Original Article

Diagnostic study of fungal and bacterial pathogens that cause

vaginal infection in women
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Heba Hadid Rashed, Ibtihal Muiz Al-Hussaini, Ali H. Al-Marzoqi

Department of Biology, College of Science, University Babylon, Babil, Iraq
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Abstract
Background: Vaginal infection is an inflammation of the vagina that can result in discharge, itching, and pain. A specific kind of
vaginal inflammation known as bacterial vaginosis is roughing on by an overabundance of naturally occurring bacteria in the vagina,
which throws the body’s delicate equilibrium out of whack. Vaginal candidiasis is a fungal infection that affects the vagina and the
vulva, the tissues at the vaginal opening, causing irritation, discharge, and severe itching. Objectives: Isolation, identification, and
partial purification of bacteria and fungi that cause vaginal infection. Materials and Methods: In this study, 150 vaginal samples were
collected from women aged 15–55 who did not have any chronic diseases in the city of Hilla, Iraq. Cultivation of samples of bacteria on
MacConkey, mannitol salt, and Eosin-methylene blue culture media and fungi on potato dextrose agar medium and chromo agar for
Candida and conducting diagnostic tests for them. Results: The samples were culture on different medium. Bacteria have a percentage
(55.3%) and fungi percentage (54.7%). Types of bacteria that appeared are Escherichia coli 24.3%, Klebsiella 20%, and Staphylococcus
aureus 55.7%. Fungi are Candida albicans 52.6%, Candida glabrata 23.7%, and Candida krusie 23.7% have all been identified as fungi in
clinical cases. Biochemical tests were performed for each sample, whether bacteria or fungi and included an examination (indol, citrate,
urease, catalase, oxidase, coagulase, and H2S). Conclusion: Through results appeared S. aureus papered was found to be more common
than bacteria species and fungi in clinical cases. C. albicans was found to be more common than other Candida species.

Keywords: Bacteria pathogens, Candida, catalase test, oxidase test, coagulase test, fermentation test, biochemical tests

Introduction their lifetime. The vaginal disease can be infectious,

noninfectious, and chronic in nature. Infectious vaginitis
Vaginitis is an inflammation of the vagina that can cause
is most commonly grouped into bacterial vaginitis and
discharge, itching, and pain. This is due to the constant
vaginal candidiasis.[3]
change in vaginal bacteria. It is also caused by a drop in
estrogen levels after menstruation and certain skin types. The polymorphic opportunistic fungus Candida albicans is
primarily responsible for the extremely common mucosal
Lactobacilli predominate in the bacterial flora of a healthy
infection of the lower female reproductive tract known
reproductive-age female’s vagina.[1] In a healthy woman,
as vulvovaginal candidiasis (VVC). C. albicans, a typical
vaginal microbes are mutually antagonistic and dependent
component of the human microbiota, frequently colonizes
on one another, maintaining a dynamic equilibrium that
the vaginal lumen asymptomatically. However, exuberant
is controlled by the endocrine system, the local immune
mucosal inflammation that is predominantly brought
system, and the internal environment of the vagina.[2] The
on by fungal overgrowth in the vagina and followed by
age of the woman, how her hormones are managed, her
sexual activity, and her exposure to hygiene agents used in
vaginal irrigation can all have a significant impact on the
Address for correspondence: Dr. Heba Hadid Rashed,
microbiota makeup. Vaginitis is one of the most common Department of Biology, College of Science, University Babylon, Hilla, Iraq.
diseases that affect women’s health; over 50% of women E-mail: hebahaded1@gmail.com; sci.heba.haded@uobabylon.edu.iq
experience at least one episode of vaginal infection in
Submission: 31-Jan-2023 Accepted: 13-Mar-2023 Published: 09-May-2024

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DOI: How to cite this article: Rashed HH, Al-Hussaini IM, Al-Marzoqi AH.
10.4103/MJBL.MJBL_104_23 Diagnostic study of fungal and bacterial pathogens that cause vaginal
infection in women. Med J Babylon 2024;21:71-8.

