CHAPTER ONE

INTRODUCTION

1.1 Background of the Study

The presence of bacteria in the urine is medically referred to as bacteriuria (but not if the

bacteria's presence is due to contamination from urine sample collection). It is usually

regarded as significant when the urine contains 10 5 organisms or more per ml of urine.

Bacteriuria becoming a global emergency concerned challenging as persistent bacterial

colonization of the urinary tract (usually by Escherichia coli) without symptom.

Bacteriuria may be asymptomatic or symptomatic (Samaga, 2016). Asymptomatic

bacteriuria is bacteriuria without accompanying symptoms of a urinary tract infection

(such as frequent urination, painful urination or fever) (AMDA, 2014; Das et al., 2017;

Sendi et al., 2017). Asymptomatic bacteriuria is a common bacterial infection of the

urinary tract requiring medical treatment in pregnancy. If left untreated, it is a risk factor

for acute cystitis (40%), pyelonephritis (25-30%) in pregnancy; and will develop a

symptomatic urinary tract infection (Alfred et al., 2013).

Symptomatic bacteriuria is bacteriuria with the accompanying symptoms of a urinary

tract (such as frequent urination, painful urination, fever, back pain) (Das et al., 2017;

Sendi et al., 2017).

Urinary tract infections (UTIs) are one of the most common bacterial infections/clinical

diseases in humans both in the community and in established hospital setting worldwide

(Ajide et al., 2016). It is the commonest bacterial infectious disease in community

practice with a high rate of morbidity and financial cost (Mahajan et al., 2014). It

1
generally estimated that millions of people are affected yearly (HDAHIS, 2012;

Zeyaullah& Kaul, 2015; Ajide et al., 2016) with a large proportion of the infections being

in apparent, many also manifest with obvious clinical features while others still show

complications in addition (Ojo et al., 2010). The infection may involve the kidney and

collecting system (Pyelonephritis), bladder (cystitis), prostate (prostatitis) and the urethra

(urethritis). It is an infection of the upper or lower urinary tract or even includes both.

Recurrent infections are common and can lead to irreversible damage to the kidneys,

resulting in renal hypertension and renal failure in severe cases (Gernohonska et al.,

2010). During pregnancy, UTIs contributes significantly to maternal and perinatal

morbidity including preterm delivery, low birth weight, foetal mortality, hypertension,

anaemia and renal insufficiency (Cunningham et al., 2010; Foxman, 2010; Imade et al.,

2010; Pegu et al., 2014; Samaga, 2016). This infection is common in pregnant women

because of unique changes in their anatomic and physiological changes in the urinary

tract (Samaga, 2016). In addition, the apparent reduction in immunity of pregnant women

appears to encourage the growth of bacteria, to increases the plasma volume and decrease

the urine concentration and up to 70% pregnant women develop glucosuria which

encourages bacterial growth in the urine; therefore, these factors lead to urinary stasis and

uretero-vesical reflux as to increase urinary progestins and estrogens which leads to a

decreased ability of the lower urinary tract to resist invading bacteria (Imade et al., 2010;

Alfred et al., 2013; Samaga, 2016; Ghimire et al., 2017). Hence, the growth of bacteria in

the urine of an individual is refer to as the bacteriuria which may be symptomatic or

asymptomatic (Ghimire et al., 2017).

2
Escherichia coli have been reported as the most common etiological agent of UTIs in

pregnant women (Samaga, 2016; Ghimire et al., 2017). Prevalence in both symptomatic

and asymptomatic bacteriuria was reported to be 2-10% in both pregnant and among non-

pregnant women (Cunningham et al., 2010; Sandhyarami et al., 2014; Samaga, 2016;

Ghimire et al., 2017).

Antibiotic such as ẞ-lactam, quinolones, aminoglycosides and others have been reported

as the most common antibiotics used for the treatment of UTIs, especially in women. A

high-level antibiotic resistance has been reported in E. coli causing UTIs (Zalmanovici et

al., 2010; Manjunath et al., 2011; Raza et al., 2011; Hertz et al., 2016).

Pathogenic bacteria that have become resistant to antibiotic drug therapy have increased

the problems of public health all over the world, and it is an ever-increasing global health

threat (Makut et al., 2014). Escherichia coli resistance to ẞ-lactam antibiotic as result of

ESBL produced resistance to quinolones and aminoglycosides antibiotics due to enzymes

and modification of using target site has also be reported (Biradar et al., 2013; Nivas et

al., 2014; Zuyaullah & Kaul, 2015; Wireko et al., 2017).

The emergence of antibiotic resistance in E. coli causing UTIs in pregnant women have

been reported due to inappropriate use of antibiotics for treatment of UTI, and this

however have had high morbidity and incurred burden to the pregnant women as well as

health care provider. Surveillance of antibiotic resistance of E. coli and other bacteria

causing UTIs have been evaluated by many researchers (Iregbu et al., 2013; Omar et al.,

2013; Niranjan & Malini, 2014; Timothy et al, 2014; Bassetti et al., 2016; Howladar &

Gandhi, 2016; Hertz et al., 2016; Igwe et al, 2016; Maria et al.,2016; Mishra et al., 2016;

Kulkarni et al., 2017; Morill et al., 2017; Eghieye et al., 2018; Hitzenbichler et al., 2018).

3
1.2 Statement of the Problem

Many individuals with bacteriuria might have had symptomatic infection in the past or

develop overt disease, and not that all individuals whose urine is contaminated with

microorganisms develop cystitis or pyelonephritis, but rather, that most cases of this

disease emerge from a larger group of bacteriurics as in view that bacteriuria is the most

common denominator of urinary tract infections (UTIs).

