CHAPTER ONE

1.1 INTRODUCTION

1.2 Background of the Study

Antimicrobial resistance is a global concern, particularly pressing in developing nations

where infectious diseases, poverty and malnutrition are endemic. Infections caused by resistant

bacteria have been shown to be more frequently associated with increased morbidity and

mortality than those caused by susceptible pathogens. In areas of concentrated use, such as

hospitals, antimicrobial resistance lead to hospital stays, increased health care costs and in

extreme cases untreatable infections. The lack of clinical microbiology laboratories to identify

the specific etiologic agents and their antimicrobial susceptibility testing has increased empirical

therapy which in turn leads to emergence of AMR. Moreover, self-antibiotic prescription, lack of

access to local antibiogram data and poor awareness of prescriber about AMR were the leading

local factors for AMR development in Ethiopia (Aberaet al., 2014).

Studies have shown that besides the temporal changes in profile of infecting

microorganisms and pattern of resistance over time, antimicrobial resistance profile of bacteria

varies among population because of difference in geography, local antimicrobial prescribing

practices and prevalence of resistant bacterial strains. Such differences are never stable and may

change rapidly especially in places where misuse of antibiotics are common particularly in

developing countries. A systematic review in Ethiopia has also indicated a trend towards an

increasing resistance rates among pathogens such as Escherichia coli, Proteus, Klebsiella,

Pseudomonas,Citrobacter and Acenotobacter to commonly prescribed antibiotics, including

Ampicillin, Amoxicillin, Amikasin, Imipenem, Cefixime and Ciprofloxacin (Moges et al.,2014).

1
Thus, up to date information on microbial resistance is needed at local level to guide the rational

use of the existing antimicrobials.

The adult human vagina is a complex biota containing a profusion of microorganisms. These

can be either unicellular or multicellular and are present everywhere in nature. They include

bacteria, fungi, archaea, protists, some microscopic plants such as green algae and animals

such as planktons and palanarian. On account of their nature, viruses may or may not be

included. Bacteria and yeast form normal flora of this ecosystem, which is normally

found on the skin and every opening of the body such as mouth, ears, rectum and vagina. Even a

neonate carries specific flora of his/ her mother and soon develops own floral community.

This flora persists till death of the individual. An adult human carries normal flora consisting of

more than 200 bacterial species. Normally these are harmless and are involved in benefiting

their hosts. Yet some are parasitic in nature, living at the expense of their host, and some are

even pathogenic.These pathogenic microbes, after getting a chance, invade their hosts and

lead to opportunistic infection. These diseases caused by normalflora are termed endogenous

diseases (Khan et at.,2002).

Resistance of bacteria to antimicrobial agents is an imminent threat to patient

management all over the world. This issue has plagued policy makers and clinicians everywhere

but there seems to be no simple way of circumventing the problem. Rapidly rising antibiotic

resistance is a challenge to comprehensive patient care in all branches of medical science. The

interaction between various clinical bacteria and the antimicrobial agents is a complex issue

involving the prokaryotic adaptive mechanisms and genetic changes. This complex interaction

must be studied in depth in order to achieve a sustainable and effective solution to the looming

threat of antibiotic resistance. Earlier, the problem of antibiotic resistance was primarily a

2
concern for not so comical infections. But now, even community acquired infections are caused

by organisms with high levels of antibiotic resistance. As a report had demonstrated, such multi-

drug resistant community acquired infections can be a cause of significant.

Earlier, such drug resistant organisms were said to infect mainly patients with identifiable

risk factors or profound immune suppression. But now, reports are showing such infections in

seemingly normal healthy persons. Also, such drug-resistant infections may complicate the

newly emerging infectious diseases. For example, influenza epidemics are sometimes reported to

be complicated by superadded infection with drug-resistant bacteria (Hageman et al., 2004). The

issue of drug resistance in clinical bacteria is such a vital threat that the UN held a special

assembly in 2016 to address only this issue. In that assembly, the issue was said to be of as much

importance as climate change and it was deemed to require a global response (Farr, 1994) and

non-pregnant women attending the University of Maiduguri Teaching Hospital (UMTH),

Maiduguri, Nigeria”.

1.3 Antibiotic Sensitivity

Antibiotic sensitivity is a term used to describe the susceptibility of bacteria to

antibiotics. Antibiotic sensitivity testing (AST) is usually carried out to determine which

antibiotic will be most successful in treating a bacterial infection in vivo. Testing for

antibiotic sensitivity is often done by the Kirby-Bauer method while other methods

include the Stokes method, E-test (also based on antibiotic diffusion) and Agar and

Broth dilution methods (for Minimum Inhibitory Concentration determination). Muller

Hinton agar is most frequently used in this antibiotic susceptibility test. Our study was aimed at

the isolation, identification and antibiotic sensitivity testing of URINARY TRACT

INFECTION (UTI) causing bacteria.

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 1.4 AIM

To isolate, identify and check antibiotic sensitivity of bacteria implicated in URINARY TRACT

INFECTION (UTI)s.

1.5 OBJECTIVES OF THE STUDY

The main objective is to determine the effect of anti-biotics on bacteria isolate from human vaginal
discharge. The specific objective are:

 To isolate bacteria from the urinary tract of women of child-bearing age using vaginal

swab.

 To identify the isolated bacteria.

 To determine the antibiotic sensitivity of bacteria isolated.

1.6 Research Questions

To guide this study, the following research questions will be addressed:

i. What are the predominant bacterial species present in vaginal discharge?

ii. How do these bacterial isolates respond to commonly prescribed antibiotics?

iii. Are there variations in bacterial susceptibility to antibiotics among different isolates?

1.7 Research Hypothesis

1.8 Significant of the Study

This study will focus on the isolation and sensitivity of bacteria from vaginal discharge samples

obtained from women presenting with symptoms of bacterial infections. The research will

encompass the identification of bacterial species and the assessment of their susceptibility to a

range of antibiotics commonly used in clinical practice.

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1.9 Limitation of the Study

The thesis will be organized into distinct chapters to facilitate a comprehensive exploration of the

topic. Chapter Two will review relevant literature, providing a foundation for understanding the

microbial ecology of the vagina and the current landscape of antibiotic resistance. Subsequent

chapters will delve into the methodology employed, the presentation and analysis of results, and a

discussion of findings. The thesis will conclude with Chapter Five, summarizing key insights and

proposing potential implications for clinical practice and future research.

