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IJBPAS, May, 2015, 4(5): 2448-2457
ISSN: 2277–4998
A STUDY ON THE PATTERN OF CANDIDA SPECIES SENSITIVITY TO
ANTIFUNGAL DRUGS AND ZNO NANOPARTICLE
AZADEHA BEDZADEH HAJAR
Department of Microbiology, Qom branch, Islamic Azad University, Qom, Iran
*Corresponding Author: [email protected]
ABSTRACT
In recent years, systemic fungal infections caused by Candida species have been regarded as one
of the most important factors of death. The outbreak of fungal infections has led to increasing
use of antifungal drugs and considerable increase resistance of Candida species against them.
Therefore, the present study was carried out to investigate the drug sensitivity of Candida agents
isolated from the vaginal samples of women who referred to Qom’s health centers, and the effect
of ZnO nanoparticle on fungal agents.
In this laboratory-descriptive study was conducted on 150 patients susceptible to vaginitis. The
samples were directly examined and cultured. The culturing was done on the Sabouraud
Dextrose Agar medium containing Cloramphenicol, Corn Meal Agar medium including
Tween80, Candida Crom agar medium and the test of germ tube and sugar assimilation test
(API20C) to isolate and identify Candida species. Then the effect of different antifungal drugs
and ZnO nanoparticles on them was examined by using disk diffusion method.
Among 150 samples examined, 83 cases (55.3%) were identified as havingCandida species. The
statistical analysis of isolated samples showed that Candida albicans (62.7%), Candida krusei
(33.7%) and Candida tropicalis (%3.6) have the highestfrequency. Sensitivity pattern of these
species to different antifungal drugs were different. Furthermore, ZnO nanoparticle showed a
considerable no-growth halo diameter in some isolated Candida species, in comparison with
other antifungal drugs. The research findings show that ZnO nanoparticle has antifungal effect
and can be used as a proper alternative to removeCandida species in medical field.
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IJBPAS, May, 2015, 4(5)
Azadeha Bedzadeh Hajar
Research Article
Keywords: Candida Species, Antifungal Drugs, ZnO Nanoparticle
INTRODUCTION
Candidiasis is one of the most epidemic
grow in the form of yeast, pseudo- mycelium
fungal infections in women, which is caused
and real mycelium. For example, Candida
by different Candida species, especially
glabrata grows only in the form of yeast
Candida albicans. Today, this infection is
while Candidaalbicans forms a real mycelium
considered the fourth common cause of
on host’s tissue. These microorganisms are
hospital infections. The equivalent terms for
found commensally in oral mucus, vagina,
Candidiasis
and
are
“Moniliasis”
“Candidal
and
Vaginitis”,
“Aphtha
(or
gastrointestinal
tract
[Falahati
M,
Sharifinia S, Foroumadi AR, Bolouri F,
thrush)”[Rajabibazl M, Javad Rasaee M,
Akhlagh L, Yazdan Parast A, et al., 2009].
Nouri Fard M, Farahnejad Z. 2013]. This
In recent years, the number of organisms
infection is observed on skin, nail, vaginal
resistant
mucus, bronchus, lungs and gastrointestinal
increased and caused problems in treatment
tract (digestive system) acutely, sub-acutely
process. Nearly 10% of patients suffering
or chronically. It sometimes spreads and
from vaginal Candidiasis do not respond to
damages kidney, lungs, liver, heart, etc. The
initial treatment [Pádua RAFd, Guilhermetti
host’s reactions to this disease range from a
E, Svidzinski TIE 2003]. This disease has
slight itch and inflammation to acute chronic
been found and reported in all cities in Iran in
septic form or Granulomatosis[Nozari S,
different forms depending on the strength and
Moghaddam AS, khoshdel A, Noorifard M,
weakness of immunity system [Nozari S,
Moosavi AA. 2013]. The results of different
Moghaddam AS, khoshdel A, Noorifard M,
studies show that the most common species
Moosavi AA. 2013].
causing Candidiasis is Candida albicans and
Nanoparticles are the preliminary principles
10-20 percent of other cases are caused by
in nanotechnology. Among nanoparticles,
Candida
and
non-organic metal oxides such as TiO2, Cao,
Candida parapsilosis [Nasrollahi Omran A,
MgO and ZnO have a strong antimicrobial
Vakili L, Jafarpur M 2010].