Although the great bulk of UTIs appear to arise from infection of the lower tract, a small

group of patients with hematogenous may not demonstrate bacteriuria. A number of

factors can lead to urinary infection. Even a few bacteria that manage to evade the urinary

tract defenses and enter the bladder can multiply to high levels during this time causing

infection. Urine provides abundant nutrients for many species of bacterial. Both

asymptomatic and symptomatic are equally important in the aetiological aspects of the

infection although symptomatic infection is only important to the woman.

The combination of mechanical, hormonal and physiological changes during pregnancy

contributes to significant changes in the urinary tract, which has a profound impact on the

acquisition and natural history of bacteriuria during pregnancy. These changes include

dilation of the ureter, decrease in urethral peristalsis, and decrease in bladder tone (Alfred

et al., 2013; Samaga, 2016). In many developing countries, the clinician and health care

providers usually focus on the presence of glucose and protein in urine specimens with

less attention on possible asymptomatic infection (Imade et al., 2010). Apart from

socioeconomic condition, ignorance, poor hygienic practices and financial constraints

may restrict the feasibility of introducing general screening of all pregnant women.

4
 The course of UTIs often runs over several decades, and to follow patients over such long

periods is difficult. It is likely for recur or reinfections since the individuals are

inadequately treated and non-compliance with the treatment as they lack the knowledge

of the infections and environmental management with risk factors involved. The

increasing ability of E. coli to cause UT'ls and the difficulty encountered in treating these

infections due to multidrug antibiotic resistance necessitates updating the knowledge of

their presence and drug resistance in a given environment (Olorunmola et al., 2013).

This is particularly necessary in a suburban community like Nyanya, Abuja, where all

kinds of antibiotics are available across the counter with or without prescription.

1.3 Justification of the Study

It is imperative that culture and susceptibility tests are carried out on infecting pathogen

prior to treatment, in order to reduce morbidity, avoid treatment failure and reduce

selective pressure that could result in the spread and proliferation of antibiotic resistant

and virulent uropathogenic E. coli in the environment.

The study is very important to an individual, community, environment, couples and

medical personnel in basic function of lifestyle and sanitation personal hygiene exercises

and to ensuring that a woman attains the highest possible level of health and much more

so, a pregnant woman as this goes a long way in determining the outcome of her

pregnancy.

To create awareness on the implications of infections and possibly management, and

make better life regarding the personal habits to the healthy living that will enhance their

immunity.

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1.4 Aim and Objectives of the Study

This study investigates the occurrence of, and molecular characterization of quinolone

resistance in Escherichia coli isolated from urine of pregnant women attending selected

hospitals in Nyanya, Abuja, Nigeria.

The specific objectives are:

1) To isolate and identify Escherichia coli from urine of healthy pregnant women attending

selected hospitals in Nyanya, Abuja, Nigeria.

2) To determine the antibiotic susceptibility profile of the E. coli isolates.

3) To evaluate the antibiotic resistance phenotypes in the resistant isolates.

4) To evaluate the multiple antibiotic resistance (MAR) index of the isolate.

5) To classify the antibiotic resistances into different categories of resistance.

6) To detect plasmid-mediated quinolone resistance genes in the ciprofloxacin-resistant E

coli isolates.

1.5 Research Hypothesis

1.5.1 Null Hypothesis (Ho)

(i) Escherichia coli cannot be isolated from urine of healthy pregnant women attending

Nyanya General Hospital and Pan Raf Hospital in Nyanya, Abuja, Nigeria.

(ii) Escherichia coli, if isolated from urine of healthy pregnant women are not resistant to

quinolone and other antimicrobial agents commonly used to treat E. coli infections.

6
 (iii) Escherichia coli, if isolated and resistant to quinolone and other antimicrobial agents

commonly used to treat E. coli infections, do not carry qnrB, qnrS, oqxAB and aac-(6') -

Ib-cr plasmid-mediated quinolone resistance genes.

1.5.2 Alternate Hypothesis (Ha)

(i) Escherichia coli can be isolated from urine of healthy pregnant women attending

Nyanya General Hospital and Pan Raf Hospital in Nyanya, Abuja, Nigeria.

(ii) Escherichia coli, if isolated from urine of healthy pregnant women are resistant to

quinolone and other antimicrobial agents commonly used to treat E.coli infections.

(iii) Escherichia coli, if isolated and resistant to quinolone and other antimicrobial agents

commonly used to treat E.coli infections, carries qnrB, qnrS, oqxAB and aac-(6')-Ib-cr

plasmid-mediated quinolone resistance genes.

7
 CHAPTER TWO

LITERATURE REVIEW

2.1 Anatomy of the Urinary Tract

The urinary tract exits to a body surface area that is densely populated by a wide range of

microbes. Yet, under most normal circumstances, it is typically considered sterile, that is,

devoid of microbes, a stark contrast to the gastrointestinal and upper respiratory tracts

where many commensal and pathogenic microbes call home. Infection of the urinary tract

over a healthy person's lifetime is relatively infrequent, occurring once or twice or not at

all for most people. For those who do experience an initial infection, the great majority

(70% to 80%) thankfully do not go on to suffer from multiple episodes.

