycline, gentamicin,
amikacin,
tobramycin,
ciprofloxacin,
levofloxacin and moxifloxacin in 83.3%, 55.6%,
38%, 33.3%, 33.3%, 22%, 22% and 16.7%
respectively. Resistance to amoxicillin/ K.
clavulanate and piperacillin / tazobactam was
55.6% for each (10/18). The least resistance was
to imipenem, meropenem and ertapenem was
11.1% for each one (2 out of 18); it could be
due to production of metallo β lactamases
(MBL), that are usually the cause of resistance
to this group (carbapenems) and could be
transferred usually from pseudomonas spp to
enterobacteriacae. These results are consistent
with many previous studies that showed that
high and multiple antimicrobial resistances are
present
with
these
enzymes
production(4,12,13,15,27,28,35). So, we should be
careful about ESBLs in VAP especially
critically ill patients and study more genes by
more molecular techniques. Optimum choice of
chemotherapeutic agents by clinicians is very
important. Our results are consistent with
previous studies(36,37) that concluded that
Carbapenems are the drug of choice for
treatment of ESBLs but after lab diagnosis and
Vol. 22, No. 2
antibiotic susceptibility testing and we should
restrict use of tigecycline and colistin for serious
infections with ESBLs to avoid emergence of
resistant strains.
We can conclude that the wise use of
antimicrobial agents is recommended especially
third generation cephalosporines inside ICU as
critically ill patients are more susceptible to
infection by these types of isolates. Also, more
concern about infection control measures to
prevent spread of such organisms must be done.
Further molecular studies like multiplex PCR,
genetic sequencing, restriction fragment length
polymorphism (RFLP), isoelectric focusing are
needed to study other genes that mediate
production of these enzymes.
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‫‪Vol. 22, No. 2‬‬
‫دور ﺟﻴﻨﺎت )اس‪ .‬اﺗﺶ‪ .‬ﭬﻰ‪ (.‬ﻣﻦ ﻣﻴﻜﺮوب اﻟﻜﻠﻴﺒﺴﻴﻼ اﻟﺮﺋﻮﻳﺔ اﻟﻤﻜﺘﺴﺒﺔ ﻣﻦ ﻋﺪوى اﻟﻤﺴﺘﺸﻔﻴﺎت‬
‫واﻟﻤﻔﺮزة ﻻﻧﺰﻳﻢ اﻟﺒﻴﺘﺎﻟﻜﺘﺎﻣﻴﺰ اﻟﻮاﺳﻊ اﻟﻤﺪى اﻟﻤﻌﺰوﻟﺔ ﻣﻦ اﻻﻟﺘﻬﺎب اﻟﺮﺋﻮى اﻟﻤﺮﺗﺒﻂ ﺑﺠﻬﺎز اﻟﺘﻬﻮﻳﺔ‬
‫اﻻﻟﻴﺔ ب ﻳﻦ ﻣﺮﺿﻰ اﻟﻌﻨﺎﻳﺔ اﻟﻤﺮآﺰة ﺑﺎﺳﺘﺨﺪام ﺗﻔﺎﻋﻞ ﺳﻠﺴﻠﺔ اﻟﺒﻠﻤﺮة اﻟﻤﺘﻌﺪدة‬