effect
Most of Candida species are considered
consideration[Oladiran AA, Olabisi IA-M.
pathogenic
of
2013]. The main mechanism of the effect of
thesemicroorganisms havetwo shapes and
nanoparticles on microorganisms is that they
glabrata,
in
Candida
human.
krusei
Some
against
and
have
antifungal
been
drugs
taken
has
into
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IJBPAS, May, 2015, 4(5)
Azadeha Bedzadeh Hajar
Research Article
damage protein and DNA and destruct cell
province, Iran, including Izadi Hospital,
wall[Pasquet J, Chevalier Y, Couval E,
ShahidBeheshti
Bouvier D, Noizet G, Morlière C, et al. 2013,
Hospital.
Higa LH, Schilrreff P, Perez AP, Morilla MJ,
transmitted into a tube containing one ml of
Romero EL. 2013]. Among these mixtures,
physiological sterile serum and sent to
ZnO nanoparticle plays an important role in
laboratory.
damaging microbic cell by generating free
Isolation of CandidaSpecies
radicals. In Escherichia coli bacterium, these
All samples were cultured on Sabouraud
nanoparticles
Dextrose
increase
penetrability
of
Hospital
Swaps
Agar
and
were
Al-Zahra
immediately
medium
containing
membrane and destruction of cell and have
Cloramphenicol in order to isolate yeasts.
inhibitory effect on such fungi as Phozarium
Cycloheximide
[Jehad M Y, Enas N D. 2012].
sensitivity of some Candida species such as
Given the ever increasing resistance of
Candida krusei, Candida parapsilosis and
microorganisms, the present paper seeks to
Candida tropicalis to this antibiotic. The
study the sensitivity of isolated Candida
plates were incubated for 24-48 hours in
agents to drugs, report the results to health
30⁰C. Then a slide was made from produced
centers to help them prescribe drugs better,
colonies and studied in terms of their
find
yeastiness, after the gram was stained [Zaini
proper
ways
to
overcome
these
was
not
used
due
to
microorganisms, and observe the better effect
F, Mehbod A, Emami M. 1999].
of ZnO nanoparticle in comparison to other
Identification of Candida Species
antifungal drugs.
After the yeasts had grown, they were
MATERIALS AND METHODS
cultured on Candida Crom agar medium,
Sample Collecttion
using streak method, and incubated for 24
This is a laboratory-descriptive study which
hours in 30⁰C in order to isolate the samples
was conducted by gynecologists on 150
including more than one type of yeast
patients susceptible to vaginitis in February
[Hashemi S. 2011]. Then one loop was taken
and March 2014, using sterile swaps. The
from every stained colony and cultured on
research
women
Corn Mill Agar medium containing Tween
susceptible to Candidal infection with clinical
80, through streak and linear culturing and
symptoms of vulvovaginitis and without these
pour plate and surface methods, and a steak
symptoms, who referred to hospitals in Qom
was put between streak and linear cultures to
population
includes
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IJBPAS, May, 2015, 4(5)
Azadeha Bedzadeh Hajar
Research Article
provide difficult conditions for Chlamido
Using
conidia
be
antibiogram test was conducted for 83
produced. The plates were heated for 48 hours
positive samples. These tests were conducted
in 30⁰C. Finally, the existence or non-
on Mueller Hinton Agar e medium which was
existence of Chlamido conidia was examined
inoculated by fungus suspension (equal to 0.5
by using weak lensing magnification of
McFarland). The plates were heated for 24
microscope
JW,
hours in 30⁰C and the diameter of halos was
Boekhout T. 2011]. A part of yeast colony
interpreted with regard to the table presented
was transferred into a tube containing 0.5 ml
by the factory that made the disks[Bauer A,
of human serum and produced suspension
Kirby W, Sherris JC, turck, Turck M. 1966].
was heated for 2-3 hours in 37⁰C in order to
The strain ATCC10231 of Candidaalbicans
examine germ tube formation. Then a drop of
was used for positive control.
the suspension was put between slide and
The Evaluation of the Effect of ZnO
steak and examined by microscope [Kelley D.