The mammalian urinary tract is a contiguous hollow-organ system whose primary

function is to collect, transport, store, and expel urine periodically and in a highly

coordinated fashion. In so doing, the urinary tract ensures the elimination of metabolic

products and toxic wastes generated in the kidneys. The process of constant urine flow in

the upper urinary tract and intermittent elimination from the lower urinary tract also plays

a crucially important part in cleansing the urinary tract, ridding it of microbes that might

have already gained access. When not eliminating urine, the urinary tract acts effectively

as a closed system, inaccessible to the microbes. Comprised, from proximal to distal, of

renal papillae, renal pelvis, ureters, bladder, and urethra, each component of the urinary

tract has distinct anatomic features and critical functions (Hickling et al., 2015).

8
The Upper Urinary-Collecting System

The renal papilla, into which each renal tubule-rich pyramid drains, is considered the first

gross structure of the upper collecting system. In humans and other higher mammals,

renal papillae are individually cupped by a minor calyx, which in turn narrows into an

infundibulum. Infundibuli vary in number, length, and diameter but consistently combine

to form either 2 or 3 major calyces. These branches are termed upper, middle, and lower-

pole calyces depending upon which pole of the kidney they drain. The renal pelvis

represents the confluence of these major calyceal branches and itself can vary greatly in

size and location (intra-renal vs extra-renal). In rodents, there is only one renal papilla

with a corresponding calyx. The ureters are bilateral fibromuscular tubes that drain urine

from the renal pelvis to the bladder. They are generally 22 - 30cm in length and course

through the retroperitoneum. They originate at the Ureteropelvic Junction (UPJ) behind

the renal artery and vein and then progress inferiorly along the anterior portion of the

psoas muscle. As the ureters enter the pelvic cavity they turn medially and cross in front

of the common iliac bifurcation. The ureters pierce the bladder wall obliquely (termed the

Ureterovesical Junction or UVJ and travel in this orientation for 1.5 to 2.0cm within the

bladder wall to terminate in the bladder lumen as ureteral orifices (Anderson & Cadeddu,

2012). The intramural ureter is compressed by the bladder wall passively during storage

and dynamically during emptying. This, in effect, prevents vesicoureteric reflux during

steady state and micturition. Along the length of the ureter there are three segments that

physiologically narrow: the ureteropelvic junction, the ureterovesical junction, and where

the ureters cross the common iliac vessels. These areas are clinically relevant as they

9
represent the most common locations where ureteral calculi become trapped, causing

obstruction. (Hickling et al., 2015).

Bladder and Urethra

The bladder is a hollow, distensible pelvic viscus that is tetrahedral when empty and

ovoid when filled. It is composed primarily of smooth muscle and collagen and, to a

much lesser degree, elastin. Its superior portion is defined by the urachus, a fibrous

remnant of the allantois. The urachus attaches the bladder apex to the anterior abdominal

wall. In males the bladder lies between the rectum and pubic symphysis and in females,

between the rectum and uterus/vagina. Anterioinferiorly and laterally, the bladder is

surrounded by retropubic and perivesical fat and connective tissue. This area is termed

the space of Retzius. The trigone of the bladder is a triangular region of smooth muscle

between the two ureteral orifices and the internal-urethral meatus. Grossly, thickened

muscle between the ureteral orifices (interureteric crest) and between each ureteral orifice

and the internal-urethral meatus (Bell's muscle) distinguishes the trigone from the rest of

the bladder. The classic view of bladder and trigone development proposes that the

trigone originates from the mesoderm-derived Wolffian ducts and the remainder of the

bladder is formed from the endoderm-derived urogenital sinus. Recent molecular

developmental studies challenge this concept. Thus, the Wolffian ducts have been shown

to undergo apoptosis during ureteral transposition and therefore do not contribute to

trigone formation (Hickling et al., 2015). In males, the bladder base rests on the

endopelvic fascia and the pelvic floor musculature, and the bladder neck is 3 to 4 cm

behind the symphysis pubis and is fixed by the endopelvic fasciae and the prostate. Here,

there is a layer of smooth muscle that surrounds the bladder neck and forms what is

10
known as the involuntary internal-urethral sphincter. In females, the base of the bladder

and urethra rest on the anterior wall of the vagina. The internal-urethral sphincter is not as

well developed in females (Chung & Sommer, 2012). The urethra is contiguous with the

bladder neck and begins at the distal end of the internalurethral sphincter.

In males the urethra is typically between 13 and 20 cm in length and is divided into

prostatic, membranous, and penile portions. The prostatic urethra is 3 - 4cm in length and

runs vertically through the length of the prostate. The membranous urethra spans 2 to 2.5

cm from the apex of prostate to the perineal membrane. This portion of the urethra is

completely surrounded by striated muscle known as the external-urethral sphincter. The

penile portion of the urethra is contained within the corpus spongiosum. It is on average

15- cm long, it dilates slightly in the glans penis (fossa navicularis) and terminates at the

external-urethral meatus. The female urethra, 3.8 to 5.1cm long, is considerably shorter

than the male one, and passes obliquely from the bladder neck to external-urethral meatus

along the anterior vaginal wall. The distal two-thirds of the female urethra are invested by

a slow twitch striated muscle termed the external-urethral sphincter (Chung & Sommer,

2012).