‫ﻋﺒﻴﺮ ﻋﺰت اﻟﺴﻴﺪ )‪ , (٢ ,١‬ﺳﺎﻣﻴﺔ رﺟﺐ اﻟﻌﺰب‬
‫)‪(٤,٣‬‬
‫ﻗﺴﻢ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻰ واﻟﻤﻨﺎﻋﺔ ‪ -‬آﻠﻴﺔ اﻟﻄﺐ ‪ -‬ﺟﺎﻣﻌﺔ ﻗﻨﺎة اﻟﺴﻮﻳﺲ اﺳﺘﺸﺎرى ﻣﻴﻜﺮﺑﻴﻮﻟﻮﺟﻰ – رﺋﻴﺲ ﻣﻜﺎﻓﺤﺔ اﻟﻌﺪوى‬
‫ﺑﺮﻳﺪة اﻟﻤﺮآﺰى اﻟﺴﻌﻮدﻳﺔ )‪ ، (٢،١‬ﻗﺴﻢ اﻟﺘﺨﺪﻳﺮ واﻟﻌﻨﺎﻳﺔ اﻟﻤﺮآﺰة ‪ -‬آﻠﻴﺔ اﻟﻄﺐ ‪ -‬ﺟﺎﻣﻌﺔ اﻻزهﺮ ﺑﻨﺎت اﺳﺘﺸﺎرى ﺗﺨﺪﻳﺮ‬
‫)‪()٤,٣‬‬
‫و ﻋﻨﺎﻳﺔ ﻣﺮآﺰة‪ -‬اﻟﻤﻠﻚ ﻓﻬﺪ اﻟﺘﺨﺼﺼﻰ اﻟﺴﻌﻮدﻳﺔ‬
‫اﻟﺨﻠﻔﻴﺔ‪ :‬ﻳﻌﺘﺒﺮ ﺟﻨﺲ اﻟﻜﻠﻴﺒ ﺴﻴﻼ اﻟﺮﺋﻮﻳ ﺔ ﻣ ﻦ اﻟﻤﻴﻜﺮوﺑ ﺎت اﻟﻬﺎﻣ ﺔ اﻟﺘ ﻰ ﺗ ﺴﺒﺐ ﻋ ﺪوى اﻻﻟﺘﻬ ﺎب اﻟﺮﺋ ﻮى اﻟﻤ ﺮﺗﺒﻂ ﺑﺠﻬ ﺎز اﻟﺘﻬﻮﻳ ﺔ‬
‫اﻻﻟﻴﺔ‪ .‬ﺗﻌﺪ ﻣﺠﻤﻮﻋﺔ اﻟﺒﻴﺘﺎ ﻟﻜﺘﺎم ﻣﻦ اآﺜﺮ اﻟﻤﺠﻤﻮﻋﺎت اﺳﺘﺨﺪاﻣﺎ ﻣﻦ ا ﻟﻤﻀﺎدات اﻟﺤﻴﻮﻳ ﺔ ‪ .‬ﺗﻌ ﺪ اﻧﺰﻳﻤ ﺎت اﻟﺒﻴﺘ ﺎ ﻟﻜﺘﻤﻴ ﺰ اﻟﻮاﺳ ﻌﺔ‬
‫اﻟﻤﺪى ﻣﻦ ااﻻﻧﺰﻳﻤﺎت اﻟﻬﺎﻣﺔ ﻟﻠﻤﻘﺎوﻣﺔ ﻟﻤﺠﻤﻮﻋﺎت اﻟﺒﻨﺴﻠﻴﻦ و اﻟﺠﻴﻞ اﻟﺜﺎﻟﺚ ﻣﻦ اﻟﻜﻔﺎﻟﻮﺳﺒﻮرﻳﻦ واﻻزﺗﺮﻳﻮﻧﺎم‪.‬ﻳﻠﻌﺐ هﺬا اﻟﻤﻴﻜﺮوب‬
‫دورا آﺒﻴﺮا ﻓﻰ اﻟﻌﺪوى اﻟﻤﻜﺘﺴﺒﺔ ﻣﻦ اﻟﻤﺴﺘﺸﻔﻴﺎت ﺧﺎﺻﺔ اﻟﻌﻨﺎﻳﺔ اﻟﻤﺮآﺰة‪ .‬وﺗﻌﺘﺒﺮ ﻣﺼﺪرا هﺎم ﻟﻠﻮﻓﻴ ﺎت واﻟﻤﺮاﺿ ﺔ داﺧ ﻞ اﻟﻌﻨﺎﻳ ﺔ‬
‫اﻟﻤﺮآﺰة ﺣﻴﺚ اﻧﻬﺎ اﻧﺰﻳﻤ ﺎت ﻣﺘﻌ ﺪدة وﺗﺤ ﺪث ﺑﻬ ﺎ ﻃﻔ ﺮات ﺑﺎﺳ ﺘﻤﺮار ﻧﻈ ﺮا ﻟﻠ ﻀﻐﻂ اﻟ ﺸﺪﻳﺪ ﻣ ﻦ اﻻﺳ ﺘﺨﺪام اﻟﻌ ﺸﻮاﺋﻰ ﻟﻠﻤ ﻀﺎدات‬
‫اﻟﺤﻴﻮﻳﺔ‪ ٠‬هﺪﻓﺖ هﺬﻩ اﻟﺪراﺳﺔ اﻟﻰ دراﺳﺔ ﻧﺴﺒﺔ هﺬا اﻟﻤﻴﻜﺮوب اﻟﻤﻨﺘﺞ ﻟﻬﺬﻩ اﻻ ﻧﺰﻳﻤﺎت ﻟﻤﺮﺿﻰ اﻻﻟﺘﻬﺎب اﻟﺮﺋ ﻮي اﻟﻤ ﺮﺗﺒﻂ ﺑﺠﻬ ﺎز‬
‫اﻟﺘﻬﻮﻳ ﺔ و دور ﺟﻴﻨ ﺎت )اس‪ .‬اﺗ ﺶ‪ .‬ﭬ ﻰ‪ (.‬آﻨ ﻮع ﻣ ﻦ اﻟﺠﻴﻨ ﺎت اﻟﻬﺎﻣ ﺔ ﻻﻧﺘ ﺎج ه ﺬﻩ اﻻﻧﺰﻳﻤ ﺎت وه ﻮ اﻟ ﺴﻠﻔﻬﻴﺪرﻳﻞ اﻟﻤﺘﻐﻴ ﺮ وذﻟ ﻚ‬
‫ﻟﻠﻤﺴﺎهﻤﺔ ﻓﻰ اﻟﺤﺪ ﻣﻦ اﻧﺘﺸﺎر هﺬﻩ اﻟﺴﻼﻻت داﺧﻞ اﻟﻌﻨﺎﻳﺔ اﻟﻤﺮآﺰة‪.‬‬
‫اﻟﻄﺮق‪ :‬ﺗﻢ ﺗﺠﻤﻴ ﻊ ﻋﻴﻨ ﺎت اﻓ ﺮازات اﻣﺒﻮﺑ ﺔ اﻟﻘ ﺼﺒﺔ اﻟﻬﻮاﺋﻴ ﺔ ﻣ ﻦ اﻟﻤﺮﺿ ﻰ اﻟﻤﺸﺨ ﺼﻴﻦ اآﻠﻴﻨﻴﻜﻴ ﺎ ﺑﺎﻻﻟﺘﻬ ﺎب اﻟﺮﺋ ﻮى اﻟﻤ ﺼﺎﺣﺐ‬
‫ﻟﺠﻬ ﺎز اﻟﺘﻬﻮﻳ ﺔ اﻻﻟﻴ ﺔ ﻃﺒﻘ ﺎ ﻟﻤﻌ ﺎﻳﻴﺮ ﻣﺮآ ﺰ اﻟ ﺘﺤﻜﻢ ﻓ ﻰ اﻻﻣ ﺮاض‪ .‬ﺗ ﻢ ﺗ ﺸﺨﻴﺺ ﻣﻴﻜ ﺮوب اﻟﻜﻠﻴﺒ ﺴﻴﻼ اﻟﺮﺋﻮﻳ ﺔ ﺑﺎﺳ ﺘﺨﺪام اﻟﻄ ﺮق‬
‫اﻟﺘﻘﻠﻴﺪﻳﺔ وﻣﺎآﻴﻨﺔ اﻟﻤﻴﻜﺮوﺳﻜﺎن اﻻوﺗﻮﻣﺎﺗﻴﻜﻴﺔ ﻣ ﻊ اﺧﺘﺒ ﺎر اﻟﺤ ﺴﺎﺳﻴﺔ ﻟﻠﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﺔ اﻟﻤﺨﺘﻠﻔ ﺔ‪ .‬ﺗ ﻢ ﺗ ﺸﺨﻴﺺ اﻟﺒﻜﺘﺮﻳ ﺎ اﻟﻤﻨﺘﺠ ﺔ‬
‫ﻻﻧﺰﻳﻤ ﺎت اﻟﺒﻴﺘ ﺎﻟﻜﺘﻤﻴﺰ اﻟﻮاﺳ ﻌﺔ اﻟﻤ ﺪى ﺑﺎﺳ ﺘﺨﺪام ﻃﺮﻳﻘ ﺔ اﻻﺧﺘﺒ ﺎر اﻟﻤ ﺰدوج ﻟﻼﻧﺘ ﺸﺎر ﻓ ﻰ اﻻﺟ ﺎر وﺗ ﻢ ﺗﺄآﻴ ﺪهﺎ ﺑﺎﺳ ﺘﺨﺪام ﻃﺮﻳﻘ ﺔ‬
‫اﻟﺘﺮآﻴﺰ اﻻﻗﻞ ﻟﺘﺜﺒﻴﻂ ﻧﻤﻮ اﻟﺒﻜﺘﺮﻳﺎ‪ .‬ﺗ ﻢ ﺗ ﺸﺨﻴﺺ ﺟﻴﻨ ﺎت )اس‪ .‬اﺗ ﺶ‪ .‬ﭬ ﻰ‪ (.‬اﻟﺨﺎﺻ ﺔ ب اﻟﺒﻜﺘﺮﻳ ﺎ اﻟﻤﻨﺘﺠ ﺔ ﻻﻧﺰﻳﻤ ﺎت اﻟﺒﻴﺘ ﺎﻟﻜﺘﻤﻴﺰ‬
‫اﻟﻮاﺳﻌﺔ اﻟﻤﺪى ﺑﺎﺳﺘﺨﺪام ﺗﻔﺎﻋﻞ ﺳﻠﺴﻠﺔ اﻟﺒﻠﻤﺮة اﻟﻤﺘﻌﺪدة‪.‬‬
‫اﻟﻨﺘﺎﺋﺞ‪ :‬ﺗﻢ ﻋﺰل ‪ ٢١‬ﺳ ﻼﻟﺔ ﻣ ﻦ اﻟﻤﻴﻜ ﺮب ﻣ ﻨﻬﻢ ‪ ١٨‬ﺳ ﻼﻟﺔ )‪ (%٨٥,٧‬ﻣﻨﺘﺠ ﺔ ﻻﻧﺰﻳﻤ ﺎت اﻟﺒﻴﺘ ﺎﻻآﺘﻤﻴﺰ واﺳ ﻌﺔ اﻟﻤ ﺪى ﺑﺎﻟﻄﺮﻳﻘ ﺔ‬
