Probiotics as treatment for bacterial vaginosis

@
Probiotics as Treatment for
Bactenal Vaginosis
NUTR 302 L
Jeniffer Beadles, Alexandra Salcedo, Ashley Corbett, Mandy Bakker
/bc
Probiotics as Treatment for Bacterial Vaginosis
I.
Specific Aims
A.
Purpose: The purpose of this study is to examine the effects of the probiotic
Lactobacillus acidophilr.rs (found in yogurl) on restoring the vaginal microbiota, maintaining
a desirable vaginai
pH of 3.5-4.8, and treating bacterial vaginosis in women of reproductive
age.
B. Objectives:
To determine if eight weeks of probiotic (Lactobacillus acidophilus) treatment restores the
1.
rnicrobial flora of women suffering from bacterial vaginosis.
To determine if eight weeks of probiotic treatment notmalizes vaginal pH to the range of
2.
3.5-4.8.
To detennine if consuming probiotics regularly is an effective treatment for bacterial
J.
vagmosls.
C. Hypothesis: It is exPected
that consuming the Lactobacillus acidophilrrs found in
yogurt for eight weeks will effectively treat bacterial vaginosis by restoring the vaginal microbial
flora and pH.
II.
Background & Significance
A. Women's Health: The vaginal microbiota is crucial for every woman's general
reproductive health. The vagina's microbial flora is predominantly made of a variety of
Lactobacillus bacteria (such as Lactobacillus acidophilas), but when pathogenic vaginal bacteria
replace the Lactobacillus species, uritogenital infections can occur (3). Additionally, pathogenic
bacteria
will raise vaginal pH to optimal levels for their own growth (pH 6-7), thus rnaking it
difficult for Lactobacilli to grow and re-colonize the area (7). Around one million women
are
infected with urogenital infections annually (6). Furthermore, according to Ling et.al, "bacterial
vaginosis is the most common disorder in women of reproductive age" (5). Because of this, it is
important to research different possible treatments and detennine their efficacy
in
treating
bacterial vaginosis and preventing recurring infections. Research has shown that probiotics can
','A''
be used to treat urogenital infections by introducing Lactobacillus rlrumno.\us and Lactobacillus
.fennentunt into the vagina (3). These probiotics
will
colonize the sunounding tissues and attack
uropathogens such as Gardnet"ella vaginalrs (an organism that commonly causes bacterial
vaginosis) in order to elirninate the infection
(5). This study aims to detemrine if olal ingestion
of Lac:tobacillus acidophilus in yogurt is an effective method to nonnalize vaginal
pH, restore
the vaginal microbiota, and treat bacterial vaginosis.
B.
Probiotics: Thc health benefits of regularly consuming yogurt are numerous. For
example, many consume yogurt because the probiotics it contains aid gastrointestinal function
(1). The effect of yogurt
and probiotics on other areas of the human body has also been
studied. Research has demonstrated that adrninistering probiotic pills aids in preventing and
trcating urovaginal infections (8,6,5,3). Curently, there is not enough evidence or research to
conclude that bacterial vaginosis can be treated and prevented by consuming Lactobacillus
species orally in yogurt products
(8). This study aims to explore this area of research, and
detennine
if
the probiotics found
yogurt (such
as
Lactobacillus ac'idoplilus) can be used
as
an effective treatment for bacterial vaginosis.
C.
Benefits: Antibiotic resistant bacteria
public health
that has continued to
rapidly grow over the years (4). Due to the overuse and misuse of antibiotics by the general
public, pathogens have evolved into antibiotic resistant forms that make medical advances
difficult (4). For example, one study recorded that pathogens in
560/o
of bacterial vaginosis
patients treated with Clindarnycin exhibited resistance to the antibiotic after treatment
(2). In
order to avoid further propagation of this issue, it is important to conduc't more research in
altemative methods of treating bacterial vaginosis and other urogenital infections in women.
Probiotics in yogufi may be a practical and effective way for women to treat and prevent
recuring infections, while also avoiding the overuse of antibiotics.
III.
Research Design & Methods
A.
Subjects: The subjects used for this study will include women between 20-50
years of age. This age group was selected because
it includes most women of reproductive
age,
who are more likely to develop recurring uritogenital infections. In addition, subjects must
cur:rently have bacterial vaginosis. Women who are pregnant, smoke, drink rnore than 4
alcoholic beverages weekly, or are significantly immunocompromised will be excluded from this
study.