nanoparticle
2006]. The colonies which had produced
Disk diffusion method and sterile paper disks
green color on Candida Cromagarmedium and
moistened with 50 mg density were used to
Chlamidoconidia on Corn Mill Agar medium
evaluate the effect of ZnO nanoparticle. First,
containing
microscopic
0.5 McFarland suspension of isolated species
examination of colonies were considered as
was densely cultured on Mueller Hinton Agar
Candida albicans, the blue colonies which
environment containing 2% glucose and 0.5
had produced long pseudo-mycelium and
mcg/ ml of Methylene Blue and paper disks
many
and
pseudo-
mycelium
[Kurtzman
Tween
80
C,
in
to
Fell
standard
disk
diffusion
method,
with
pseudo-
moistened with ZnO nanoparticle were put on
as
Candida
the cultures by sterile forceps. Then plates
tropicalis and those violet-pink colonies with
were heated for 24 hours in 30⁰C and the
jagged edges as Candidakrusei [Hashemi S.
results were measured by ruler and recorded
2011]. Furthermore, sugar assimilation test
[Jehad M Y, Enas N D. 2012].
(including
Statistical Analysis
blastopores
mycelium
were
along
considered
glucose,
maltose,
sucrose,
trehalose, cellobiose and raffinose) was
Charts were drawn by using Excel software.
conducted by using AP120C kit [Evans
T-test was used to identify the significant
EGV, Richardson MD. 1989].
difference between the effect of antifungal
AntibiogramTest
drugs and that of ZnO nanoparticle.
2451
IJBPAS, May, 2015, 4(5)
Azadeha Bedzadeh Hajar
Research Article
RESULTS
andZnO nanoparticle were examined, which
Isolation and Identification of Candida
showed that these species were resistant
Species
against some antifungal drugs and sensitive
150 patients susceptible to Candidal infection
and semi-sensitive to some others. The
with clinical symptoms of vulvo vaginitis and
resistance of isolated Candida species from
without these symptoms were studied in this
patients against different antifungal drugs was
research, among whom 83 Candida species
different.
(55.3%) were isolated and 67 negative cases
ZnO nanoparticle showed a considerable no-
(44.7%) were reported.
growth halo diameter in some isolated
As Figure 1 shows, among 83 cases of
Candida species, in comparison with other
isolated Candida species, 52 cases were
antifungal
identified as Candida albicans (62.7%), 28
nanoparticle had a significant difference, in
cases as Candida krusei (33.7%) and 3 cases
comparison
as Candida tropicalis (3.6%).
Fluconazole, Ketoconazole, and Caspofungin
The Results of Antibiogram
(p<0.05).Figure 2 shows resistance and
Resistance and/or sensitivity of isolated
sensitivity pattern of isolated Candida species
Candida species to some antifungal drugs
to mentioned antifungal drugs.
70
60
drugs.
with
Furthermore,
Amphotricin
ZnO
B,
number
62.6
percent
52
50
40
33.7
28
30
20
10
3
3.6
0
Candida albicans
Candida krusei
Candida tropicalis
Figure 1: Distribution of number and percentage based on isolated yeast species
2452
IJBPAS, May, 2015, 4(5)
Percent
Azadeha Bedzadeh Hajar
120
100
80
60
40
20
0
Research Article
100
100
97.6
98.8
97.6
Resistance
Intermediat
45.8 45.8
0 0
0 0
8.4
Sensitive
1.2 1.2
0 2.4
1.9 0
Antifungal drugs
Figure 2: The resistance and sensitivity pattern of isolated Candida species to antifungal drugs
DISCUSSION
albicans had the highest frequency (62.7%) in
Given the growing epidemic of vaginitis
this study. Candida krusei and Candida
infection among Iranian women (about 75%)
tropicalis constituted 33.7% and 3.6% of
and the difference between the pattern of
cases, respectively. In 2008, Falahati et al
yeast species and that of antibiogram in each
reported that 80 patients out of 150 patients
district, the present paper seeks to examine
suffering
these patterns in Qom province in order to
vaginitis, with Candida albicans having the
identify causing factors and obtain their
highest frequency and Candida krusei and
resistance pattern. Furthermore, it tries to
Candida gilermondi the lowest frequency
provide physicians with proper pathological
[Falahati M, Sharifinia S, Foroumadi AR,
techniques, by using such new sciences as
Bolouri F, Akhlagh L, Yazdan Parast A, et
nanotechnology and ZnO nanoparticle. The
al. 2009]. In 2011, Nazeri et al and
findings showed that ZnO nanoparticle has
Nasrollahi et al., found that
antifungal effect and can be used as a proper
albicans and Candida krusei had the highest
alternative to remove Candida species in
and
medical domain.