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 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Conceptual Framework

Antibiotic resistance was reported to occur when a drug loses its ability to inhibit bacterial growth

effectively. Bacteria become ‘resistant’ and continue to multiply in the presence of therapeutic

levels of the antibiotics. Bacteria, which replicate even in the presence of the antibiotics, are called

resistant bacteria.

Antibiotics are usually effective against them, but when the microbes become less sensitive or

resistant, it requires a higher than the normal concentration of the same drug to have an effect. The

emergence of antimicrobial resistance was observed shortly after the introduction of new

antimicrobial compounds. Antibiotic resistance can occur as a natural selection process where

nature empowers all bacteria with some degree of low-level resistance. For example, one study

confirmed that ampicillin and tetracycline that were commonly used in yesteryears, but now have

no longer role in treating non-cholera diarrhea disease in Thailand (Hoge et al.,1998). At the same

time, another study conducted in Bangladesh showed the effectiveness of the same drugs in treating

them effectively (Rahman et al., 2017). In fact, resistance was documented even before the

beginning of the usage of the antibiotics in fighting the infection (Abraham and Chain, 1940). Non-

judicial use of antibiotic is responsible for making microbes resistant. Since the introduction of

sulfonamides in 1937, the development of specific mechanisms of resistance had provoked their

therapeutic use. However, sulfonamide resistance was reported in the 1930s, which reveals the

same mechanism of resistance that still operates even now, more than 80 years later). Within six

years of the production of the aminoglycosides, aminoglycoside-resistant strains of

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2.1.2 Conceptual Review

Staphylococcus aureus was developed (Gootz , 1990). Introduced in 1961, Methicillin was the first

of the semisynthetic penicillinase-resistant penicillin to target strains of penicillinase- producing

Staphylococcus aureus. However, resistance to methicillin was reported soon after its initiation (Br

Med , 1961). Further, although fluoroquinolones were introduced for the treatment

of Gram-negative bacterial diseases in the 1980s, fluoroquinolones resistance later revealed that

these drugs were also used to treat Gram-positive infections. Most recently, the clinical isolates of

Vancomycin-resistant Staphylococcus aureus (VRSA) were found in 2002, after 44 years of

Vancomycin introduction to the market. Antibiotics used in agriculture are often the same or

similar to antibiotic compounds used clinically (McEwen and FeDorka-Cray , 2002), this over-

usage could also invite drug resistance. The food chain can be considered the main route of

transmission of antibiotic-resistant bacteria between animal and human populations (Witte , 1998).

In some developed countries, animals receive antibiotics in their food, water, or parenterally which

may be responsible for carrying microbe resistance to that specific antibiotic (McEwen and Fe

Dorka-Cray, 2002). For example, the use of antibiotics in cattle feed as growth promoters increase

antibiotic resistance (Levy , 1993). Recent evidence suggests that poultry or pork might be a

possible source of quinolone resistant-Escherichia coli in the rural villages in Barcelona, where

one-fourth of children were found to be fecal carriers of these organisms. However, these kids were

never exposed to quinolones (Garan, et al.,1998)

2.1.3 Development of antibiotic resistance

Antibiotics fight to eliminate bacteria. Hence, bacteria tend to have a natural process that

encourages resistance. The resistance process occurs via gene level mutations. (Laxminarayan and

Brown, 2001). Antibiotics induce selective pressure and the genes act in association with selective

7
pressure (Levy, 1993). Bacteria possess the quality to directly transfer genetic material between

each other by transferring plasmids, which signifies that natural selection is not the only

mechanism by which resistance evolves. Broad spectrum antibiotics are prescribed in hospitals

as a solution for nosocomial infections; however, it increases resistance (Lowey, 2003).

Antibiotics can generally eliminate the majority of bacteria in a colony. However, there may exist a different

colony of bacteria that are genetically mutated which can lead to resistance (Alanis, 2005). The level of antibiotic-

resistant infections was found to be strongly correlated with the degree of antibiotic consumption (Goossens, et al.,

2005). Development of resistance may also likely to occur if users fail to take their full course of prescribed

antibiotic treatment. The bacteria subsequently remain untouched gaining more strength against the antibiotics

(Levy, 1993). Bacteria may collect multiple resistance traits over time and can become resistant to multiple classes

of antibiotics (Avon, et al., 2001). For example, resistance was found in Staphylococci from the chromosomal

mutations, ineffective transport of aminoglycosides into the bacteria as well as enzyme modification (Lowey,

2003). A single antibiotic may not only select resistance to one particular drug. Resistance can occur with other

structurally related compounds of the same class. For example, resistance to tetracycline may incur resistance to

oxytetracycline, chlortetracycline, doxycycline, and minocycline (Chopra and Roberts, 2001). Antimicrobials

possessed resistance genes that defend their antimicrobial products and these genes developed antibiotic resistance

even long ago before the antibiotic started working for treatment purpose (Chadwick and Goode, 1997).

Antimicrobials – including antibiotics, antivirals, antifungals, and antiparasitics – are medicines used to

prevent and treat infectious diseases in humans, animals and plants. Antimicrobial Resistance (AMR)

occurs when bacteria, viruses, fungi and parasites no longer respond to antimicrobial medicines. As a

result of drug resistance, antibiotics and other antimicrobial medicines become ineffective and infections

become difficult or impossible to treat, increasing the risk of disease spread, severe illness, disability and

death. AMR is a natural process that happens over time through genetic changes in pathogens. Its

emergence and spread is accelerated by human activity, mainly the misuse and overuse of antimicrobials

to treat, prevent or control infections in humans, animals and plants.

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Antimicrobial medicines are the cornerstone of modern medicine. The emergence and spread of drug-

resistant pathogens threatens our ability to treat common infections and to perform life-saving procedures

including cancer chemotherapy and caesarean section, hip replacements, organ transplantation and other

surgeries.

AMR has significant costs for both health systems and national economies overall. For example, it creates

need for more expensive and intensive care, affects productivity of patients or their caregivers through

prolonged hospital stays, and harms agricultural productivity. AMR is a problem for all countries at all

income levels. Its spread does not recognize country borders. Contributing factors include lack of access

to clean water, sanitation and hygiene (WASH) for both humans and animals; poor infection and disease

prevention and control in homes, healthcare facilities and farms; poor access to quality and affordable

vaccines, diagnostics and medicines; lack of awareness and knowledge; and lack of enforcement of

relevant legislation. People living in low-resource settings and vulnerable populations are especially

impacted by both the drivers and consequences of AMR

2.1.4 Colonization and Transmission of urinary tract infection (UTI)

Urinary tract is regularly flushed with sterile urine and its acidity, there it makes difficulty for

microbes to access and establish here but anterior urethra is inhabited by relatively constant flora

such as Staphylococcus epidermidis, Enterococcus faecalis and some Alpha-hemolytic streptococci

(Pubus and Enderdonk, 1999)

Occasionally, some enteric bacteria like E. coli, Proteusand corynebacteria may be found there.