B.
Design & Measurement: The study will be conducted as a parallel intervention,
using a control group (pasteurized yogufi placebo) and an intervention group (yogurt containing
Lactobacillus acidophilus). Tbe intelentions will be carried out during eight weeks in order to
ensure that the following parameters are accurately measured: Lactobacillus acidophilus colony
fonning units (CFU's/ml-) within the vaginal flora, Gardnerella vaginalis colony forning units
(CFU's/ml-) within the vaginal flora, and overall pH of the vaginal area.
Data collection
will be conducted according to the
schedule outlined in the following table:
,'V
Table 1. Tirning of Data Collection
DATA COLLECTED/TESTS PERFORMED
WEEK
One week prior to week
I of thc interuention
will
be collected'rn order to incubate
selective medium petri dishes and detenline
CFU's. pH tests will also be perfonned.
Swabs
Week l-8
Subjects will consume 17 .6 oz of yogurt or
placebo daily, as instructed.
Week 9
Subjects will be swabbed and microorganlsms
incubated as perfonned previously, in order to
determine CFU's/ml. Another pH test will be
performed. This data will be compared to the
pre-intervention data in order to determine resu
and analyze the outcomes obtained.
D.
Collection Processing/Storage: The swabs collected will be inoculated onto
selective mediums (within,petri dishes) imrnediately after collection. The inoculated petri dishes
will
be incubated at 35oC for 48 hours in order to allow the organisms to grow for accurate CFU
estimates. After data has been collected, the petri dishes must be discarded and labeled as
biohazardous material. This procedure
post-intervention samples.
will be followed for both pre-intervention
and
I
xl"u''
E.
Procedure: Subjects will be screened in order to ensure they meet the specified
criteria by requiring refenals from rnedical practitioners or lab results indicating the subjects are
infected with bacterial vaginosis. In addition, the subjects
will
regarding their medical history and demographics. Participants
complete a brief questionnaire
will
also sign waivers in order to
remind them of the procedures they are agreeing to follow during the study and what these
procedures consist of. Subiects
will participate as volunteers for the study,
and
will
be recruited
from local health centers and hospitals. Once test subjects have been determined (total of 20)
their vaginal areas will be swabbed twice in order to inoculate petri dishes. The petri dishes will
be incubated at 35"C for 48 hours. Lactobacillus-MRS Agar and HBT Agar
will
be used in the
petri dishes to isolate Lactobacillus acidophilus and Gardnerella vaginalis colonies, respectively,
and detennine
CFU'S/nL in order to estimate the number of each organism present within the
area. Colony fonning units
will
be determined by entering pictures of each incubated petri dish
into the computer program OpenCFU. Additionally, a pH test of the vaginal area will be
performed (using pHEM-ALERT vaginal pH test strips) aftcr the subjccts are swabbed.
At the beginning of week
The twenty subjects
will
1, subjects
will begin consuming the items they were
be divided into two groups of ten people, each assigned a specific item
they must consume daily for the duration of the testing. Group fomrations
and placebo/test yogurt
groups
will
assigned.
will
be randornized
will be administered using the double-blind mcthod. In addition, both
receive the placebo/test yogurt in the same kind of packaging, so neither thc
participants nor the administrators can distinguish between them. The placebo
will
consist of pasteurized
(0% fat FAGE) yogurt which eliminates live cultures, and further ensures that botlt
sarnples have the same appearance. Group
A (intervention group) will consume
fat plain FAGE yogurt per day. Group B (control group)
fat plain FAGE yogurl per day. Both groups
will
will
consum e 17 .6 oz
17
.6 oz of
0o/o
of pasteurized
0o/o
consume a total of 260 calories, 469 protein,
and 18g carboliydrate daily fiom the yogurt. Only Group A
will consume live
and active
L.acidophilas cultures, at a minimum of 100 million cultures per gram, as indicated by the "Live
and
Active" seal on the packaging (a seal issued by the National Yogurt Association to products
that contain the required minimum arnount of live cultures). At the end of.the 8 week period,
subjects
will return for further analysis. Vaginal
areas
will be swabbed, bactcria isolated, CFU's
detennined, and pH tests conducted as described previously.
F.