Vagina

Although not part of the urinary tract, the vagina plays an integral role in UTI

pathogenesis. It is a fibromuscular tube lined by epithelial cells. It extends from the

opening of the labia minora (vestibule) to the uterus with its anterior wall approximately

7.5-cm long and its posterior wall approximately 9-cm long. The anterior wall is related

to the bladder base superiorly and urethra inferiorly. Posteriorly, the vaginal wall is

separated from the rectum by the recto-uterine pouch superiorly and Denonvillier's fascia

11
 and the perineal body inferiorly. The inner vagina is covered by a non-keratinized,

stratified, squamous epithelium. With the onset of puberty, the vaginal epithelium

thickens and its superficial cells accumulate glycogen. There are no mucous glands, but

transudate from the underlying lamina propria and mucus from the cervical glands

lubricate the vagina. The muscular layers are composed of smooth muscle found in both

longitudinal and circular orientation (Hickling et al., 2015).

Normal vagina in reproductive women is populated by the lactobacilli which produces

lactic acid, producing a low pH condition highly unfavorable for the growth and

colonization by uropathogenic microbes (Stapleton et al., 2011). This constitutes one of

the major host defenses, as alterations in vaginal flora are considered a key predisposing

factor to UTIs.

2.2 Urinary Tract and Pregnancy

Urinary tract represents a sterile space with the exception of the distal urethra. Urinary

tract includes organs that collect, store and release urine from the body which include:

kidneys, ureters, bladder, urethra and accessory structures (Samaga, 2016). The urinary

tract has several systems to prevent infection (Amdekar et al., 2011). Most commonly, a

urinary tract infection occurs when gastrointestinal bacteria (bacteria in the gut) enter

through the urethra and start multiplying in the bladder. Our defense system is designed

to keep such germs out, but sometimes they fail, and bacteremia may take hold and

multiply into an infection. People with diabetes or problems with the body's natural

defense system are more likely to get UTI (Lane & Takhar, 2011) Despite the presence of

several antibacterial factors in urine such as pH, urea concentration, osmolarity, various

organic acids, salt content of the urine, urinary inhibitors to bacterial adherence (such as

12
Tamm-Horsfall protein or THP, bladder mucopolysaccharide, low molecular weight

oligosaccharide, secretory IgA and lactoferrin), the uropathogenic bacteria are able to

adhere, grow and resist against host defenses which finally result in colonization and

infection of the urinary tract (Cunha, 2010).

Pregnancy is a fundamental stage in life of every woman in reproductive age. The

immunologic features of normal pregnancy are unique because the mother tolerates the

semi allogenic embryo. The immunologic changes during pregnancy are very important

not only in normal tissues of the female reproductive tract but also in embryo-related

tissues created during pregnancy such as fetal membrane and placenta (Amirchaghmaghi

et al. 2013). Pregnancy presents with significant risk of complications which may lead to

morbidity and mortality for mother-baby pairs. Globally, more than 50% of neonatal

deaths occur in Sub-Saharan Africa because of infections and limited health resources

(UN, 2014; WHO, 2014).

Pregnancy causes numerous changes in the physiology of a woman's body. The

combination of mechanical/immunological, hormonal and physiologic/metabolic changes

during pregnancy contributes to aimed at supporting the growth of the fetoplacental unit

(Newbern &Freemark, 2011), or causes significant changes in the urinary tract, which

has a profound impact on the acquisition and natural history of bacteriuria during

pregnancy. Interestingly, a healthy pregnancy also induces dramatic changes in the

maternal gut microbiota over the course of gestation, with a great expansion of diversity

between individuals, an overall increase in Proteobacteria and Actinobacteria, and

reduced diversity within each microbiome. Similarly, the vaginal microbiome in

pregnancy is different from that of non-pregnant women, with lesser diversity and

13
richness, and with a dominance of Lactobacillus species, Clostridiales, Bacteroidales, and

Actinomycetales (Aagaard et al., 2012). At around 6th week of pregnancy, due to the

physiological changes of pregnancy the ureters begin to dilate ("hydronephrosis of

pregnancy"), which peaks at 22-26 weeks and continues to persist until delivery. Both

progesterone and estrogens levels increase during pregnancy and these will lead to

decreased ureteral and bladder tone. Increased plasma volume during pregnancy leads to

decrease urine concentration and increased bladder volume. The combination of all these

factors lead to urinary stasis and uretero-vesical reflux also, the apparent reduction in

immunity of pregnant women appears to encourage the growth of both commensal and

non-commensal microorganisms (Girishbubu et al., 2011; Samaga, 2016). The

physiological increase in plasma volume during pregnancy decreases urine and up to 70%

pregnant women develop glucosuria, which encourages bacterial growth in the urine.

Girishbubu et al., 2011; Samaga, 2016; Ghimire et al., 2017).

These changes, along with an already short urethra (approximately 3-4 cm in females)

increase the frequency of UTI in pregnant women (Johnson & Kim, 2012).

In general, pregnant women are considered immunocompromised UTI hosts because of

the physiological changes associated with pregnancy (Boye et al., 2012) which leads to

increase the risk of infections that could be either symptomatic or asymptomatic

(Girishbubu et al., 2011; Ghimire et al., 2017). It seems that the TLRs family during

normal or complicated human pregnancies as one of the main regulators of the innate

immunity, is not only involved in protecting the female reproductive tract against

invading pathogens, but also is a key regulator in immunologie events during stages of

normal pregnancy such as implantation or labor (Amirchaghmaghi et al., 2013). Maternal

14
 pregnancy complications significantly influence the bacterial composition and diversity

of the stool microbiota of premature infants, with these changes persisting during the first

year after birth (Chernikova et al., 2016). To manage the complications associated with

pregnancy and motherhood, many medicines are employed. Antibiotics remain important

in pregnancy and may be second to only iron and food supplement (de Tejada, 2014).