Vagina is an available space for certain microbes. It is colonized with Corynebacteria,

Staphylococci, Streptococci, E. coli, and Doderlein’s bacillus (Lactobacillus acidophilus).

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During reproductive life vaginal epithelium contains glycogen which is metabolized by

Lactobacillus acidophilus. The lactic acid and other products of metabolism inhibit colonization

of offending agents in that area and allow only Lactobacillus acidophilus and some other lactic

acid producing species to grow(Noskan et at., 2005)

Oral contraceptives, steroids, and antibiotics disrupt either the normal flora or naturally acidic Phof

the urinary tract. Reduction in lactic acid leads to high pH of vaginal epithelium which

encouragesother offending agents to grow and results in embarrassing vaginal odour (sometimes

described as smelling “fishy”), abnormal discharge (often thin and white-grey in colour) and

discomfort (normally irritation or soreness in and around vagina). Vaginal discharge is a term

given to the biological fluids contained within or expelled from vagina. Normally this discharge

demonstrates various phases of menstrual cycle but it may also be due to vaginal infections. That

is why vaginal infection is checked by the presence or absence of these offending microorganisms

in a vaginal discharge. The three major kinds of vaginitis are vaginal candidiasis, bacterial

vaginosis(BV) and Trichomoniasis.

At a time, a woman may have any vaginal co-infections. Untreated vaginal infection may

lead to any complication especially in pregnant women (Momoh et tal., 2007)

2.1.5 Consequence of antibiotic resistance

Antibiotic resistant organisms are known as superbugs. These are not only a laboratory concern but

have become a global threat responsible for high death tolls and life-threatening infections (Lipp, et

al., 2002). Consequences of these infections are aggravated enormously in volatile situations such

as civil unrest, violence, famine and natural disaster (Fact Sheet, 2015). World Health Organization

(WHO) (Fact Sheet, 2015) has warned that a post-antibiotic era will result in frequent infections

and small injuries may result in death if we fail to act against antibiotic resistance; Multi-drug
10
resistant bacteria causing more deaths worldwide. More than 63,000 patients from the United States

of America (USA) die every year from hospital-acquired bacterial infections (Aminov and

MackieI, 2007). Every year, an estimated 25,000 patients die due to multiple drug resistance

(MDR) bacterial infections in Europe (Freire-Moran ,et al., 2011). Many countries are facing

the burden of nosocomial Staphylococcus aureus (S. Aureus) infections as waves of clonal

dissemination. Methicillin-resistant Staphylococcus aureus (MRSA) strains are rapidly spreading

globally (Lowey, 2003). Estimated costs due to multidrug- resistant bacterial infection might result

in extra healthcare costs and productivity losses (Freire- moran, et al., 2011). It has been a standard

practice for most of the pharmaceutical companies to distribute antibiotics that may no longer be

effective or lacking regulatory approval (Levy and Marshall, 2004). Evidence shows that increased

antibiotic use may result in a positive association with a higher prevalence of resistant

microorganisms, while reduced antibiotic use showed lower resistance rates. There is clear

evidence that patients historically treated with antibiotics are more likely to have antibiotic

resistance (Laximinarayan R and Brown, 2001).

Further, re-administration of antibiotics from the initial cycle accelerates resistance mechanisms

(Anderson, 1991). Antibiotics encourage selective pressure for bacteria to evolve when

administered frequently or irrationally. Individuals and states play a role in the evolution of

antibiotic resistance (Laxminarayan and Brown, 2001). For example, Clarithromycin consumption

and its resistance similarly increased fourfold in Japan between 1993 and 2000 in comparison to

other countries (Perez et al., 2002).

2.1.6 Regulatory issues related to antibiotic resistance

Congruent international management guidelines for daily antibiotic practices are yet unavailable.

Hence, regulatory guidelines vary in different countries. Some countries have acted swiftly offering

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guidance e.g. United Kingdom, while other nations have yet to move toward interventions. The

WHO has offered recommendations such as children in developing countries that antibiotics should

only be used for the treatment of severe bloody diarrhea and cholera (WHO. CAH. Geneva, 1995).

Since the beginning of the industrial revolution, we have dumped increase amounts of organic and

inorganic toxins into streams, rivers, oceans, land, and air. In the personal care industry, there are

insufficient guidelines for monitoring the home hygiene products which are likely to cause more

risk for resistance because these products contain a high concentration of antibacterial ingredients

(Laxminarayan and Brown, 2001).

With an abundance of evidence, there is no scope to ignore global antibiotic resistance. Antibiotic

resistance can be more prevalent where antibiotic consumption is found to be higher. Lack of

regulation and control in using antibiotics is prominent and needs to be targeted at a global

capacity. Developing nations are at the greatest risk. Low prices of antibiotics, ease of availability

and unnecessary use of antibiotics are causing more burdens in developing countries (Levy and

Marshall, 2004). Antibiotic use is relatively uncontrolled among the countries where there is no

universal health coverage for its citizens (Zaman and Hossain, 2017). Hence, irrational use of

drugs has become a major concern. According to a study done in the United Kingdom, among the

participants, 11.3% reported that they did not finish their last antibiotic course as prescribed. When

asked about the reason why not comply with the course, 65% of the respondents stated that they felt

better or forgot to take an antibiotic in time (Woodhead and Finch, 2007).

We are all affected by this multi-face ted public health issue. An all-encompassing problem that

doesn’t just pertain to clinical personnel and microbiologists, but service personnel, industry

stakeholders, specialists and the general public. We have to take necessary steps to tackle this

complicated challenge. Social awareness, motivation, commitment in responsible sectors, stringent

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rules and regulation have to be prioritized. Further, we need the coupled action for the proper

Urinary Tract Infection(UTi)lization of antibiotics, best management practices, and behavioral

shifts across all industries that we can then combat against this public health burden. Application of

modern technology can help the patient to take the antibiotic timely (Zaman , 2017). At present, the

most notorious superbug is the Gram-positive organism Staphylococcus aureus (Lowey , 2003).