‫اﻟﺘﺎآﻴﺪﻳﺔ ﻟﺠﻬﺎز اﻟﻤﻴﻜﺮوﺳﻜﺎن وﻗ ﺪ ﻓ ﺸﻠﺖ ﻃﺮﻳﻘ ﺔ اﻻﺧﺘﺒ ﺎر اﻟﻤ ﺰدوج ﺑﺎﻻﻧﺘ ﺸﺎر ﻓ ﻰ اﻻﺟ ﺎر ﻓ ﻰ ﺗ ﺸﺨﻴﺺ ﺳ ﻼﻟﺔ واﺣ ﺪة ﻓﻘ ﻂ ﻣ ﻦ‬
‫اﻟﺴﻼﻻت اﻟﺘﻰ ﺗﻢ ﺗﺎآﻴﺪهﺎ ﺣﻴﺚ اﻋﻄﺖ ﻧﺘﻴﺠﺔ ﺳﻠﺒﻴﺔ زاﺋﻔﺔ ‪ .‬آﺎن ﻣﻌ ﺪل اﻟﻼﺻ ﺎﺑﺔ ﺑﺎﻻﻟﺘﻬ ﺎب اﻟﺮﺋ ﻮى ‪٥,٦‬ﺣﺎﻟ ﺔ ﻟﻜ ﻞ اﻟ ﻒ ﻳ ﻮم ﻣ ﻦ‬
‫اﻳﺎم اﻟﺒﻘﺎء ﻋﻠﻰ ﺟﻬﺎز اﻟﺘﻬﻮﻳﺔ اﻟﺮﺋﻮﻳﺔ اﻟﺼﻨﺎﻋﻰ ﺧﻼل ﻋ ﺎم واﺣ ﺪ‪ ٠‬وآ ﺎن ﻣﺘﻮﺳ ﻂ ﺑﻘ ﺎء اﻟﻤﺮﺿ ﻰ ﻋﻠ ﻰ ﺟﻬ ﺎز اﻟﺘ ﻨﻔﺲ اﻟ ﺼﻨﺎﻋﻰ‬
‫‪ ٨,٤‬ﻳﻮم‪ ٠‬ﺗﻢ ﻋﺰل ‪ ٢١‬ﺳﻼﻟﺔ ﻣﻦ اﻟﻜﻠﻴﺒﺴﻴﻼ اﻟﺮﺋﻮﻳﺔ ﺧﻼل هﺬا اﻟﻌﺎم ﻣ ﻦ ﻣﺴﺘ ﺸﻔﻰ ﺑﺮﻳ ﺪة اﻟﻤﺮآ ﺰى واﻟﻤﻠ ﻚ ﻓﻬ ﺪ اﻟﺘﺨﺼ ﺼﻰ و‬
‫ﻣﺜﻠﺖ اﻟﺴﻼﻻت اﻟﻤﻨﺘﺠﺔ ﻻﻧﺰﻳﻤﺎت اﻟﺒﻴﺘﺎﻻآﺘﻤﻴﺰ اﻟﻮاﺳﻌﺔ اﻟﻤﺪى ﻧﺴﺒﺔ ‪ ( ٢١/ ١٨) %٨٥,٧‬ﻣﻦ ﻣﺠﻤﻮع اﻟﺴﻼﻻت اﻟﻤﻌﺰوﻟﺔ ﻣ ﻦ‬
‫اﻟﺤﺎﻻت اﻟﻤﻨﻮﻣﺔ واﻟﻤﺼﺎﺑﺔ ﺑﻌﺪوى اﻻﻟﺘﻬﺎب اﻟﺮﺋﻮى اﻟﻤﺮﺗﺒﻂ ﺑﺠﻬﺎز اﻟﺘﻨﻔﺲ اﻟﺼﻨﺎﻋﻰ ﻟﻜﻼ اﻟﻤﺴﺘﺸﻔﺘﻴﻦ ﺑﺎﻟﻌﻨﺎﻳﺔ اﻟﻤﺮآﺰة‪ ٠‬وآﺎن‬
‫اﺟﻤﺎﻟﻰ ﻧﺴﺒﺔ اﻟﺴﻼﻻت اﻟﻤﻨﺘﺠﺔ ﻻﻧﺰﻳﻤﺎت اﻟﺒﻴﺘﺎﻻآﺘﻤﻴﺰ اﻟﻮاﺳ ﻌﺔ اﻟﻤ ﺪى ‪ %٢٦,٥‬ﻣ ﻦ ﻣﺠﻤ ﻮع ﺣ ﺎﻻت ﻋ ﺪوى اﻻﻟﺘﻬ ﺎب اﻟﺮﺋ ﻮى‬
‫اﻟﻤﺼﺎﺣﺐ ﻟﺠﻬ ﺎز اﻟﺘﻬﻮﻳ ﺔ اﻟ ﺼﻨﺎﻋﻰ) ‪ ٠(٦٨/١٨‬و ﺑﺎﺳ ﺘﺨﺪام ﻃﺮﻳﻘ ﺔ ﺗﻔﺎﻋ ﻞ ﺳﻠ ﺴﻠﺔ اﻟﺒﻠﻤ ﺮة اﻟﻤﺘﻌ ﺪدة ﻟﻠﻜ ﺸﻒ ﻋ ﻦ ﺟﻴﻨ ﺎت )اس‬
‫‪.‬أﺗﺶ‪.‬ﻓﻰ( اﻟﻤﻨﺘﺠﺔ ﻻﻧﺰﻳﻢ اﻟﺒﻴﺘﺎﻻآﺘﺎﻣﻴﺰ اﻟﻮاﺳﻌﺔ اﻟﻤﺪى وﺟﺪ ان ‪ ١٢‬ﺳﻼﻟﺔ ﻣﻨﻬﻢ اﻳﺠﺎﺑﻴ ﺔ ﻟﻬ ﺬا اﻟﻨ ﻮع ﺑﻨ ﺴﺒﺔ ‪ %٦٦,٧‬ﻣﻤﺎﻳﻮﺿ ﺢ‬
‫ﻣﺪى اهﻤﻴﺘﻪ و ارﺗﻔﺎع ﻧﺴﺒﺘﻪ ﻓﻰ هﺬﻩ اﻟ ﺴﻼﻻت‪ ٠‬وآﺎﻧ ﺖ ﺟﻤﻴ ﻊ اﻟ ﺴﻼﻻت اﻟﻤﻨﺘﺠ ﺔ ﻟﻬ ﺬﻩ اﻟﻼﻧﺰﻳﻤ ﺎت ﻣﻘﺎوﻣ ﺔ ﻟﻠﺠﻴ ﻞ اﻟﺜﺎﻟ ﺚ‪,‬اﻻول‬
‫واﻟﺜﺎﻧﻰ واﻟﺮاﺑﻊ ﻣﻦ اﻟﻜﻴﻔﺎﻟﻮﺳﺒﻮرﻳﻦ )‪ (١٠٠ %‬وآﺬﻟﻚ ﻟﻼﻣﺒﺴﻴﻠﻠﻴﻦ‪ ,‬اﻟﺒﻴﺒﺮاﺳﻴﻠﻴﻦ‪ (١٠٠ %) ,‬ﺑﻴﻨﻤﺎآﺎﻧ ﺖ ﺣ ﺴﺎﺳﺔ ﻟﻠﺴﻴﻔﻮآ ﺴﻴﺘﻴﻦ‬
‫ﻓﻴﻤﺎ ﻋﺪا ﺳﻼﻟﺘﻴﻦ ﻓﻘﻂ آﺎﻧﺖ ﻣﻘﺎوﻣﺔ ﻟﻪ ‪ %٠١١,١‬وآﺎﻧﺖ ﺟﻤﻴﻊ اﻟﺴﻼﻻت ﺣﺴﺎﺳﺔ ﻟﻠﺘﻴﺠﻴﺴﻴﻜﻠﻴﻦ واﻟﻜﻮﻟﻴ ﺴﺘﻴﻦ )‪ (١٠٠ %‬ﺑﻴﻨﻤ ﺎ‬
‫آﺎﻧ ﺖ ﻣﻘﺎوﻣﺘﻬ ﺎ ﻟﻼﻣﻴﻜﺎﺳ ﻴﻦ ‪,‬اﻟﺠﻴﻨﺘﺎﻣﻴ ﺴﻴﻦ ‪,‬اﻟﺘﻮﺑﺮاﻣﻴ ﺴﻴﻦ ‪,‬اﻟﺴﺒﺮوﻓﻠﻮآ ﺴﺎﺳﻴﻦ ‪,‬اﻟﻠﻴﻔﻮﻓﻠﻮآ ﺴﺎﺳﻴﻦ‪ ،‬اﻟﻤﻮآﺴﻴﻔﻠﻮآ ﺴﺎﺳﻴﻦ‪،‬‬
‫اﻟﺘﺘﺮاﺳ ﻴﻜﻠﻴﻦ و اﻟﺘ ﺮاي ﻣﻴﻴﺜ ﻮﺑﺮﻳﻢ ﺳﻠﻔﺎﻣﻴﺴﻮآ ﺴﺎزول ه ﻰ‪% ٨٣,٣ , % ١٦,٧ , %٢٢ ,%٢٢,% ٣٣,٣ ,%٣٨ ,%٣٣,٣‬‬
‫ﻋﻠ ﻰ اﻟﺘ ﻮاﻟﻰ ‪ ٠‬وﻟﻘ ﺪ آﺎﻧ ﺖ اﻗ ﻞ ﻧ ﺴﺒﺔ ﻟﻠﻤﻘﺎوﻣ ﺔ ﻟﻬ ﺬﻩ اﻟ ﺴﻼﻻت ﻟﻠﺘﻴﻨ ﺎم ‪ ,‬اﻟﻤﻴ ﺮوﺑﻴﻨﻢ ‪,‬اﻻرﺗ ﺎﺑﻴﻨﻢ ‪٠‬ﺑﻨ ﺴﺒﺔ ‪ %١١,١‬ﻓﻘ ﻂ‬
‫)‪٠(١٨\٢‬وآﺎﻧ ﺖ اﻟﻤﻘﺎوﻣ ﺔ ﻟﻼوﺟﻤﻨﺘ ﻴﻦ و اﻟﺘﺎزوﺳ ﻴﻦ ‪ (١٨/١٠) % ٥٥,٦‬ﻳﻤﻜ ﻦ ﺑ ﺴﺒﺐ اﻟ ﻀﻐﻂ اﻟ ﺸﺪﻳﺪ ﻻﺳ ﺘﺨﺪاﻣﻬﻢ‬
‫ﺑﺎﻟﻤﺴﺘﺸﻔﻴﺎت‪٠‬‬
‫اﻟﺨﻼﺻﺔ‪ :‬وﻧ ﺴﺘﻨﺘﺞ ﻣ ﻦ هﻨ ﺎ ﻣ ﺪى دور ه ﺬﻩ اﻟ ﺴﻼﻻت ﻓ ﻰ اﻟﻌ ﺪوى اﻟﻤﻜﺘ ﺴﺒﺔ ﻣ ﻦ اﻟﻤﺴﺘ ﺸﻔﻴﺎت ﺧﺎﺻ ﺔ ﻋ ﺪوى اﻻﻟﺘﻬ ﺎب اﻟﺮﺋ ﻮى‬