Due to limited health resources, such as laboratories to carry-out culture and sensitivity

test, the extent of exposure of pregnant women and babies to antibiotics and the effects

on their health may be underestimated. Antibiotic usage during pregnancy undoubtedly

affects the bacterial environment of the mother and of the fetus (Mensah et al., 2017).

However, medication use among pregnant women must focus not only on the intended

subject, the pregnant woman, but also on the unintended subject, the fetus, who is placed

at potential risk for a wide range of adverse effects. While a number of antenatal

medication exposures are known to cause birth defects, there is insufficient information

on the risks and safety for the vast majority of medications, whether they are obtained by

prescription or over-the-counter (OTC).

As a result, pregnant women may unknowingly take a medication that poses risk to their

fetus; on the other hand, anxiety about the potential teratogenic effects of medications

may discourage women from adhering to beneficial treatments (Mitchell et al., 2011).

2.3 Urine

Urine is a liquid by-product of metabolism in the bodies of many animals, including a

human which is produced by the kidneys during the process of filtration of blood.

Whilethe kidneys retain the essential salts and nutrients in the blood, they filter out the

15
unwanted substances. These unwanted substances are carried from the kidneys through

narrow tubes called ureters and stored as urine in the bladder. Urine is then flushed out

from the body through a duct called urethra. Urine is usually sterile in nature; however,

bacteria may sometimes travel to any part of the urinary tract. This may lead to the

growth of bacteria (Okorondu et al., 2013; //en.m.wikipedia.org.wiki/urine) Urine is fluid

with a variety of molecules and salts, some of which are waste products. It does not

usually have bacteria as a normal component, therefore when a bacterium by any means

get into the bladder and subsequently multiply, it may cause UTIs (Boye et al., 2012).

Urine formed in the kidney is a sterile fluid that serves as a good culture medium for

proliferation of bacteria (Samaga, 2016). Cellular metabolism generates numerous by-

products, many nitrogenous (rich in nitrogen), that require clearance from the

bloodstream. These by- products are eventually expelled from the body during urination,

the primary method for excreting water-soluble chemicals from the body. These

chemicals can be detected and analyzed by urinalysis. Of the many such substances that

exist, the three main nitrogenous wastes of the mammalian body are urea, uric acid, and

creatinine. Other dissolved ions, inorganic and organic compounds (proteins, hormones,

metabolites substances may be excreted due to injury or infection of the glomeruli of the

kidneys, which can alter the ability of the nephron to reabsorb or filter the different

components of the blood plasma, varying by what is introduced into the body. Average

urine production in adult humans is around 1.4L of urine per person per day with a

normal range of 0.6 to 2.6L per person per day, produced in around 6 to 8 urinations per

day depending on state of hydration, activity level, environmental factors, weight, and the

16
individual's health (Rose et al, 2015). Producing too much or too little urine needs

medical attention.

The physical characteristics of urine include:

(i) Colour- which varies in appearance, depending principally upon a body's level of

hydration, as well as other factors. Normal urine is a transparent solution ranging

from colourless to amber but is usually a pale yellow. In the urine of a healthy

individual the colour comes primarily from the presence of urobilin.

(ii) Odour - odour of normal human urine can reflect what has been consumed or

specific diseases (presence of glucose and ketones) or indicates the age of the urine.

(iii) Turbidity cloudy/transparency urine may be a symptom of a bacterial infection or

obstruction.

(iv) pH (acidity-alkalinity) - often influenced by diet, normally is within the range of 5.5

to 7 with an average of 6.2.

(v) Specific Gravity (density) Human urine has a specific gravity of 1.003-1.035. Any

deviations may be associated with urinary disorders (Rose et al., 2015). However,

the amount of urine a person produces depends on many factors, such as the

amounts of liquid and food a person consumes and the amount of fluid lost through

sweat and breathing, certain medications, medical conditions.

Despite the presence of several antibacterial factors in urine such as pH, urea

concentration, osmolarity, various organic acids, salt content of the urine, urinary

inhibitors to bacterial adherence (such as Tamm-Horsfall protein or THP, bladder

mucopolysaccharide, low molecular weight oligosaccharide, secretory IgA and

lactoferrin), the uropathogenic bacteria are able to adhere, grow and resist against host

17
 defenses which finally result in colonization and infection of the urinary tract (Cunha,

2010).

2.4 Bacteriuria

Bacteriuria is presence of bacteria in the urine and can be classified as symptomatic or

asymptomatic bacteriuria. A patient with asymptomatic bacteriuria is defined as having

colonization with one or more organisms in a urine specimen without symptoms or

infection. Symptomatic bacteriuria is associated with an infection in the urinary tract,

usually by a single organism. Bacteriuria without symptoms is not an infection. (Samaga,

2016; Crader&Lesile, 2021).

Asymptomatic bacteriuria (ASB) is defined as the "presence of actively multiplying

bacteria within the urinary tract excluding the distal urethra", at a time when the patient

has no urinary symptoms. Asymptomatic bacteriuria (ASB) is a microbial diagnosis

based on the isolation of a specified quantitative count of bacteriuria (that is greater than

or equal to 10 bacteria/ml) in a properly collected specimen of urine from pregnant

women without signs or symptoms of UTI. Thus, urine culture is the gold standard

screening technique for asymptomatic bacteriuria during pregnancy (Gayathree et al.,

2010; Girishbabu et al., 2011).