This pathogen is frightening as its resistance to antibiotics is dramatically increasing. With an

intimate history so closely tied to humans, Staphylococcus aureus is feared and at times,

misunderstood (Lowey , 2003). These tendencies are causing higher resistance rates resulting in

imminent hazards in human health. Notably, irrationality is observed in using antibiotics in

livestock. Animals are given antibiotics for faster growth and disease prophylaxis. Strict and

enforced regulations in the agricultural industry are needed to curb the harmful ripple effects.

Treatments for bacterial infections are becoming intensified every day. Infections remain as

antibiotics gain resistance; treatment failure is common due to antibiotic resistance and multi- drug

resistance, for e.g. tuberculosis. Newer and effective antibiotics that have no known resistance to

bacteria are in high demand. Alternative treatment procedures are under consideration to fight

bacterial infection. Passive immunization or administration of antibodies to non-immunized to

prevent bacterial infections have been found effective (Keller and Stiehn, 2000). Another effective

intervention is phage therapy, whereas bacteriophages are used to treat pathogenic bacterial

infections (Monk et al., 2010). Many newer classes of antimicrobials to fight antibiotic

resistance are in the pipeline for clinical trials (Devasahayam et al., 2010). Intervention strategies

are aimed not only at targets but rather at the biological networks that may help to create new

antibacterial therapies (Kohanski et al., 2010). Combination therapies coupling antibiotics with

antibiotic-enhancing phage have demonstrated the potential to be a promising antimicrobial

intervention (Aminov, 2010).

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2.1.7 Mechanism of antimicrobial resistance

There are number of ways by which microorganisms are resistant to antimicrobial agents. These

include:

1. Bacteria produce enzymes which destroy the antimicrobial agents before it reaches its targets e.g.
Beta lactamase enzyme hydrolyses beta lactam drugs which develop resistance.

2. Impermeable cell for antimicrobial drugs e.g. Gram negative bacteria may become resistant to

Beta lactam antibiotics by developing permeability barrier.

3. Mutation e.g. Ribosome methylation of ribosomal RNA develop macrolide resistant.

4. Bacterial efflux pump that expels antimicrobial drugs from cell before it can reach its targets.

5. Specific Metabolic pathways in the bacteria are genetically altered so that antibacterial

agents cannot exert an effect. (Marie et al., 2005; Rice et al., 2007).

2.2 Theoretical Review

The adult human vagina is a complex biota containing a profusion of microorganisms. Microbes

are microscopic organisms. These can be either unicellular or acellular and are present

everywhere in nature. They include bacteria, fungi, archaea, protists, some microscopic plants

such as green algae and animals such as planktons and palanarian.

On account of their nature viruses may or may not be included. Bacteria and yeast form normal

flora of this ecosystem, which is normally found on the skin and every opening of the body such

as mouth, ears, rectum and vagina. Even a neonate carries specific flora of his/ her mother and

soon develops own floral community. This flora persists till death of the individual. An adult

human carries normal flora consisting of more than 200 bacterial species. Normally these are

14
harmless and are involved in benefiting their hosts. Yet some are parasitic in nature, living at the

expense of their host, and some are even pathogenic, disease causing.

These pathogenic microbes, after getting a chance, invade their hosts and lead to opportunistic

infection. These diseases caused by normal flora are termed as endogenous diseases

Urinary tract is regularly flushed with sterile urine and its acidity, there it makes difficulty for

microbes to access and establish here but anterior urethra is inhabited by relatively constant flora

such as Staphylococcus epidermidis, Enterococcus faecalis and some Alpha-hemolytic

streptococci. Occasionally some enteric bacteria like E. coli, Proteus and Corynebacteria may be

found there.

2.2.1 Antibiotic Culture Sensitivity

Antibiotic sensitivity is a term used to describe the susceptibility of bacteria to antibiotics.

Antibiotic sensitivity testing (AST) is usually carried out to determine which antibiotic will be

most successful in treating a bacterial infection in vivo. Testing for antibiotic sensitivity is often

done by the Kirby-Bauer method while other methods include the Stokes method, E-test (also

based on antibiotic diffusion) and Agar and Broth dilution methods (for Minimum Inhibitory

Concentration determination). Muller Hinton agar is most frequently used in this antibiotic

susceptibility test. Our study was aimed to isolate, identify and check the antibiotic sensitivity

against infection causing offending pathogens in females by using swabs. It is a handy study for

informing doctors about resistance and sensitive strains these microbes while epidemiologically a

handy one to aware female community to adopt protective measures against these offending

agents.

2.3 Theoretical Framework

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The Kirby-Bauer Disc Method

This method is also called the agar diffusion method or the disk diffusion method. The procedure

followed is simply that a filter disk impregnated with an antibiotic is

applied to the surface of an agar plate containing the organism to be tested and the plate is

incubated at 37°C for 24-48 hours. As the substance diffuses from the filter paper into the agar,

the concentration decreases as a function of the square of the distance of diffusion. At some

particular distance from each disk, the antibiotic is diluted to the point that it no longer inhibits

microbial growth. The effectiveness of a particular antibiotic is shown by the presence of growth-

inhibition zones. These zones of inhibition (ZOIs) appear as clear areas surrounding the disk from

which the substances with antimicrobial activity diffused. The diameter of the ZOI can be

measured with a ruler and the results of such an experiment constitute an antibiogram. The agar

diffusion method uses commercially available filter paper disks, each containing a defined

concentration of a specific antibiotic. The relative effectiveness of different antibiotics provides

the basis for a sensitivity spectrum of the organism.

This information, together with various pharmacological considerations, is used in the selection of

an antibiotic for treatment. It should be emphasized that chemotherapeutic agents are not chosen

simply on the basis of the drug producing the widest ZOI. This is because of the nature of the

growth-inhibition substances. The size of the zone may be affected by the density or viscosity of

the culture medium, the rate of diffusion of the antibiotic, the concentration of the antibiotic on

the filter disc, the sensitivity of the organism to the antibiotic, and the interaction between the

antibiotic and the medium. In addition, an agent that has been found to have a significant

antibiotic effect may not be therapeutically useful because it may also have significant adverse

effects in the system for which it is intended. The disk diffusion method represents a simple

16
procedure for screening substances to determine if they have significant antibiotic activity.