‫اﻟﻤﺼﺎﺣﺐ ﻟﺠﻬﺎز اﻟﺘﻬﻮﻳﺔ اﻟﺼﻨﺎﻋﻰ وﻓ ﻰ اﻟﻤﻘﺎوﻣ ﺔ ﻟﻠﻤ ﻀﺎدات اﻟﺤﻴﻮﻳ ﺔ ﻣﻤ ﺎ ﻳ ﻮءدى اﻟ ﻰ ﻓ ﺸﻞ اﻟﻌ ﻼج و ارﺗﻔ ﺎع اﻟﺘﻜﻠﻔ ﺔ واﻧﺘﻘ ﺎل‬
‫اﻟﻤﻘﺎوﻣ ﺔ اﻟ ﻰ ﻣ ﻀﺎدات ﺣﻴﻮﻳ ﺔ اﺧ ﺮى ﻣﻤ ﺎ ﻳﺠﻌ ﻞ ﻟﻼﻃﺒ ﺎء ﻋ ﺪدا ﻣﺤ ﺪودا ﻓﻘ ﻂ ﻣ ﻦ اﻻﺧﺘﻴ ﺎرات اﻟﻌﻼﺟﻴ ﺔ‪٠‬وﻟ ﺬا وﺟ ﺐ اﻟﻨ ﺼﺢ‬
‫ﺑﺎﻻﺳﺘﺨﺪام اﻟﺤﻜﻴﻢ ﻟﻠﻤﻀﺎدات اﻟﺤﻴﻮﻳﺔ ﺧﺎﺻﺔ اﻟﺠﻴﻞ اﻟﺜﺎﻟﺚ ﻣﻦ اﻟﻜﻴﻔﺎﻟﻮﺳ ﺒﻮرﻳﻦ وﻻﺑ ﺪ ﻣ ﻦ اﺳ ﺘﺨﺪام ﻃ ﺮق ﻓﻌﺎﻟ ﺔ و دﻗﻴﻘ ﺔ ﻟﻠﻜ ﺸﻒ‬
‫ﻋﻦ اﻟﺠﻴﻨﺎت اﻟﻤﺴﺌﻮﻟﺔ ﻋﻦ اﻧﺘﺎج هﺬﻩ ا ﻻﻧﺰﻳﻤﺎت ﻟﻤﻨﻊ اﻧﺘﺸﺎر هﺬا اﻟﻨﻮع ﺧﺎﺻﺔ ﻟﻠﺤﺎﻻت اﻟﺤﺮﺟﺔ داﺧ ﻞ اﻟﻌﻨﺎﻳ ﺔ اﻟﻤﺮآ ﺰة‪٠‬ﻳﻮﺻ ﻰ‬
‫ﺑﻌﻤﻞ دراﺳﺎت ﻣﺴﺘﻘﺒﻠﻴﺔ ﺟﺰﻳﺌﻴﺔ ﻟﺪراﺳﺔ ﺟﻴﻨﺎت اﺧﺮى ﺗﺴﺎهﻢ ﻓﻰ اﻧﺘﺎج هﺬﻩ اﻻﻧﺰﻳﻤﺎت ﻟﻠﺤﺪ ﻣﻦ اﻟﻤﻘﺎوﻣﺔ ﻟﻠﻤﻀﺎدات اﻟﺤﻴﻮﻳﺔ‪.‬‬
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Vol. 22, No. 2
Characterization and Kinetics Study for Rhamnolipids Produced by
an Environmental Pseudomonas aeruginosa isolate
1
M. Kassem1*, N. Fanaki1, H. Abou-Shleib1, F. Dabbous1, Y. R. Abdel-Fattah 2
Department of Microbiology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt;
2
Department of Bioprocess Development, Genetic Engineering and Biotechnology Research
Institute, City for Scientific Research and Technology Applications SRTA City,
New Borg El-Arab, Alexandria, Egypt
ABSTRACT
Pseudomonas aeruginosa produces glycolipidic surface-active molecules (rhamnolipids) which have
potential biotechnological applications. A previously identified biosurfactants-producing environmental
isolate Pseudomonas aeruginosa strain 6 (Ps. 6) was selected. Comparing the kinetics of cell growth and
biosurfactants production by Ps. 6 in bioreactor with that of shake flask, the bioreactor was characterized
by about 35% higher cell density than that of shake flask. However, the production of rhamnolipids was
higher in shake flask culture than in bioreactor culture, where their minimum surface tension values
obtained were 12 mN/m and 13.9 mN/m, respectively. The biosurfactants solution produced by Ps. 6 was
stable to all tested temperatures and pH range 2-14. However, the surface tension of the biosurfactants
solution increased with increasing salinity. Moreover, its emulsification indexes EI24 varied from 33.3%
to 58%. It was also demonstrated that this biosurfactants solution form water in oil emulsion.