[Nasrollahi omran A, Vakili L, Jafarpur
Having conducted necessary tests on 150
M. 2011, Nazeri M, Mesdaghinia E,
patients susceptible to Candida, 83 positive
Moravej
cases were found. Like most studies, Candida
Soleymani F 2012]. In 2013, Panchal et al.
from
lowest
vaginitis
frequencies,
SAR,
had
Candidal
Candida
respectively
Atabakhshiyan
R,
2453
IJBPAS, May, 2015, 4(5)
Azadeha Bedzadeh Hajar
Research Article
reported that out of 100 samples of vaginal
examined the sensitivity of eight Candida
swab 40 cases were positive, 22 of which
species in 106 local isolates to Fluconazole
were related to Candida albicans and 18 cases
and reported that 69.8% of isolates was
were non-Candidal [Panchal P. 2013].
sensitive to this antifungal drug [Pakshir K,
Different isolated Candida species showed
Akbarzadeh
different sensitivity to antifungal drugs.
Mohagheghzadeh AA. 2010]. In 2009,
45.8% of all Candida species was resistant
Pakshir et.al found that 94% of all samples
against Ketoconazole and Candida albicans
were sensitive to Clotrimazole, 55% to
species was reported as the most resistant one.
Fluconazole and 99% to Nystatin [Pakshir K,
All Candida species isolated from patients
Akbarzadeh
were
Mohagheghzadeh AA. 2010].
completely
(100%)
sensitive
to
M,
M,
Bonyadpour
Bonyadpour
B,
B,
Fluconazole and Amphotricin B. Although
Furthermore, the results of this study showed
some studies show that the degree of
that ZnO nanoparticle had antifungal effect
sensitivity to Fluconazole is less than 100%,
and
this difference may be due to difference in
antifungal drugs such as Amphotricin B,
population under study or drug purity degree.
Fluconazole, Ketoconazole, and Caspofungin
Moreover, all Candida species showed 97.6%
(p<0.05) and showed
of sensitivity to Nystatin and Itraconazole and
growth halo diameter in some isolated
98.8% to Caspofungin. In 2003, Padua et al
Candida species. In 2011, Husseini et al.
examined antifungal activity of drugs on 400
examined and compared the antifungal effect
secretory samples and reported that sensitivity
of ZnO nanoparticle on inhibiting the growth
pattern of isolates to Fluconazole, Nystatin
of standard strain of Candida albicans with
and Amphotricin B was 96%, 71% and
that of Fluconazole. MIC limit for ZnO
98.8%,
RAFd,
nanoparticle was 1.013-296 mg/ml and for
Guilhermetti E, Svidzinski TIE. 2003]. In
Fluconazole 0.062-128 mg/ml [Hosseini SS ,
2007, Pfaller et al examined 205329 fungus
Roudbar Mohammadi Sh , Joshaghani HR
samples in 40 countries and reported that
, Eskandari M 2011]. This study showed that
nearly 90.1% of all Candida species are
ZnO nanoparticle had antifungal effect and
sensitive
M,
can be used as a proper alternative to remove
Diekema D, Gibbs D, Newell V, Meis J,
Candida species. In 2013, Pasquet et al.
Gould I, et al. 2005]. In 2007, Shirzi et.al
introduced ZnO as a new preservative, with
respectively
to
[Pádua
Fluconazole
[Pfaller
significant
difference
with
other
a considerable no-
2454
IJBPAS, May, 2015, 4(5)
Azadeha Bedzadeh Hajar
Research Article
effective antimicrobial activity on bacteria
nanoparticle will provide physicians with
and fungi [Pasquet J, Chevalier Y, Couval
proper pathological techniques.
E, Bouvier D, Noizet G, Morlière C, et al.
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