17
 CHAPTTER THREE

3.1 MATERIALS AND METHODS

This is a study conducted at Department of Microbiology of The Polytechnic, Ibadan. A total of

6 high vaginal swabs were collected both from indoor and outdoor patients from Apollos

laboratory which were presented with symptoms of virginal discharge such as: malodor, dysuria,

dyspareunia, itching and fever. Patients who have vaginal instrumentation recently and history of

usage of past two weeks antibiotics were excluded from the study. Sterile swabs were labeled

with a unique identifier. The collection of sample was done using cottonswab which was inserted

deep into the vagina. The samples were brought to Godfrey Okoye University for further

analysis. Collected sample was inoculated in blood agar and chocolate agar by streaking method.

Plates were incubated aerobically for 18-24 hours. Preliminary identification was done on the

basis of morphological characteristics (shape, color, and spore formation), Gram stain and

biochemical reactions (catalase test, oxidase test, IMViC test). Antibiotic sensitivity testing was

done using (modified Kirby-Bauer’s) disc diffusion method. Antimicrobials tested for sensitivity

were Amikacin, Ampicillin, Amoxicillin Clavulanic acid, Imipenem, Ciprofloxacin and Sulzone.

After overnight incubation plates were examined to read the susceptibility zone. Data obtained

were presented as biochemical tests result, antibiotic sensitivity of bacteria isolated from vaginal

swab and number and percentage of patients from which the microorganisms were isolated. The

frequency and antimicrobial sensitivity patterns of microbes were presented in percentages.

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3.2 PREPARATION OF MACCONKEY AGAR

MacConkey Agar media was prepared in flask by dissolving 1.68gm of powdered agar media in

1litre of distilled water or the equivalent of this in smaller volumes. The flask was shaken for

some time, heat on a Burnsen flame to dissolve the medium completely and then sterilized

inautoclave (at 121°C temperature for 15 minutes).

3.3 PREPARATION OF BLOOD AGAR

Nutrient ager was prepared by dissolving 3.46 grams of powdered ager into a liter of distilled

water, in a flask shaken for some time, heated to dissolve the medium and then sterilized by

autoclaving at 121°C for 15 minutes. It was transferred to a 50°C water bath. When the agar base

was cooled to 50°C, sterile blood agar was added aseptically and mixed well gently. Formation

of air bubbles was avoided. The blood was warmed to room temperature at the time of

dispensing to molten ager base. A volume of 15 ml was dispensed into sterile-Petri plates

aseptically. The medium was labeled with the date of preparation and name of agar. The plates

are stored at 2-8°C, preferably in sealed plastic bags to prevent loss of moisture.

3.4 SUBCULTURE

Isolates were separately sub cultured onto blood and chocolate agar plates using a sterile wire

loops and incubated at 37oc for 24 hours.

3.5 AGAR SLANT SUB CULTURE IN BIJOU BOTTLES

Nutrient agar was prepared by dissolving the nutrient agar into distilled water. It was boiled for

sixty seconds to dissolve the agar completely, dispensed into the Bijou bottles up to halve of

each bottle and sterilized by autoclaving at 121ocfor 15 minutes. It was then allowed to cool in a

slant manner. Isolates were individually sub cultured onto agar slants, incubated for 18h and

subsequently stored in the refrigerator at 4oC as stock culture

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3.6 GRAM STAINING

Using a sterile wire loop, a loopful of colony of microorganisms was collected and fixed on a

sterile glass slide and smeared then allowed to air dry. It was then gently flooded with crystal

violet, tilting the slide and allowed to stand for 60seconds then rinsed with water then blot dry.

Gently flood the smear with Grams iodine and again allowed for 60seconds. Then rinsed with

water. Decolorize using Acetone, tilting the side slightly and immediately flushed with water.

Finally it was flooded with safranin and allowed to stand for 45seconds then rinsed. Then it was

blot dry and viewed under the light microscope under oil immersion.

3.7 BIOCHEMICAL TESTS

3.7.1 CATALASE TEST

An aliquot, 2ml of hydrogen peroxide solution was put into a test tube. A sterile wire loop was

used to collect a loop full of the test organism and immersed in hydrogen peroxide solution and

was observed for production of bubbles.

3.7.2 OXIDASE TEST

Procedure for oxidase test using dry filter paper method

Filter paper was soaked with 1% solution of reagent. Little culture is smeared on it with a sterile

wire loop and is checked for result.

3.7.3 UREASE TEST

Procedure

A broth medium was inoculated with a loop full of pure culture of the test organism and was

streaked on the surface of the agar slant. The cap was loosely covered and incubated at 35 oc for

24hours.

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3.7.4 METHYL RED TEST

Procedure

Prior to inoculation, allow medium to equilibrate to room temperature. Using organisms taken

from an 18-24 hour pure culture, lightly inoculate the medium.

Incubate aerobically at 37 degrees C. for 24 hours. Following 24 hours of incubation, aliquot 1ml

of the peptone glucose broth to a clean test tube. Reincubate the remaining broth for an

additional 24 hours. Add 2 to 3 drops of methyl red indicator to aliquot. Observe for red color

immediately.

3.7.5 INDOLE TEST

Procedure using conventional tube method

A culture of the microorganism was sterilized in a test tube containing tryptophan broth and

incubated at 37oc for 24 hours. 0.5 ml of kovac’streagent was added to the broth culture and

observed for the presence or absence of red ring.

3.7.6 VOGES PROSKAUER TEST

Procedure

Voges proskauer broth was inoculated with a pure culture of the test organism and incubated for

24 hours at 37oC . 1.0ml of the incubated broth was removed to a separate tube for vp testing.

The reagent was allowed to warm to room temperature prior to use and 9 drops of Naphthol

followed by 0.2ml of KOH the tube was shaken gently to expose the medium to atmospheric

oxygen and allow the tube to remain undisturbed for 10 to 15minutes..

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3.7.7 CITRATE TEST

Procedure

Simmons citrate agar lightly inoculated on the slant by streaking it to the microbial colony. It

was then incubated at 37oC for 24 hours and observed for colour development

3.8 ANTIBIOTIC CULTURE SENSITIVITY

In third stepa 100 ml of muller hinton was prepared under sterile condition and poured into

sterile test plates. A sterile cotton swab was dipped into a sample from well-mixed colonies

in distilled water and applied onto nutrient agar plate. Sensitivity was checked by using 6

different commercially available anti-microbial discs (wafers) that were, Amoxicillin,

Oflaxacin, Ciproflaxin and Gentamycin, and, were placed on the plate by means of multi-

disc dispenser and pressed firmly onto agar plate with sterile forceps by using the agar-disc

diffusion. The inoculated plates containing the antibiotics were incubated at 37oC for 24 hours

after which the diameter of zone of inhibition around each antibiotic disc were then measured

to the nearest millimeter (mm). Zones of inhibition of growth were examined around the disc;

susceptible: zone of inhibition of ≥15mm. Resistant: zone of inhibition of ≤15mm. The discs

were dispensed onto agar surface aseptically by forceps and firm contact was ensured. A distance

of 24mm was maintained between the discs. After overnight incubation at 37 OC the plates were

examined for the zone of inhibition. Zone diameters were measured by calipers and strains were

reported sensitive or resistant according to the chart supplied.