Furthermore, the biosurfactants had greater antimicrobial activity against Bacillus spp. than S. aureus
and M. flavus with undetectable activity against Gram negative bacteria and an experiment was done to
know whether rhamnolipids act on the lipid part of cell membrane or the outer protein. On testing the
efficiency of the biosurfactant solution as preservative using challenge test, it was found that <0.3 g% of
the crude biosurfactants solution was not sufficient for preservation up to 1 month. Finally, the antibiofilm activity of the produced biosurfactants reduced the biofilm formation of S. aureus ATCC 6538p by
more than 2 log units.
Keywords: Rhamnolipids – kinetics – biosurfactants production – biofilm formation
Due to their biological properties,
rhamnolipids demonstrated many applications
including medical, biological, pharmaceutical
and even environmental. Rhamnolipids were
also being used as cosmetic additives and have
been patented to make some liposomes and
emulsions both of which are important in the
cosmetic industry(7). They also showed
inhibitory
activity
against
different
Furthermore,
the
microorganisms(8).
rhamnolipids biosurfactants isolated from P.
aeruginosa strains were able to form reverse
micelles and were involved in the synthesis and
stabilization of silver nanoparticles (9).
Consequently, study of kinetics of
rhamnolipids for large scale economical
production was the main goal of the present
study. In addition, testing some of their
characters such as; emulsification, antimicrobial
and antibiofilm activities could support their use
especially in medical, pharmaceutical and
cosmetics manufacture as multifunctional
materials for the new century.
INTRODUCTION
Biosurfactants means surface active
products of microbial origin; they are
amphiphilic compounds, produced mostly as
secondary metabolites. Biosurfactants have
several advantages over synthetic surfactants
including; low toxicity, are biodegradable, are
effective
under
extreme
environmental
conditions-such as temperature, pH and ionic
strength-show
strong
surface
activity,
emulsifying ability and have antimicrobial and
anti-adhesive properties (1-3).
Pseudomonas aeruginosa produces and
secretes
rhamnose-containing
glycolipid
biosurfactants called rhamnolipids. The
production of rhamnolipids by P. aeruginosa
was first reported by Jarvis et al. in 1949 (4). The
formation of rhamnolipids by P. aeruginosa
occurs during the late-exponential and
stationary phases of growth and on the genetic
level, biosynthesis of rhamnolipids, mono- and
di-rhamnolipid, occurs through three sequential
steps encoded by the three genes rhlA, rhlB and
rhlC (5,6).
123
Vol. 22, No. 2
Determination of Biosurfactants Activity as
Emulsifying Agent:
Equal volumes of the rhamnolipids and
tested oils were mixed and vortexed, then left
for 24 h and for 7 days to allow phase
separation. The emulsification index EI24% and
EI7%, were calculated according to the
following equation:
(Height of emulsified layer/total height of both
layers)*100
Moreover, in order to know type of emulsion
formed, rhamnolipids and paraffin oil were
admixed in different ratios and (EI24%) was
measured (16).
Antimicrobial susceptibility testing:
MICs were determined by the agar dilution
method against selected strains (16 bacterial and
fungal microorganisms) by incorporating serial
doubling concentrations (from 43.44 to 3000
μg/mL) of the rhamnolipids stock solution,
sterilized either by autoclaving or by membrane
filtration, in Mueller-Hinton agar (Oxoid)
according to the CLSI (formerly NCCLS)
standards(17). The plates inoculated with
properly diluted overnight cultures were
incubated at 35ºC for 18 h.
Moreover, the growth patterns of standard S.
aureus ATCC 6538p was monitored in the
absence and presence of its corresponding subMICs of rhamnolipids solution. The number of
survivors in the original system was estimated
by the surface viable count technique (18).
Effect of Rhamnolipids Solutions on the
Cytoplasmic Membrane:
Bacterial suspensions of standard Gram
positive strains were prepared and treated with
600 μg/mL biosurfactants solution or left
untreated. After incubation, the absorbance of
the clear supernatant was estimated at 260 and
280 nm using UV-Visible spectrophotometer
(Thermospectronic, Helios alpha, England) and
the corresponding net leakage due to treatment
was determined (19).
Evaluation of the Anti-biofilm Activity of
Biosurfactants:
The effect of sub-MIC of the biosurfactants
on biofilm formation on membrane filters (0.2
μm pore size and 13 mm diameter) by selected
microorganisms; S. aureus ATCC 6538p (St),
C. albicans ATCC 10231(Ct), P. aeruginosa
ATCC 9027 (Pt), Staphylococcus sp. isolate
(S75), E. coli isolates (E11 and E14), P.
aeruginosa isolate (P25) and Klebsiella spp
isolate (K103) was tested. After incubation, the
filters were washed with saline to remove the
planktonic cells. The formed biofilms were
examined by surface viable count and by
MATERIALS AND METHODS
Microorganisms:
The biosurfactants-producing P. aeruginosa
(Ps. 6) was isolated from the sewage treatment
unit of Kabbary station in the governorate of
Alexandria, Egypt and was identified by both
biochemical and molecular techniques in a
previous work in the microbiology department,
Faculty of Pharmacy, Alexandria University (10).
Determination of Growth and Production
Kinetics in Shake Flask and Bioreactor:
The kinetics of growth and biosurfactants
production for Ps. 6 were compared in both
shake flask and bioreactor (Bioflo 110, New
The
Brunswick Scientific, Canada)(11).
optimized media(12) was inoculated with 1%
inocula . The culture temperature, aeration and
agitation rate were adjusted to 35°C, 1 vvm and
200 rpm, respectively. At 4 h intervals, samples
were withdrawn and the OD600 was measured
using cell density meter while the surface
tension (ST) was measured using Tensiometer
(TD1, Lauda, Germany). Moreover, glucose
concentrations in the culture supernatants of
some samples were monitored using FreeStyle®
glucose monitoring system. Calculation of the
kinetics parameters was done according to
Monod equation (13). Partial recovery and
purification of the biosurfactants was done as
previously reported (14).
Study of the Stability of the Surface Activity
of Rhamnolipids to Different Factors:
Temperature:
The ST of the rhamnolipids solution was
measured after exposure to different
temperatures including autoclaving, 70°C,
50°C, 35°C, room temperature (RT), 4°C, 20°C, each for 1 h, and many successive times
of freezing and thawing. The ST of the solution
at RT was considered as the standard.
pH:
The rhamnolipids solution pH was adjusted
to 2, 6, 7, 10 and 14 using 2N HCl and 2N
NaOH. The solutions of different pH(s) were
then left for 24 h and the ST was then measured
where the solution with pH 7 was considered as
the standard.
Salinity:
Equal volumes of the biosurfactants and sea
water or double strength NaCl solution of
different concentrations (4-20%) were mixed.
After 1 h, the ST was measured where the
sample mixed with water was considered as the
standard (15).
124
scanning electron microscope(20,21) (Scanning
Microscope, JEOL, JSM-5300, Japan).
Vol. 22, No. 2
the concentration of NaCl increased, the ST
increased indicating instability of rhamnolipids
(data not shown).
Emulsifying Activity of the Rhamnolipids
Solution and Type of Emulsion Formed:
The EI24% of the rhamnolipids produced
ranged from 33.3% to 58%, Table 2. The lower
the hydrophilic/lipophilic balance (HLB), i.e.
<7, the more lipophilic/hydrophobic is the
compound. Recently, HLB of rhamnolipids
produced by P. aeruginosa isolates of 24.1 and
10.07 were reported(29,30), denoting that
rhamnolipids emulsions are formed in the oily
layer. Moreover, as the paraffin oil:
rhamnolipids ratio increased the EI24%
increased denoting emulsions formation in the
oily layer, i.e. w/o surfactant type (data not
shown).