      ©
 2024 Medical Journal of Babylon | Published by Wolters Kluwer - Medknow 71  
 Rashed, et al.: Fungal and bacterial that cause Vaginitis
epithelial invasion and the synthesis of virulence effectors
can result in symptomatic infection. Itching, burning,
soreness, and redness in the vagina are typical illness
signs. These frequently come with a vaginal discharge that
contains immune cells, yeast, sloughed epithelium, and
vaginal fluid. VVC is the most common human candidal
infection, affecting 75% of women at some point in their
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lifetimes; according to estimates, four risk factors for VVC
are the use of antibiotics, sexual activity, high-estrogen-
containing oral contraceptives, pregnancy, use of sodium–
glucose transporter 2 inhibitors, and uncontrolled diabetes
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mellitus.[4]
One of the most frequent vaginal disorders linked to
abnormal alterations in the vaginal microbiome is bacterial
vaginosis (BV), also known as vaginal dysbiosis.[5]
A decrease in the local Lactobacillus species that produce
lactic acid and an increase in anaerobic bacteria are two
features of BV.[6]
Materials and Methods
Sample collection
One hindered fifty clinical samples were taken from female
patients using a sterile swab. Bacteria and fungi culture
media samples were collected with the assistance of a
gynecologist. All of the women in the study were married
women between the ages of 16 and 55.[7]
Reagent and culture media
Culture media
All cultures were prepared according to the manufacturers.
Catalase reagent was prepared by mixing 100 ml of
distilled Water with 3% H2O2, then store it in a dark-
colored bottle. The reagent was used to identify the ability
of bacteria and Candida to generate catalase enzymes.[8]
Oxidase reagent
This reagent was ready-made as filter paper discs called
oxidase discs that contained tetramethyl-paraphenylene
diamine dihydrochloride. They were kept in a dark and
cool environment. When the color of the smear changed
to purple, the oxidase test was positive. A small fraction
of the bacterial colonies were dispersed on the disc with
a wooden stick. Tetramethyl-p-phenylene-diamine-
dihydrochloride, a redox dye, and the presence of a
specific bacterial oxidase that would catalyze the transit of
electrons between electron donors in bacteria are required
for the test.[9]
Coagulase test
The test is a rabbit’s plasma that has been vaccinated with
Staphylococcus bacteria. The tube is incubated at 37°C for
an hour and a half. The result is negative; the result is 18 h.
The result is positive, and the plasma will coagulate and
appear in the form of collected pieces.[10]
72 72  Medical Journal of Babylon ¦ Volume 21 ¦ Issue 1 ¦ January-March 2024
Stains
Lacto phenol stain
The dye was used to color the chlamydospores to make
them appear more visible under the microscope and to
germ tube.
Formation of germ tube test
It was taken 0.5 mL of human serum after separating
blood components in a centrifuge and blended well with
a colony of yeast isolates from the medium and incubated
at 37°C for 3–4 h, and then took a drop of the mixture
and placed on a slide with a drop of dye lacto phenol
cotton blue and examined under a microscope to detect
the species of yeast that component the germ tube, this is
a rapid diagnostic test for the C. albicans from other yeasts
species.[11]
Ethical approval
The study was conducted in accordance with the ethical
principles that have their origin in the Declaration of
Helsinki. The patients’ permission was obtained before
sampling. The study protocol, subject information, and
consent form were reviewed and approved by a local ethics
committee according to document number 7/17/1336 on
February 21, 2022.
Results
Isolation and diagnosis of the bacteria and fungi
pathogens in ``vaginitis"
The samples were culture on different medium, where 141
bacterial isolates were found. After that, it was diagnosed
whether it was gram-positive or gram-negative by
culturing it on special culture media. Then a microscopic
examination was performed for each sample where the
following types appeared that are positive for a gram stain
(Staphylococcus aureus and Staphylococcus epidermidis)
and negative for a gram stain (Escherichia coli, Klebsiella
pneumonia pneumonia). These media include blood agar,
MacConkey agar, Eosin-methylene blue (EMB) agar,
Nutrient agar.
A gram-positive bacteria such as Staphylococcus aureus
was isolated by culturing it on a Mannitol salt agar
medium.
After plotting the sample on MacConkey medium,
single, dry, circular pink colonies appeared fermented
with lactose sugar. These characteristics apply to E. coli
bacteria and 114 fungi yeasts that were isolated, including
Candida tropicalis, C. albicans, Candida glabrata, and
Candida krusie, as shown in Tables 1–4.
Figure 1 shows E. coli and Klebsiella growth on EMB.
In addition, a sample was diagnosed by culturing it on a
Mannitol salt agar medium. Figure 2 show S. aureus on
Mannitol salt agar.
 Rashed, et al.: Fungal and bacterial that cause Vaginitis