22
 CHAPTER FOUR

4.1 Study Design, Setting, And Study Population

A cross-sectional study was conducted at the gynecology clinic and microbiology laboratory of

Mbarara Regional Referral Hospital (MRRH) from December 2020 to June 2021 among reproductive-

age women (15–49 years) with abnormal vaginal discharge. MRRH is a tertiary hospital located in

Mbarara City, southwestern Uganda, approximately 250 km from Kampala, the capital city. MRRH is

the main referral hospital for all of southwestern Uganda, serving over 10 districts and receiving

patients from the neighboring countries of Tanzania, Rwanda, the Democratic Republic of Congo

(DRC) and Burundi.

4.2 Sample size and Sampling technique

Kish Leslie formula of 1965 was used for calculating the sample size for cross-sectional studies and

obtained 361 as the minimum number of respondents, whom we enrolled using consecutive sampling.

We calculated the sample size using a population proportion of 70% women with abnormal virginal

discharge based on a study from Nigeria [6] to have isolates.

4.3 Study procedure

All women aged 15–49 years attending the gynecology clinic of MRRH with a complaint of abnormal

vaginal discharge with or without other symptoms were approached to participate in the study. We

included all non-pregnant women, while women who also had vaginal bleeding and those with

confirmed genital malignancy were excluded. The research team screened the women for eligibility and

obtained written consent from the eligible participants. Gynecology examination was performed for all

participants enrolled in the study. The participants underwent a speculum examination during which

two endocervical and two high vaginal swab samples were collected. A standardized interviewer-

guided structured questionnaire was used to obtain data on sociodemographic, sexual, obstetric,

gynecological and medical characteristics of the participant, including HIV serostatus.

23
The women received their empirical treatment as guided by the syndromic management of abnormal

vaginal discharge as per the clinical care guidelines and were asked to return after three days for their

culture and susceptibility results. Participants with bacterial isolates and susceptibility tests were linked to

the clinical care team for further management.

4.4 Tests carried out

This was done on a pure culture showing significant growth following standard criteria as described by

the Kirby-Bauer disc diffusion method [7]. The medium for fastidious organisms was chocolate agar

incubated in carbon dioxide. For nonfastidious organisms, we used Muller Hinton Agar (MHA) incubated

aerobically at 37°C. The inoculum density required for susceptibility testing was 0.5%, McFarland. The

choice of antibiotic discs was based on the type of organism(s) cultured. The following antimicrobial

agents were employed: tetracycline (50 μg), ceftriaxone (30 μg), ciprofloxacin (5 μg), amoxicillin (10 μg),

and erythromycin (15 μg) for gram-positive organisms and gentamycin (10 μg), amoxicillin/clavulanate,

cefixime, cefuroxime, azithromycin, and doxycycline for susceptibility testing for gram-negative

organisms. Susceptibility testing for erythromycin was only performed against gram-positive bacteria, as

the antibiotic is not active against gram-negative bacteria. Also susceptibility testing for S. agalactiae

against Ciprofloxacin and Klebsiella pneumoniae against Amoxicillin were not done as these antibiotics

are not active against these isolates.

4.5 Results and Findings

Results of the biochemical tests indicating the presumptive identity of the isolates are shown in

tables 1, 2 and 3 below. Table one shows the sociodemographic characteristic, Table two shows the

Gynecological and medical characteristics of the study participants and Table three shows the

Antibiotic susceptibility of the bacterial isolates

24
 Bacteria Isolated P value Table 1
Variable n (%)
Yes, N = 107 n (%) No, N = 254 n (%)
Age Mean ± SD 36.42 (± 7.99) years 37 ± 7.67 yrs 36.01 ± 8.11yrs 0.933
Residence
Urban 161 (44.6) 48 (44.86) 113 0.948
Rural 200 (55.4) 59 (55.14) 141
Education level
Primary and below 216 (59.8) 66 (61.68) 160 (62.99) 0.814
Secondary and above 135 (40.2) 41 (38.32) 94 (37.01)
Occupation
Unemployed 223 (61.8) 71 (66.36) 152 (59.84) 0.245
Employed 138 (38.2) 36 (33.64) 102(40.16)
HIV status
Positive 106 (29.4) 35 (32.71) 71 (27.95) 0.365
Negative 255 (70.6 72 (67.29) 183 (72.05)
Marital status
Living with partner 252 (69.8) 74 (69.16) 178 (70.08) 0.862
Not living with partner 109 (30.2) 33 (30.84) 76 (29.92)
Sexually active in last 6 months
No 49 (13.6) 15 (14.02) 34 (13.39) 0.873
Yes 312 (86.4) 92 (85.98) 220 (86.61)
Participants’ sociodemographic characteristics, N = 361

25
A total of 373 women of reproductive age presented at the gynecology clinic of Mbarara Regional

Referral Hospital with abnormal vaginal discharge during the study period. Of these, 12 were excluded

because they had concurrent vaginal bleeding. Culture was performed on swabs collected from 361 of

these women, and bacteria were isolated in 107 of the participants. The mean age of the participants was

36.42 (± 7.99) years, as shown in Table 1 above. There was no significant difference in the

sociodemographic characteristics between women with and without bacterial isolates.