Antimicrobial Activity of the Rhamnolipids
Solution:
The inhibitory activity of the rhamnolipids
solutions was detected mainly against Gram
positive bacterial strains regardless to the
sterilization method used. The highest inhibitory
activity was against Bacillus strains (187.5-375
µg/mL), followed by S. aureus while activity
against Gram negative bacteria and fungi were
absent (>3000 µg/mL), (data not shown). This
was somewhat in agreement with Benincasa et
al.[27]. On the other hand, Abalos et al.[8]
demonstrated inhibitory activity of rhamnolipids
mixture obtained from P. aeruginosa AT10
against E. coli, Micrococcus luteus, Alcaligenes
faecalis (MIC= 32 µg/mL), Serratia arcescens,
Mycobacterium phlei (16 µg/mL) and S.
epidermidis (8 µg/mL).
Moreover, subinhibitory concentrations of the
rhamnolipids solution on S. aureus ATCC
6538p demonstrated reduction in the number of
survivors by time as shown in Figures 3.
Effect of the Rhamnolipids Solutions on the
Bacterial Cytoplasmic Membrane:
Similar to previous studies Kulakovskaya et
al.; Sotirova et al.[31,32], when comparing the
leakage of treated and untreated Gram positive
bacteria, biosurfactants enhanced the nonspecific permeability of the cytoplasmic
membrane causing a net loss of the 260 and 280
nm-absorbing materials (proteins and nucleic
acids), where the extent of leakage differed
from one organism to another, Table 3. This
may be due to their action on the lipid part of
cell membrane or the outer protein causing
structural fluctuations in the membrane.
Efficiency of the Rhamnolipids Solution as
Anti-biofilm Agent:
Subinhibitory
concentration
of
the
rhamnolipids solution enhanced the biofilm
RESULTS & DISCUSSION
Comparing Cell Growth and Production
Kinetics on Shake Flask and Bioreactor
Scales:
This comparison showed that the bioreactor
was characterized by about 35% higher cell
mass and specific growth rate (µ). The increased
growth in the bioreactor may be due to higher
aeration and agitation which reflected better
mixing between the air/liquid phase and
increased oxygen availability in culture(22). The
75% and 70% reduction of ST shown was due
to maximum rhamnolipids production, whereas,
Rashedi et al.(23) reported only 48% reduction in
ST due to production of 1.7 g/L rhamnolipids.
Hence, compared to the latter results, it may be
concluded that the produced rhamnolipids
concentration in the culture used, greatly
exceeded 1.7 g/L, based on the rule that the
lower the ST, the higher is the biosurfactants
concentration, Figure1 and 2 and Table 1.
The minimum ST for the produced
rhamnolipids in this study was 12.9 mN/m.
Although a minimum ST for rhamnolipids of
23.5 mN/m(24) and 25-30 mN/m(25) was
previously published. This may be due to
production of rhamnolipids different from those
published or production of other types of
biosurfactants besides the rhamnolipids.
Similar to previous reported studies(26,27),
the rhamnolipids production occurred during the
whole cell growth cycle, especially in the
stationary phase.
Stability of the Produced Rhamnolipids to
Different Factors:
The rhamnolipids, both crude and after
partial purification, were stable to all tested
temperatures including autoclaving and after
successive freezings and thawing. When testing
the stability of the produced rhamnolipids to
different pHs, the values of ST were almost
constant at pH range 4-14 while at pH 2
precipitations occurred. After re-dissolving the
precipitates in 0.1 M NaHCO3 solution (pH=7),
still the ST of the new solution was greatly
reduced. In a previous study, the pKa of a
mono-rhamnolipids mixture in water was
determined to be about 4.28 and 5.50 for
concentrations below and above the CMC of
this rhamnolipids, respectively(28). Therefore, at
pH 2, about 2 units below its pKa, the acidic
form predominated and precipitation occurred.
This may explain why precipitation occurred at
low pH without losing the activity. Finally, as
125
formation in the Pseudomonas and klebsiella
srains tested but not the E. coli, with almost no
effect against C. albicans (Ct). The increase in
the biofilm formed by some Gram negative
isolates could be explained by that rhamnolipids
are essential to maintain the architecture of
Pseudomonas biofilms and they are considered
as one of its virulence factors Davey et al.[33].
Similar to a previously reported study
Rodrigues et al.[34], in the presence of the
biosurfactants, formation of biofilm by S.
aureus (St) was reduced by about 2 log units,
Figure 4. This latter effect was confirmed by
Vol. 22, No. 2
capturing photos using scanning electron
microscopy, where it showed that the biofilmforming cocci were more condensed in the
control photo (A) and less condensed in photo
(B), Figure 5.
Consequently,
the
production
of
biosurfactants formed by P. aeruginosa isolate
Ps. 6 was optimized for large scale production
as they may be used as a multifunctional
material in pharmaceutical preparation for their
emulsification, anti-biofilm and antimicrobial
activities.
Figure 1: Kinetics of cell growth and biosurfactants production in shake flask, where Δ ST in mN/m,
■ pH, ● OD at 600 nm, ○ Glucose concentration (g%).
Figure 2: Kinetics of cell growth and biosurfactants production in bioreactor,
where ▼ DO (%) and the rest as in Figure 1.
126
Vol. 22, No. 2
Table 1: Kinetic parameters of cell growth and rhamnolipids production
Cultivation Vessel
Parameter
Shake flask
Bioreactor
1.34
1.81
ODmax
0.2
0.15
Growth rate [h-1]
0.337
0.451
µ [h-1]
12.9
13.9
STmin [mNm-1]
-1 -1
-6.25
-4.3
QP [mNm h ]
-0.18625
-0.12
Qglu [gL-1 h-1]
Where; ODmax: the maximum optical density at 600 nm, µ: specific cell growth rate,
STmin: minimum surface tension (mN/m), Qglu: glucose consumption rate
Qp: ST reduction rate
Table 2: The emulsifying activity of the rhamnolipids solution with different oils
Oil name
EI24 (%)
EI7days
n-hexadecane
57
57
Sesames
55.5
55.5
Camphor
58.3
58.3
Olive oil
33.2
10.2
Peppermint
36.4
18.2
Paraffin
47.2
44.4
Figure 3: Effect of sub-MICs of rhamnolipids solution on the growth pattern of
S. aureus ATCC 6538p
Table 3: Effect of rhamnolipids solution on the cytoplasmic membrane
260 nm
280 nm
Strain
control
treatment
control
0.801
1.336
0.561
S. aureus ATCC 6538p
0.217
0.917
0.171
M. flavus ATCC 9341
1.547
2.184
1.173
B. insolitus DSM 5
127
treatment
0.918
0.459
1.963
Vol. 22, No. 2
Figure 4: Effect of rhamnolipids solution on biofilm formation.
Figure 5: Effect of rhamnolipids solution on the biofilm formation using scanning electron
microscopy (A): the control, (B): treated with rhamnolipids solution
3.
Acknowledgments
This study was supported by the Faculty of
pharmacy, Department of Microbiology,
Alexandria University, with considerable
contribution of the Department of Bioprocess
Development, Genetic Engineering and
Biotechnology Research Institute, City for
Scientific
Research
and
Technology
Applications SRTACity, Alexandria, Egypt.
4.
5.
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Lebron-Paler A., J.E. Pemberton, B.A.