Table 1: Total number of samples, the kind of microorganisms, Table 3: Type of bacteria isolated from vaginal sample
and the percentage of isolates Number Percentage
Type of Number of Percentage of Gram-positive
microorganisms isolates isolates Staphylococcus 117 55.7
Bacteria 141 55.3 Gram-negative
Fungi 114 44.7 Klebsiella spp. 51 24.3
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Total microorganisms 255 100 Escherichia coli 42 20

Total number 210 100

Table 2: Gram-positive and gram-negative bacteria isolated

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from vaginal swap Table 4: Type of fungi isolated from vaginal sample
Type of bacteria Number Percentage Type of fungi Number Percentage
Gram-positive 138 50 C. krusie 27 23.7
Gram-negative 138 50 C. glabrata 27 23.7
Total number 276 100 C. albicans 60 52.6
Total number 114 100

Figure 1: Gram-negative bacteria on EMB (A) control, (B) E. coli, (c) Klebsiella

Figure 2: Mannitol salt agar (A) control, (B) S. aureus

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 Rashed, et al.: Fungal and bacterial that cause Vaginitis
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Figure 3: Blood agar (A) control, (B) E. coli, (C) Klebsiella, (D) S. aureus

Figure 4: Indol test (A) control, (B) S. aureus, (C) E. coli, (D) Klebsieala, (E) S. epdermides

Figure 3 shows the ability of bacteria E. coli, Klebsiella,
and S. aureus to hemolysis of blood.
Through this research, we found the different bacterial
species that were isolated differed in their ability to produce
the enzyme tryptophanase; that’s why we found E. coli and
S. aureus to be positive for the indol test while Klebsiella is
negative for the indole test, as shown in Figure 4.
To ascertain an organism’s capacity to transform
tryptophan into indole, a biochemical test known as an
“indole test” is carried out on a variety of microorganisms.
This division is carried out by a cascade of numerous
separate intracellular enzymes, or “tryptophanase,” which
is the name given to this system.
Through the samples that were isolated and diagnosed,
it was found that some bacterial species, Klebsiella and
S. aureus positive for Simmon citrate and E. coli, negative
for this test, as shown in Figure 5.
Figure 6 shows the Kligler iron test; all bacteria are
positive (S. aureus, E. coli, Klebsiella, and S. epidermis).
The samples were diagnosed, and all bacterial species that
were isolated and diagnosed were positive for the catalase
test, such as E. coli, S. aureus, and Klebsiella, as shown in
Figure 7.
Figure 8 shows the oxidase test; all bacteria are positive for
the oxidase test. The E. coli and Klebsiella were positive
for the oxidase test, while S. aureus was negative for this
test, as shown in Figure 8.
The diagnosis of S. aureus was made using the
coagulase test, and this test was found to be positive
only for S. aureus, while the other samples were negative
for this test, such as E. coli, Klebsiella, as shown in
Figure 9
The morphology and color of colonies on CHROMagar
were used to identify the yeast cells in Figure 10.

      
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Figure 5: Simmon citrate test (A) control, (B) S. aureus, (C) E. coli, (D) Klebsieala, (E) S. epdermides

Figure 6: Kligler iron test (A) control, (B) S. aureus, (C) E. coli, (D) Klebsieala, (E) S. epdermides

Figure 7: Catalase test (A) E. coli, (B) Klebsiella, (C) S. aureus

Figure 8: Oxidase test

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 Rashed, et al.: Fungal and bacterial that cause Vaginitis

Hemolysis test create enzymes, such as hemolysin, is a crucial virulence

The purpose of this research was to assess the hemolytic factor (C. albicans, C. krusie, C. glabrata, and C. tropical).
activity amongst Candida species, where a positive result
was found for all species [Figure 11]. Candida’s ability to Figure 12 shows all isolates are positive for the catalase test.
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Figure 9: Coagulase test

Figure 10: CHROMagar (A) C. albicans, (B) C. krusie, (C) C. tropical

Figure 11: Blood agar (A) C. krusie, (B) C. albicans, (C) C. tropical [Figure 10]