26
Table 2 Gynecological and medical characteristics of the study participants, N = 361

Bacteria Isolated
Variable n (%) P value
Yes, N = 107 n (%) No, N = 254 n (%)
Prior infertility treatment
Yes 14 (3.9) 2 (1.87) 12 (4.72) 0.199
No 347(96.1) 105 (98.13) 242 (95.28)
Barrier FP method (condoms) n = 112
Yes 8 (2.2) 4 (11.43) 4 (5.19) 0.235
No 104 (28.8) 31(88.57) 73 (94.81)
Miscarriage in prior 6 weeks
Yes 13 (3.6) 3 (2.80) 10 (3.94) 0.598
No 348 (96.4) 104 (97.20) 244 (96.06)
Prior abnormal vaginal discharge
Yes 259 (71.7) 74 (69.16) 185 (72.83) 0.479
No 102 (28.3) 33 (30.84) 69 (27.17)
AVD times in last 6 months
None 102 (28.3) 33 (30.84) 69 (27.17)
0.324
Once 137 (38.0) 44 (41.12) 93 (36.61)
> = two times 122 (33.8) 30 (28.04) 92 (36.22)
Duration of current abnormal discharge
≤ 7 days 161 (44.6) 45 (42.06) 116 (45.67)
0.02
8–14 days 104 (28.8) 41 (38.32) 63 (24.8)
More than 14 days 96 (26.6) 21 (19.63) 75 (29.53)
Prior antibiotic use
Yes 93 (25.8) 29 (27.1) 64 (25.2) 0.705
No 268 (74.2) 78 (72.9) 190 (74.8)
a
Long term drug use
Yes 109 (30.2) 35 (32.71) 74 (29.13) 0.499
No 252 (69.8) 72 (67.29) 180 (70.87)

Prior infertility treatment and duration of abnormal vaginal discharge differed among participants with

and without bacterial isolates. Bacterial isolates were more abundant in participants who had not received

prior treatment for infertility than in those who had received the treatment. The participants with a shorter

duration (≤ 7 days) of abnormal vaginal discharge were more likely to have bacteria isolated than those

with a longer duration of abnormal vaginal discharge, as shown in Table 2.

Table 3 Antibiotic susceptibility of the bacterial isolates

27
 Bacterial isolates n (%)
Total N =
Antibiotic S.aureus n = S.agalactieae n Kleb spp n E.coli n E. fecalis n 107
52(%) = 3 (%) = 32 (%) = 4 (%) = 16 (%)
S 22(42.3) 2(66.7) Not tested 2(50) 13(81.3) 39 (36.5)
Amoxicillin
R 30(57.7) 1(33.3) Not tested 2(50) 3(18.7) 68 (63.5)
S 41(78.8) 3(100) 22(68.8) 4(100) 13(81.3) 83(77.6)
Amoxicillin/Clavulanate
R 11(21.2) 0(00) 10(31.2) 0(00) 3(18.7) 24(22.7)
S 42(80.8) Not tested 26(81.3) 3(75) 16(100) 87 (81.3)
Ciprofloxacin
R 10(19.2) Not tested 6(18.7) 1(25) 0(00) 20 (18.7)
S 46(88.5) 3(100) 3(9.4) 4(100) 12(75) 68 (63.6)
Ceftriaxone
R 6(11.5) 0(00) 29(90.6) 0(00) 4(25) 39 (26.4)
S 41(78.8) 3(100) 21(65.6) 3(75) 14(87.5) 82 (76.6)
Cefixime
R 11(21.2) 0(00) 11(34.4) 1(25) 2(12.5) 25 (23.4)
S 48(92.3) 3(100) 27(84.4) 4(100) 15(93.8) 97 (90.7)
Cefuroxime
R 4(7.7) 0(00) 5(15.6) 0(00) 1(6.2) 10 (9.3)
Not
S 11(21.2) 0(00) Not tested 3(18.7) 14 (19.7)
tested
Erythromycin
Not
R 41(78.8) 3(100) Not Tested 13(81.3) 57 (80.3)
tested
S 38(73.1) 3(100) 27(84.4) 3(75) 13(81.3) 84 (78.5)
Gentamycin
R 14(26.9) 0(00) 5(15.6) 1(25) 3(18.7) 23 (21.5)
S 3(5.8) 1(33.3) 4(12.5) 0(00) 1(6.2) 9 (8.4)
Tetracycline
R 49(94.2) 2(66.7) 28(87.5) 4(100) 15(93.8) 98 (91.6)
S 7(13.5) 1(33.3) 6(18.7) 0(00) 1(6.2) 15 (14)
Doxycycline
R 45(86.5) 2(66.7) 26(81.3) 4(100) 15(93.8) 92 (86)
S 22(42.3) 2(66.7) 8(25) 2(50) 12(75) 46 (43)
Azithromycin
R 30(57.7) 1(33.3) 24(75) 2(50) 4(25) 61 (67)

The total sensitivity of all bacterial isolates was highest with cefuroxime at 90.7% and ciprofloxacin at

81.3%. The highest resistance was observed with doxycycline at 86%, as shown in Table 3.

CHAPTER 5

5.1 Discussion

28
Vaginal flora contains a range of microorganisms normally .Lactobacillus spp. play fundamental

role in maintaining acidic vaginal pH and prevent the overgrowth of potentially harmful and

opportunistic bacteria. Vaginal infections are a great threat for women’s health related to common

gynecological problem. This study demonstrates the prevalence of vaginal pathogens among the

women study group. Vaginal infections are increasing due to vagina colonization by pathogenic

bacteria other than the protective bacteria. The results of this study are similar to the study

conducted by Lakshmi et al.,(2001). They reported the prevalence of vaginal infections in India.

(Larsen, 2001)In our study E.coli was the most frequent bacteria followed by Klebsiella ,

PseudomonasCitrobacter , Proteus and Acinetobacter respectively, similar to a study conducted by

Dutta et a.,.(1995). McDonald et al.,(1994) also found E. coli to be the important bacteria

associated with bacterial vaginosis. The most useful antibiotics against gram negative rods in our

study were Imipenem (27mm) and Ciprofloxacin (28). Antibiotics like Imipenem are extremely

effective but expensive. Tariq et al.,(2017)reported similar findings. Whereas the antimicrobials

with least affectivity against gram negative rods were Penicillins (Ampicillin, amoxicillin-

clavulanic acid), Amikacin due, probably, to indiscriminate use of antibiotics.

5.2 Findings

29
Microbial-based therapeutics have recently attracted an increasing amount of interest owing to the

beneficial effects to the host health. As pivotal bacteria in the healthy vaginal microbiome,

Lactobacillus species can act as antimicrobial adjuvants due to their ability to potentiate the effect

of antibiotics (Larsson et al., 2011; Bodean et al., 2013; Recine et al., 2016; Bohbot et al., 2018;

Cohen et al., 2020). In 2020, a phase 2b trial (NCT02766023) was conducted on 228 women to

assess the efficiency of L. crispatus CTV-05 in preventing BV relapse. When L. crispatus CTV-05

was used after antibiotic treatment, a remarkably lower rate of BV recurrence was observed at 3

months compared with placebo (Cohen et al., 2020). Vaginal microbiota transplantation (VMT), in

which optimum vaginal microbiota is transplanted to patients, is another microbial-based

therapeutic strategy. In 2019, the first exploratory research has reported the viability of utilizing

VMT as a long-term therapeutic regimen for women with intractable BV (Lev-Sagie et al., 2019).