Becker, W.H. Otto, C.K. Larive and
R.M. Maier, 2006. Determination of the
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‫‪acid dissociation constant of the‬‬
‫‪biosurfactant mono-rhamnolipid in aqueous‬‬
‫‪solution‬‬
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‫‪29. Noordman W.H., J.H.J. Wachter, G.J. de‬‬
‫‪Boer and D.B. Janssen, 2002. The‬‬
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‫‪degradation by Pseudomonas aeruginosa‬‬
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‫‪Biotechnol., 94: 195-212.‬‬
‫‪30. Abalos, A., M. Vinas, J. Sabate, M.A.‬‬
‫‪Manresa and A.M. Solanas, 2004.‬‬
‫‪Enhanced biodegradation of casablanca‬‬
‫‪crude oil by A microbial consortium in‬‬
‫‪presence of a rhamnolipid produced by‬‬
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‫‪Biodegradation, 15: 249-260.‬‬
‫‪31. Kulakovskaya, T.V., E.V. Kulakovskaya‬‬
‫‪and W.I. Golubev, 2003. ATP leakage‬‬
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‫‪glycolipids of Pseudozyma fusiformata.‬‬
‫‪FEM Yeast Res., 3: 401-404.‬‬
‫‪32. Sotirova, A.V., D.I. Spasova, D.N.‬‬
‫‪Galabova, E. Karpenko and A. Shulga,‬‬
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‫‪Rhamnolipid-biosurfactant‬‬
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‫‪and Gram-negative bacterial strains. Curr.‬‬
‫‪Microbiol., 56: 639–44.‬‬
‫‪33. Davey, M.E., N.C. Caiazza and G.A.‬‬
‫‪O’Toole, 2003. Rhamnolipid surfactant‬‬
‫‪production affects biofilm architecture in‬‬
‫‪Pseudomonas aeruginosa PAO1. J.‬‬
‫‪Bacteriol., 185: 1027–1036.‬‬
‫‪34. Rodrigues L.R., I.M. Banat and H.C.‬‬
‫‪Van der Mei, 2006. Interference in‬‬
‫‪adhesion of bacteria and yeasts isolated‬‬
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‫‪rubber by rhamnolipid biosurfactants. J.‬‬
‫‪Appl. Microbiol., 100: 470-480.‬‬
‫دراﺳﺔ ﺣﺮآﻴﺔ ﻧﻤﻮ اﻟﺨﻼﻳﺎ وﺗﻮﺻﻴﻒ ﻋﺰﻟﺔ ﺳﻮدوﻣﻮﻧﺎس إﻳﺮوﺟﻴﻨﻮزا‬
‫ﺑﻴﺌﻴﺔ ﻣﻨﺘﺠﺔ ﻟﻠﺮاﻣﻨﻮﻟﻴﺒﻴﺪز‬
‫ﻣﻴﺮﻓﺖ أﻣﻴﻦ ﻗﺎﺳﻢ ‪ - ١‬ﻧﻮرهﺎن ﺣﺴﻴﻦ ﻓﻨﺎآﻰ ‪ – ١‬ﺣﻤﻴﺪﻩ أﺑﻮ ﺷﻠﻴﺐ ‪ – ١‬ﻓﺎﻃﻤﺔ دﺑﻮس ‪ – ١‬ﻳﺎﺳﺮ رﻓﻌﺖ‬
‫‪٢‬‬
‫‪ .١‬ﻗﺴﻢ اﻟﻤﻴﻜﺮوﺑﻴﻠﻮﺟﻰ اﻟﺼﻴﺪﻟﻴﺔ – آﻠﻴﺔ اﻟﺼﻴﺪﻟﺔ – ﺟﺎﻣﻌﺔ اﻷﺳﻜﻨﺪرﻳﺔ – ﺟﻤﻬﻮرﻳﺔ ﻣﺼﺮ اﻟﻌﺮﺑﻴﺔ‬
‫‪ .٢‬ﻣﻌﻬﺪ ﺑﺤﻮث اﻟﻬﻨﺪﺳﺔ اﻟﻮراﺛﻴﺔ واﻟﺘﻜﻨﻮﻟﻮﺟﻴﺎ اﻟﺤﻴﻮﻳﺔ – ﻣﺪﻳﻨﺔ اﻷﺑﺤﺎث اﻟﻌﻠﻤﻴﺔ واﻟﺘﻄﺒﻴﻘﺎت اﻟﺘﻜﻨﻮﻟﻮﺟﻴﺔ ﺑﻤﺪﻳﻨﺔ ﺑﺮج‬