Bacteriuria may persist after delivery and may result into overt symptomatic infections

and chronic infections (Nabbugodi et al., 2014). Pregnant woman diagnosed with

bacteriuria are thus offered antibiotics to prevent complications (Sabir et al., 2014; Ayub

et al., 2016). Kerure et al., (2013) observed that the prevalence of bacteriuria in

18
 pregnancy may rise between 2-10%. Other studies in India had reported prevalence as

high as 8% (Balamurugan et al., 2012).

2.4.1 Asymptomatic Bacteriuria in Pregnancy

Asymptomatic bacteriuria is a common bacterial infection of the urinary tract requiring

medical treatment in pregnancy (Alfred et al., 2013). It gives a clear predisposition to the

development of symptomatic UTI, which in turn pose risk to mother and fetus

(Girishbabu et al., 2011). Among the pregnant women, approximately 4% to 10% will

have asymptomatic bacteriuria (ASB), and 1% to 4% will develop acute cystitis and 1%

to 2% may develop severe acute pyelonephritis during the second half of pregnancy

(Samaga, 2016). It has been associated with pre-term labour and low birth weight infants,

intrauterine growth restriction and fatal death, hypertension, preeclampsia, maternal

sepsis, respiratory insufficiency and anaemia (Moyo et al., 2010; Mohammed &Fareid

2012). The prevalence of asymptomatic bacteriuria has previously been reported to be 2-

13% (Amala&Nwokah 2015). The reasons are increased rate of urine formation during

pregnancy as a result of increase load of secretory products, the rate of glomerular

filtration which may increase up to 50% or more in pregnancy, progesterone and

relaxation hormone secreted during pregnancy, these have direct effect on the relaxation

of the ureter and renal pelvis with marked decrease in urethral peristalsis. Contamination

of the female urethra is enhanced by sexual intercourse pressure which can introduce

bacteria into the bladder consequently, coupled with honeymoon cystitis. The use of

contraceptives, diaphragm and spermatocides alters the normal flora of introitus causing

colonization by E. coli and other bacteria thereby predisposing to bacteriuria

19
 (Amala&Nwokah, 2015). The relative high prevalence of asymptomatic bacteriuria

during pregnancy and the consequences on women and their pregnancies justify

screening of pregnant women for bacteriuria to avoid the squeals with treatment

(Balamurugan et al., 2012; Patel et al., 2015).

2.5 Urinary Tract Infections

Urinary Tract Infections (UTIs) refer to the presence of a certain number of bacteria in

the urine (generally 10/ml) and symptomatic UTIs are classified in order of severity as

urosepsis syndrome, pyelonephritis (or upper UTI, with infection in the kidney) and

cystitis (or lower UTI, with bacteria into the bladder (Foxman, 2014; Smelov et al.,

2016).

Clinically, UTIs classification comprises either uncomplicated or complicated cases,

depending on the presence of structural or neurological urinary tract abnormalities

(Zacché & Giarenis, 2016). Complicated UTIs require prolonged therapy and occur in

patients with renal failure, anatomical urinary tract abnormalities such as urinary

obstruction and retention or in patients that use medical devices such as a catheter.

Complicated UTIs are also associated with immunosuppression and previous antibiotic

exposure. This category of UTIs increases the risk of chronic and/or recurrent infections.

Uncomplicated UTIs are found in patients who have no anatomical urinary tract

abnormalities and do not use the urinary tract instrumentation. In uncomplicated UTIs,

host immune response may successfully fight infection without antibiotic therapy.

Urinary tract infection is a social problem, widespread and affects people all over the

world human population. About 150 million people worldwide develop UTI each year,

20
 with high social costs (Flores-Mireles et al., 2015). It is estimated that 40% of women

develop at least one UTI episode during their lifetime (Braumbaugh et al., 2013; Micali

et al., 2014) and that 11% of women over 18 years have an episode of UTI per year

(Foxman, 2014), roughly eleven- million cases reported and treated due to UTIs in the

sole U.S. each year generating the costs estimated $5-6 billion annually (Foxman, 2014;

Mann et al., 2017).

Urinary tract infections (UTIs) are the most frequent bacterial infection in women

(Colgan et al., 2011). The organisms causing UTIs during pregnancy are the same as

those found in non- pregnant patients. Escherichia coli accounts for 80%-90% infections,

about 85% of community acquired UTIs, 50% of nosocomial UTIs and more than 80% of

uncomplicated pyelonephritis. This E. coli may be endogenous flora of the colon, first

colonize the periurethral area and vaginal introitus, then ascend to the bladder and from

the bladder to the renal pelvis by receptor mediated ascending process. The process

involves both host and bacterial factors, namely tissue receptors and expression of

bacterial attachment factors. Increase in concentration of amino acids and lactose during

pregnancy also encourages the growth of E. coli (Samaga, 2016).

2.5.1 Risk factors of Urinary Tract Infections

Anything that reduces bladder emptying or irritates the urinary tract can cause UTIs.

There are many factors which put someone at risk. Untreated asymptomatic bacteriuria is

a risk factor for acute cystitis (40%) and pyelonephritis (25-30%) in pregnancy (Alfred et

al., 2013).

21
 Gender - Women are more likely to get UTIs because their urethras are shorter, its

proximity to the vagina and anus and the inability of women to empty their bladder

completely.

Recurrent UTI- Three or more urinary tract infections within 12 months define the

recurring UTI, as well as two or more recurrences within 6 months. The same bacterial

species that caused previous infection is typically responsible for relapses.