Statistical Analysis: The collected data frorn both groups will include: Pre and Post
interuention CFU's/rnl of Lactobacillus acidophilus,Pre and Post interuention CFU's/ml- of
Gardnerella vaginalis, Pre and Post intervention pH. The values obtained for Group A and
Group B
will be averaged
and compared in order to detennine
if the intervention
caused
signihcant changes in the subjects. Analysis of the data will allswer the following questions
regarding the paraneters studied:
Did treatment increase/decrease the CFU's/mL of L.acidophilus within the vaginal area?
2) Did treatment increase/decrease CFU's/ml- of G.vaginalrs within the vaginal area?
3) Were vaginal pH values restored to normal, healthy levels? (ph 3.5-a.8)
1)
Significance
will be dctennined at a p-value of p < 0.05 , in order to ensure
obtained as a results of the study, and not by
chance.
data
was
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ry.
Budget:
Item
ltems required
Total Price
Vaginal pH testing strips
(pHEM-ALERT)
4 boxes (12 count box),
$45.95 each
$
Puritan cotton Ob/Gyn swabs
I box (50 count)
$7.89
Fage yogurl, plain, lYo fat, 17.
50 containers, 65 per
person, $5.99 each
$6,888.5
OZ
1,1
Lactobacillus MRS Agar Plate 4 packs (10 count), $10.45
183.8
$41.80
each
4 packs (10 couut) $102.60
HBT Agar Plates
$410.40
each
I
s519.00
Latex powder free-gloves
3 boxes (100 count) $9.25
each
s27.7s
Employee salaries
8 employees, $10.00/hr, 160
$
Therrno scientific
microbiological incubator
12.800
hours total
40 days, M-F during 8 weeks,
$100 daily
$4.000
SDSU Computer Lab Space
40 days, M-F during 8 weeks,
$75 daily
$3,000
H&M commercial Dairy Milk
Pasteurizer HMIX 30Cp-236w
I
$1 1,798
I
$91.7s
SDSU Microbiology Lab
Space (including microscopes
and facilities)
10 Gallon Biohazard
Bin
Total Cost: $39,768.39
Total Budget: $45,000
IV.
References
l. Adolfsson
Oskar, Meydani Simin, and Russel Robert. Yogurt and Gut Function. The
American Journal of Clinical Nutrition. 2004; 80(2):245 -25 6.
Available fiom: ajcn.nutrition.org, Accessed: February I 8th
2. Beigi Richard, Austin Michelle, Meyn Leslie, et al. Antimicrobial resistance associated with
the treatment of bacterial vaginosis. American Journal of Obstetrics and Gynecology. 2004;
19l(4):1124-1129.
Available from: ScienceDirect, Accessed: February
l8th
i
3. Burton Jeremy P., Peter A. Cadieux, and Gregor Reid. Improved Understanding of the
Bacterial Vaginal Microbiota of Women before and after Probiotic Instillation. Applied and
Environmental Microbiology. 2003 ; 69 (l)
:97 - I 0 |
.
Available from: American Society for Microbiology, Accessed: February 4th
4. Levy Stuart. Factors impacting on the problem of antibiotic resistance . Journal of
Antimicrobial Chemotherapy. 2002;a9 ()
:25 -3 0
.
Available on: Oxford Journals, Accessed: February 18th.
5.
Ling Zongxin, Chen Weiguang, Luo Yueqiu, et al. The Restoration of the Vaginal Microbiota
After Treatment for Bacterial Vaginosis with Metronidazole or Probiotics. Microbial Ecology.
2013; 65(3):773-780.
Available on: Springer Link, Accessed: February 4th
6. Reid G, Owen J, Heinemann C, Henning B, Fraser N, Bruce
AW.
Oral probiotics can resolve
urogenital infections. FEMS Immunology & Medical Microbiology. 200I;30(l):49-52.
Available on: PubMed, Accessed: February 3rd
7. Robinson Taylor. The relation between vaginal pH and the microbial status in vaginitis.
International Journal of Obstetrics and Gynecology. 1985;92(12):1267 -1271 .
Available on: Wiley Online Library, Accessed: February 18th
8. Ya Wang
MD, Reifer Cheryl PhD, MillerLarry PhD. Efficacy of vaginal probiotic capsules
for recurrent bacterial vaginosis: a double-blind, randomized, placebo-controlled study.
American Journal of Obstetrics and Gynecology. 20 l0 ; 203(2):120-121
Available on: PubMed, Accessed: February 5th
.