‫اﻟﻌﺮب اﻟﺠﺪﻳﺪة – ﺟﻤﻬﻮرﻳﺔ ﻣﺼﺮ اﻟﻌﺮﺑﻴﺔ‬
‫إن اﻟﻤﺸﺘﺘﺎت اﻟﺤﻴﻮﻳﺔ اﻟﺮﺋﻴﺴﻴﺔ ﻟﻤﻌﻈﻢ ﺳﻼﻻت اﻟﺴﻮدوﻣﻮﻧﺎس إﻳﺮوﺟﻴﻨﻮزا )ﺗﺴﻤﻲ راﻣﻨﻮﻟﻴﺒﻴﺪز( اﻟﻤﻜﻮﻧﺔ ﻣ ﻦ ﺑﻴﺒﺘﻴ ﺪات اﻟ ﺴﻜﺮﻳﺎت‬
‫اﻟﺒﺴﻴﻄﺔ ﻟﻬﺎ ﺗﻄﺒﻴﻘﺎت هﺎﻣﺔ ﻓﻰ ﻣﺠﺎل اﻟﺘﻜﻨﻮﻟﻮﺟﻴﺎ اﻟﺤﻴﻮﻳﺔ‪ .‬وﻟﻘﺪ وﻗﻊ اﻷﺧﺘﻴﺎر ﻋﻠﻰ ﻋﺰﻟ ﺔ ﺳ ﻮدوﻣﻮﻧﺎس إﻳﺮوﺟﻴﻨ ﻮزا ﺑﻴﺌﻴ ﺔ ‪Ps. 6‬‬
‫ﻣﻌﺮﻓﺔ ﻓ ﻰ ﺑﺤ ﺚ ﺳ ﺎﺑﻖ‪ .‬و ﻋﻨ ﺪ إﺟ ﺮاء ﻣﻘﺎرﻧ ﺔ ﺣﺮآﻴ ﺔ ﻧﻤ ﻮ اﻟﺨﻼﻳ ﺎ وإﻧﺘ ﺎج اﻟﻤ ﺸﺘﺘﺎت اﻟﺤﻴﻮﻳ ﺔ ﻟﻠﻌﺰﻟ ﺔ ‪ ٦‬ﻓ ﻲ اﻟﻤﻔﺎﻋ ﻞ اﻟﺤﻴ ﻮي و‬
‫اﻟﻘﺎرورة اﻟﻤﻬﺘﺰة‪ ،‬آﺎن ﻣﻌﺪل اﻟﻨﻤﻮ اﻟﻤﺤﺪد ﻓﻲ اﻟﻤﻔﺎﻋ ﻞ اﻟﺤﻴ ﻮي ﺣ ﻮاﻟﻲ ‪ ٪ ٣٥‬أآﺜ ﺮ ﻣﻤ ﺎ ﻓ ﻲ اﻟﻘ ﺎرورة اﻟﻤﻬﺘ ﺰة‪ .‬ﻟﻜ ﻦ ﻋﻨ ﺪ ﻗﻴ ﺎس‬
‫اﻟﺤ ﺪ اﻷدﻧ ﻰ ﻟﻠﺘ ﻮﺗﺮ اﻟ ﺴﻄﺤﻲ ﻟﻠﻮﺳ ﻂ ﻓ ﻲ اﻟﻘ ﺎرورة آ ﺎن ﻣﻘ ﺪارﻩ ‪ ١٢‬ﻣﻴﻠ ﻲ ﻧﻴ ﻮﺗﻦ ‪/‬م ﺑﻴﻨﻤ ﺎ آ ﺎن ﻗﻴﻤﺘ ﻪ ﻓ ﻲ اﻟﻤﻔﺎﻋ ﻞ اﻟﺤﻴ ﻮي‬
‫‪١٣.٩‬ﻣﻴﻠﻲ ﻧﻴﻮﺗﻦ ‪/‬م‪ .‬ﺗﺸﻴﺮ ه ﺬﻩ اﻟﻨﺘ ﺎﺋﺞ أن إﻧﺘ ﺎج ﻟﻠﺮاﻣﻨﻮﻟﻴﺒﻴ ﺪز ﻓ ﻲ اﻟﻘ ﺎرورة اﻟﻤﻬﺘ ﺰة أﻋﻠ ﻰ ﻣﻤ ﺎ ﻓ ﻲ اﻟﻤﻔﺎﻋ ﻞ اﻟﺤﻴ ﻮي‪ .‬أﻇﻬ ﺮت‬
‫اﻟﻨﺘﺎﺋﺞ أن ﻧﺸﺎط اﻟﻤﺸﺘﺘﺎت اﻟﺤﻴﻮﻳﺔ اﻟﻤﺨﺘﺒﺮة ﺛﺎﺑﺖ ﻓﻲ ﺟﻤﻴﻊ درﺟﺎت اﻟﺤﺮارة اﻟﻤﺨﺘﺒ ﺮة و ﻓ ﻲ اﻟﻘ ﻴﻢ اﻟﻤﺨﺘﻠﻔ ﺔ ﻟ ﻸس اﻟﻬﻴ ﺪروﺟﻴﻨﻲ‬
‫ﻟﻠﻮﺳﻂ ﻣﻦ ‪ ٢‬إﻟﻰ ‪ ،١٤‬آﻤﺎ ﻟﻮﺣﻆ زﻳﺎدة اﻟﺘﻮﺗﺮ اﻟﺴﻄﺤﻲ ﻟﻤﺤﻠﻮل اﻟﻤﺸﺘﺘﺎت اﻟﺤﻴﻮﻳﺔ ﻣﻊ زﻳﺎدة ﺗﺮآﻴﺰ ﻣﺤﻠﻮل آﻠﻮرﻳ ﺪ اﻟ ﺼﻮدﻳﻮم‪.‬‬
‫ﻟﻘ ﺪ ﺗ ﻢ ﻗﻴ ﺎس ﻣﺆﺷ ﺮ اﺳ ﺘﺤﻼب ‪ EI24‬ﻟﻠﻤ ﺸﺘﺘﺎت‪ ،‬و ﻟﻘ ﺪ ﺗﺮاوﺣ ﺖ ﻣ ﻦ ‪ ٪ ٣٣.٣‬إﻟ ﻰ ‪ .٪ ٥٨‬ﺑﺎﻹﺿ ﺎﻓﺔ إﻟ ﻰ ذﻟ ﻚ ﻟﻘ ﺪ ﺗﺒ ﺖ أن‬
‫اﻟﺮاﻣﻨﻮﻟﻴﺒﻴﺪز ﻗﺎدر ﻋﻠﻰ ﺗﻜﻮﻳﻦ ﻣﺴﺘﺤﻠﺐ اﻟﻤﻴﺎﻩ ﻓ ﻲ اﻟﺰﻳ ﺖ‪ .‬و ﻗ ﺪ اﺳ ﺘﻜﻤﻠﺖ اﻟﺪراﺳ ﺔ ﺑﺒﺤ ﺚ ﻧ ﺸﺎط اﻟﻤ ﺸﺘﺘﺎت اﻟﺤﻴﻮﻳ ﺔ آﻤ ﻀﺎدات‬
‫ﺣﻴﻮﻳ ﺔ ‪ ،‬و ﻟﻘ ﺪ أﻇﻔ ﺮت اﻟﻨﺘ ﺎﺋﺞ أن أآﺒ ﺮ ﻧ ﺸﺎط آﻤ ﻀﺎدات ﻣﻴﻜﺮوﺑ ﺎت ﻟﻠﺮاﻣﻨﻮﻟﻴﺒﻴ ﺪز آ ﺎن ﺿ ﺪ ﺳ ﻼﻻت اﻟﺒﺎﺳ ﻴﻠﺲ اﻟﻤﺨﺘﺒ ﺮة ﺛ ﻢ‬
‫ﺳ ﺘﺎﻓﻴﻠﻮآﻮآﺲ وﻣﻴﻜﺮوآ ﻮآﺲ ﻓﻼﻓ ﺲ و ﻟﻜﻨﻬ ﺎ ﻟ ﻢ ﺗﻈﻔ ﺮ ﻋ ﻦ أي ﻧ ﺸﺎط ﺿ ﺪ اﻟﺒﻜﺘﻴﺮﻳ ﺎ ﺳ ﺎﻟﺒﺔ اﻟﺠ ﺮام‪ .‬وﻋﻨ ﺪ دراﺳ ﺔ ﻓﺎﻋﻠﻴ ﺔ ه ﺬﻩ‬
‫اﻟﻤﺸﺘﺘﺎت اﻟﺤﻴﻮﻳﺔ ﻟﻤﻌﺮﻓﺔ إﻣﻜﺎﻧﻴ ﺔ اﺳ ﺘﺨﺪاﻣﻬﺎ آﻤ ﺎدة ﺣﺎﻓﻈ ﺔ ﻓ ﻲ اﻟﻤﺴﺘﺤ ﻀﺮات اﻟ ﺼﻴﺪﻟﻴﺔ‪ .‬وﺟ ﺪ أن اﻟﺘﺮآﻴ ﺰ اﻟﻤﺨﺘﺒ ﺮﻣﻦ ﻣﺤﻠ ﻮل‬
‫اﻟﻤﺸﺘﺘﺎت اﻟﺤﻴﻮﻳﺔ اﻟﺨﺎم ‪ ،‬ﻷﻗﻞ ﻣﻦ ‪ ، ٠.٣ ٪‬ﻟﻢ ﻳﻜﻦ آﺎﻓﻴﺎ آﻤﺎدة ﺣﺎﻓﻈﺔ ﻟﻠﻤﺴﺘﺤﻀﺮ أﺛﻨﺎء ﺣﻔﻈﻪ ﻟﻤ ﺪة ﺷ ﻬﺮ‪ .‬أﺧﻴ ﺮًا ‪ ،‬ﺗﻤ ﺖ ﺑﺤ ﺚ‬
‫اﺧﺘﺒ ﺎر ﻧ ﺸﺎط اﻟﺮاﻣﻨﻮﻟﻴﺒﻴﺪزاﻟﻤ ﻀﺎد ﻟﻠﻐ ﺸﺎء اﻟﺤﻴ ﻮي اﻟﺒﻜﺘﻴ ﺮي وﻗ ﺪ ﺗﺒ ﻴﻦ أن ﻣﺤﻠ ﻮل اﻟﺮاﻣﻨﻮﻟﻴﺒﻴ ﺪز أدى إﻟ ﻰ ﺗﻘﻠﻴ ﻞ ﻋ ﺪد اﻟﺨﻼﻳ ﺎ‬
‫اﻟﻤﻜﻮﻧﺔ ﻟﺘﻜﻮﻳﻦ اﻟﻐﺸﺎء اﻟﺤﻴﻮي ﺑﻮاﺳﻄﺔ ﺳﺘﺎﻓﻴﻠﻮآﻮآﺲ أورﻳﺲ ‪ ATCC 6538p‬ﺑﻤﻘﺪار ﻣﺎﺋﺔ ﻣﺮة ﺗﻘﺮﻳﺒﺎ‪.‬‬
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April 2013 Volume 22, No 2 - Egyptian Journal of Medical

Transcription

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