Approximately 20-30% of adult women with an initial UTI will experience a recurrence

within 3-4 months; whereas, in children, about one third experiencing a UTI before the

age of one, will experience a recurrence within 3 years, and 18% of them will have a

recurrence within a few months. Recurrent UTIs can be introduced from different sources

and the same or different UTI- causing strains in the gut are able to (re)inoculate the

bladder. Alternatively, bacteria residing in the bladder epithelium are able to re-emerge

periodically and cause UTI recurrence (Silverman et al., 2013). In patients suffering from

recurrent UTIs, maintenance is ensured by antibiotic prophylaxis; however, in some cases

UTI needs to be treated by surgery (TolgandBagli, 2012). During pregnancy, recurrent

UTIs may be frequent and can cause severe adverse outcomes for the mother and the

baby, including preterm birth. The interventions in this setting can be pharmacologic

(antibiotics) or non-pharmacological (alternative remedies) (Schneeberger et al., 2012).

2.5.2 Clinical Manifestations and Pathogenesis

The clinical manifestation of UTI depend upon the portion of urinary tract involved,

etiological organism, the severity of infection and patient's ability to mount and immune

response to it. (Sibi et al., 2011; Ajide et al., 2016). Three common clinical

22
manifestations of UTIs in pregnancy are: asymptomatic bacteriuria, acute cystitis and

acute pyelonephritis. UTI during pregnancy contributes significantly to maternal and

perinatal morbidity. (Pegu et al., 2014) UTI in pregnant women is also characterized by

fever, flank pain and tenderness in addition to significant bacteriuria. Other symptoms

may include nausea, vomiting, frequent urination, urgency, dysuria, premature birth,

preterm labour and low birth weight infants, fetal complications like intra uterine growth

retardation/restriction and fatal acute respiratory distress and prematurity (Pegu et al.,

2014) hypertension, preeclampsia, maternal sepsis, respiratory insufficiency, and

maternal anaemia (Moyo et al., 2010; Mohammed et al., 2012). Abortion, phlebitis,

thrombosis and chronic pyelonephritis. Untreated UTI had also been reported to increase

the risk of transient renal failure, acute respiratory distress syndrome, sepsis, shock, and

hematological abnormalities (Boye et al., 2012; Obirikorang et al., 2012) has been

associated/ related to asymptomatic bacteriuria and urinary tract infections during

pregnancy (Pegu et al, 2014: Ajide et al., 2016).

The pathogenesis of UTIs in women begins with the colonization of the vaginal introitus

by uropathogens from the fecal flora, followed by ascension through the urethra into the

bladder.

Pyelonephritis develops when pathogens ascend to the kidneys via the ureters.

Pyelonephritis can also be caused by seeding of the kidneys from bacteremia and possible

that some cases are associated with seeding of the kidneys from bacteria in the

lymphatics (Ajide et al., 2016). Pyelonephritis (infection of the kidney) is potentially

more serious (Zeyaullah& Kaul, 2015).

23
 Acute Pyelonephritis occurs in 1-2% of pregnancies and is the most severe form of UTIs

and most common indication for antepartum hospitalization (Alemu et al., 2012).

Acute bacterial cystitis presents 1-4% of all pregnancies (Nabbugodi, 2014).

Cystitis often referred to as a bladder infection, is the most common type of UTIs

(Zeyaullah & Kaul, 2015).

2.5.3 Urinary tract infection in Pregnancy

Urinary tract infection in pregnancy is associated with significant morbidity for both

mother and baby (Samaga, 2016) and was about 4-10 times more common in pregnant

than in the nonpregnant women because there is a change in urine chemical composition

with increase in glucose and amino acids, which facilitate bacterial growth in urine

during pregnancy and its high frequency due to the combination of mechanical, hormonal

and physiological changes in the urinary tract which has a profound impact on the

acquisition and natural history of bacteriuria during pregnancy (Alfred et al., 2013;

Nabbugodi et al., 2014; Samaga, 2016). Urinary tract infections (UTIs), which are caused

by the presence and growth of microorganisms in the urinary tract, are perhaps the single

commonest bacterial infections of mankind and in pregnancy, it may involve the lower

urinary tract or the bladder (Samaga, 2016). UTI has been reported among 20% of the

pregnant women and it is the most common cause of admission in obstetrical wards.

Urinary tract infection is more prevalent in young primigravids in the second trimester,

diabetic mellitus, sickle cell disease, Human immunity suppression as in human

24
 immunosuppression virus, chronic drug abusers, and low socioeconomic status with poor

genital and perennial hygiene (Okonko et al., 2010; Hamdam et al., 2011).

The susceptibility to develop an UTI phenotype is related to several factors, as

dysfunctions of the urinary tract and/or genetic mechanisms involved in the innate

immune response control to infections (Koves & Wullt, 2016). In particular, the innate

immune system may respond either to UPEC patterns (pathogen-associated molecular

patterns; PAMPs) or to molecules derived from damaged or dying cells (danger/damage-

associated molecular patterns; DAMPs). Pattern recognition receptors (PRRs) recognized

these patterns in specialized immune cells, epithelia, and other tissues (Purves & Hughes,

2016). Assembling in the cytosol of multimeric protein complexes(inflammasomes)

occurs after sensing PAMPs or DAMPs structures that can be formed in both upper and

lower urinary tract (Guo et al., 2015). They trigger innate immune responses through

mechanisms depending or not from the production of proinflammatory cytokines (Purves

& Hughes, 2016).