Figure 12: Catalase test

      
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Figure 13: Oxidase test
Figure 13 shows all isolates are negative for testing oxidase.
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The cytochrome C oxidase or cytochrome oxidase test is
a procedure for determining the existence of the terminal
enzyme system in aerobic respiration.
Discussion
Tables 1–4 show clearly that the vagina contains bacteria
and fungi in different proportions. This has been proven
in a number of studies include. Instead, a complex
combination of numerous factors, including the various
elements of the vaginal microbial ecosystem and their
human host, results in BV. Many of these characteristics
still need to be described because developing an effective
treatment and prophylactic for this condition depends
on having a good understanding of their respective
contributions to the genesis of BV.[12]
Most of the studies mention that C. albicans is the most
prevalent and the most more than BV. It is considered
a normal flora organism but the variation of the
gastrointestinal tract and reproductive tract, and the
different varieties of exogenous factors from predisposed
monarchal women to acute vulvovaginitis candidiasis,
pregnancy situation, lacteal phase of the menstrual cycle,
hormone change to oral estrogen with high dose and the side
effect of antibiotic and variation of diabetes mellitus.[13] The
sample was also cultured on MacConkey’s medium, so the
colonies appeared in a single violet color, moist and mucous
in texture. This differs from colonies of the intestinal coli.
The bacteria were cultivated on differential media, as
shown by Figures 1–3, as it is considered one of the
methods of diagnosis, and this was confirmed by a
lot of research, including.[14-18] Kligler test shows the
susceptibility of bacteria to produce H2S and gas on
Kligler iron agar and fermentation tests for nine different
saccharides, namely glucose, sucrose, lactose, mannitol,
xylose, rhamnose, mannose, dextrose, and maltose.[19]
A common enzyme called catalase is present in almost
all living things that are exposed to oxygen, including
bacteria, plants, and animals. It catalyzes the breakdown
of hydrogen peroxide into water and oxygen. It is a crucial
enzyme for defending the cell against reactive oxygen
species’ oxidative damage. One catalase molecule may
convert millions of hydrogen peroxide molecule.[20]
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E. coli and Klebsiella species isolates were identified with
a negative oxidase test.[21] S. aureus is Biochemically, they
are catalase-positive and oxidase-negative.[22]
S. aureus produces the coagulase enzyme, which turns
plasma’s (soluble) fibrinogen into (insoluble) fibrin. This
test helps distinguish S. aureus from other staphylococci
since they do not produce coagulase. Coagulase comes in
two varieties: free coagulase and bound coagulase. It is
discovered via various techniques. A slide test can quickly
identify the clumping factor, also known as the bound
coagulase. The creation of a clot, in turn, allows for the
detection of free coagulase in the test tube.[23]
According to the color key putted by the differentiation
in colors belonging to the containing species of this
agar, chromosomal material interacts with an enzyme
called hexosaminidase, which is secreted by the same
species, and this results in a quick diagnosis based
on the color and appearance of the colony.[24] The
advantages of CHROMagar are easy to prepare,
boiling, and dispensing in petri plates, and facilitates
the fast isolation and identification of yeast species.
CHROMagar facilitates identification between Candida
spp. From specimens containing a mixture of Candida
spp. in the present study do not affect the viability on
subsequent subcultures. CHROMagar has the advantage
of rapid identification of Candida species, technically
simple, quick, and cost-effective compared to the
technically demanding, time-consuming, and expensive
conventional method.[25]
Figure 10 shows CHROMagar medium was used to
diagnose Candida species due to its positive properties
agreed upon by a number of researchers.
Table 5 shows a biochemical test, where he explained
the ability of some types of Candida to produce certain
enzymes, and this was explained in a number of research
papers. The C. albicans, C. krusie, C. glabrata, and
C. tropical are capable of analyzing blood. An essential
component of virulence known as hemolytic Capability
enables the Candidato genus of fungi to absorb iron from
host tissues for use in metabolism, growth, and invasion
during host infection. For practically all species, both
unicellular and multicellular, iron is a necessary element[26]
ability Catalase produces agreement with.[27] The result of
77  
 Rashed, et al.: Fungal and bacterial that cause Vaginitis
Table 5: Biochemical test for some Candida species
Candida Ureaes Catalase
C. albicans – +
C. krusie – +
C. glabrata – +
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oxidase agreed with[28] that which explained the inability
of Candida to produce it.
Conclusion
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Through results appeared S. aureus papered found to be
more common than bacteria species and fungi in clinical
cases. C. albicans was found to be more common than
other Candida species. All samples have the ability to do
blood analysis and have many virulence vectors.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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