In this study, five patients with intractable BV were treated with VMT after antibiotic treatment,

and long-lasting relief was shown in four of these patients in the follow-up period of 5–21 months

after VMT. This finding suggested the remarkable alleviation of symptoms and the rebuilding of

the vaginal microbiota with Lactobacillus spp. dominance. The risks of this treatment include the

acquisition of antimicrobial-resistant microbes, sperms, undetected pathogens, and other clinically

silent phenotypes from the donors. Overall, antibiotics, biofilm-disrupting agents, probiotic

Lactobacillus, and VMT can be utilized separately or in combination to regulate the microbiome

through the reestablishment of vaginal eubiosis. The intricate and dynamic vaginal microbiome

brings the challenge for diagnosis.

The challenge for treatment is attributed to inaccurate diagnostics, biofilm, antibiotic resistance,

and the simultaneous elimination of both pathogenic bacteria and probiotics, such as Lactobacillus.

It is expected that future research should be conducted to target specific microbes, thus eliminating

more pathogenic bacteria, but without affecting probiotic microorganisms. In addition, it is

30
necessary to develop personalized diagnosis and treatment by taking individual differences in

vaginal microbiome into consideration.

Further, with increasing knowledge of BV-associated epidemiology and pathogenesis, more

improved novel therapeutic strategies will be developed in the future.

5.3 Conclusion

Bacteria were isolated in 3 of every 10 women with abnormal vaginal discharge. The majority of

the bacterial isolates were resistant to antibiotics commonly used in the treatment of women with

abnormal vaginal discharge at Mbarara Regional Referral Hospital. There is a need for routine

culture and susceptibility testing of women with abnormal vaginal discharge to guide treatment,

minimize inappropriate antibiotic use and consequently reduce antibiotic resistance.

High prevalence of gynecological infections demands that the patients who suffer from the

symptoms of gynecology must be investigated carefully. The elevated prevalence of vaginal

bacteria in the recent study stress that high vaginal swab culture provides a good laboratory means

for the identification of causative bacteria.It is therefore recommended for roUrinary Tract

Infection(UTi)ne use. There is a need for stakeholders to be aware of antibiotic resistance in order

to avoid improper use and frequent abuse of available antibiotics. Treatment schedule must be

designed subsequent to proper laboratory investigations.

The vaginal microbiome forms a homeostatic and mutualistic relationship with human host and

plays an important role in vaginal health and disease. The variations of internal and/or external

factors lead to the breakdown of a balanced ecosystem, which is also known as dysbiosis. Although

the increasing scientific knowledge has already provided insights into the characteristics of vaginal

microbiome and its correlation with diseases, such as STIs, PID, PTB, there is still inadequate

31
understanding of interactions between microbiota and the host. Efforts should be made to reveal the

mechanism of interactions between species and their impact on vaginal microbiome.

As a highly prevalent dysbiosis, BV triggers numerous adverse health outcomes and becomes a

burden to individuals and public health. G. vaginalis is the most common microorganism detected

in BV, but its presence in the vagina does not always lead to BV. Other microorganisms, such as

Atopobium and Prevotella, also have a strong relationship with BV. These BV-associated microbes

can affect immune mediators, which may serve as predictive biomarkers for dysbiosis, in the

vaginal environment. Although the epidemiology and pathogenesis of BV is not fully understood,

studies based on genomic, lipidomic, glycomic, metabolomic, and proteomic techniques may

provide further insights. More improved novel and accurate diagnosis and therapeutic strategies

will be developed based on the accumulated information on BV.

5.4 Recommendations

Although these antibiotics are effective against BV-associated bacteria and somewhat relieve

symptoms, the remission is usually temporary, and many patients relapse after treatment (Bradshaw

et al., 2006; Hay, 2009; Mayer et al., 2015). The high recurrence rate (50%–67%) may be the result

of the inability of antibiotics to eliminate the biofilm-associated bacteria of BV in vagina ( Figure

2 ) (Swidsinski et al., 2008; Swidsinski et al., 2011; Javed et al., 2019; Ahrens et al., 2020; Verwijs

et al., 2020). For example, Ahrens et al. reported that G. vaginalis and other BV-associated bacteria

are eradicated or are largely decreased in 58% of the patients treated with metronidazole.

Meanwhile, none of these bacteria are eliminated in the remaining half of the patients.

This phenomenon is attributed to the sheltering of G. vaginalis and other bacteria by biofilms

32
(Ahrens et al., 2020). In addition, Swidsinski et al. observed the persistence of G. vaginalis biofilms

after oral metronidazole therapy (Swidsinski et al., 2008). Therefore, a promising therapeutic

strategy against BV, i.e., adjuvant-based therapy with the ability to disrupt biofilms, has been

developed ( Figure 2 ). Extracellular DNA is an important integrant of G. vaginalis biofilms. The

enzymatic activity of DNase can be used to disrupt newly formed and mature biofilms.

DNase has shown its ability to release microorganisms from biofilms to supernatant fractions and

to enhance the activity of metronidazole (Hymes et al., 2013). Similarly, lysozyme can reportedly

disrupt biofilms and prevent their formation. The combination of lysozyme and antibiotic

(clindamycin or metronidazole) can potentiate both the ability of antibiotics and biofilm disruption

(Thellin et al., 2016).

In addition, the use of amphoteric tenside sodium cocoamphoacetate causes degradation of the

established biofilms and enhances the effect of metronidazole (Gottschick et al., 2016). Lauramide

arginine ethyl ester and subtilosin reportedly have great bactericidal effect on G. vaginalis biofilms

(Turovskiy et al., 2012). These two compounds have displayed a synergistic effect with antibiotics

(clindamycin and metronidazole) against G. vaginalis biofilms (Algburi et al., 2015). Besides the

abovementioned adjuvants investigated with antibiotics, many others have been studied

individually. For instance, L. reuteri RC-14 can degrade surface density, depth, and area of G.

vaginalis biofilms (Saunders et al., 2007). Thymol is effective in the inhibition of mature and native

G. vaginalis biofilms (Braga et al., 2010). Endolysin (PM-477) can selectively and effectively

eliminate Gardnerella in native polymicrobial biofilm and in cultures of isolated strains (Landlinger

et al., 2021).

33
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