2.6 Aetiology of Urinary Tract Infections

The bacteria belonging to the Enterobacteriaceae such as Escherichia coli, Klebsiella

pneumoniae, Proteus mirabilis, Citrobacter, Enterobacter, and other bacteria such as

Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus,

Staphylococcus saprophiticus, Enterococcus faecalis, Streptococcus bovis, and the

fungus Candida albicans, are the common etiological agents of UTIs (Reis et al., 2016;

Bartoloni et al., 2017; Hof, 2017; Mann et al.,2017), However, among the bacterial

species involved in UTIs, uropathogenic Escherichia coli strains (UPEC) are the most

common.

25
2.7 Escherichia coli

Escherichia coli (E.coli) is a Gram-negative, facultative anaerobic, motile and non-spore

forming bacterial species belonging to the genus Escherichia (Tenaillon et al., 2010; Yu

et al., 2014). It is a highly versatile bacterial species made up of harmless strains as well

as different pathogenic varients with the unique ability to cause either intestinal or

extraintestinal diseases (Danmap, 2012; Lupindu et al., 2017). E. coli was first identified

in 1885 by a German bacteriologist called Dr. Theodor Escherich. He identified the

organism from stools of babies with enteritis, a disease which causes nausea, vomiting,

stomach pain and diarrhea (Manning, 2010). E. coli as a human pathogen is very

significant, as it has been associated with a range of disease presentations such as

diarrhea, haemolytic uremic syndrome (HUS), surgical wound infection, haemorrhagic

colitis (HC), meningitis, septicaemia, dysentery and kidney infections. Some of these

diseases are fatal, especially in the elderly, young ones and immunosuppressed. Different

strains of E. coli are associated with different clinical outcomes (Olsen et al., 2016).

Escherichia coli is relatively easy to cultivate on ordinary laboratory media and there are

many formulations of differential and selective laboratory media that allow optimal

growth and survival of E. coli strains.

Eosine Methylene Blue (EMB) agar medium allows the differential growth of Gram-

negative bacteria, including E. coli, with the colonies being differentiated by a greenish-

metallic sheen colour in comparison to other bacterial species in the inoculum. In

MacConkey's (McC) medium, Gram-negative, lactose fermenting organisms are

differertiated based on their colour formation; E. coli is defined as a lactose fermenting

26
 organism, giving a distinct pinkish colour when grown on MacConkey medium

(Chander& Shrestha, 2013).

2.7.1 Classification

Escherichia coli belong to the Enterobacteriaceae family, and occur as pathogens and as

mammalian intestinal commensals (Riley, 2014); and has been reported to be one of the

most predominant bacterial causative organisms (El bouamri et al., 2015; Tajbakhsh et

al., 2016).

Domain: Bacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Enterobacteriales

Family: Enterobacteriaceae

Genus: Escherichia

Species: E. coli

Mobile genetic elements of E. coli can be horizontally exchanged in related bacteria or in

bacteria from different families, which allows their settlement in different environments.

E. coli strains can be classified into the following phylogenetic groups: A, B1, B2, and D

were identified based on the genomic Pathogenicity Islands (PAI) occurrence.

Commensal E. coli, with no pathogenic features, that occur, among others, on the

27
gastrointestinal tract mucosa, most often represent group A or B1. Pathogenic E. coli

cause/ responsible for a wide variety of intestinal and extraintestinal infections and

represent phylogenetic groups A, BI or D. The widespread geographical clonal

dissemination of intestinal pathogenic E. coli strains, such as E coli 0157:H7, is well

recognized, and its spread is most often attributed to contaminated food products.

Escherichia coli responsible for extraintestinal infections belong to groups B2 and D

(including 0157: H7), and strains belonging to B2 and D groups are isolated most

frequently from UTI (Tivendale et al., 2010; Köhler & Dobrindt, 2011; Totsika et al.,

2012; Riley, 2014; Kot et al 2016, Asadi Karam et al. 2019). Intestinal pathogenic E. coli

strains are generally divided into those that cause diarrhoea by: (i) expressing heat-labile

or heat-stable toxin (enterotoxigenic E. coli (ETEC)) or Shiga toxin (Shiga toxin-

producing E. coli (STEC). including enterohemorrhagic E. coli (EHEC)); (ii) invading

the intestinal mucosa (enteroinvasive E. coli (EIEC)); (iii) causing attaching and effacing

lesions in the intestinal mucosa (enteropathogenic E. coli (EPEC)); and (iv) other less

well-defined mechanisms (for example, enteroaggregative E. coli (EaggEC)). Whereas

ETEC, EIEC, EPEC, and EaggEC remain important causes of diarrhoea among children

in developing countries, the most prevalent intestinal pathogenic E. coli strains in

developed countries are STEC/EHEC. The most common EHEC, E. coli O157:H7, is

usually community-acquired via contaminated food or water.

A newly recognized clonal lineage belonging to serotype 0104:H4 that carries virulence

traits of both EHEC and EaggEC was responsible for a massive outbreak of bloody

diarrhoea, haemolytic-uraemic syndrome and death in Germany in 2011 that was traced

to contaminated bean sprouts.

28
 Widespread geographical clonal dissemination of intestinal pathogenic E. coli is

frequently detected, primarily because these organisms cause outbreaks of diarrhoea,

which can be readily investigated. These investigations often implicate food products that

enter domestic and global food distribution networks (Riley, 2014). The intestinal Ecoli

enters the urinary tract system and colonizes the periurethral and vaginal areas and the

urethra. After that, bacteria reach the bladder and attach to the surface epithelium using

fimbrial and nonfimbrial adhesins (Mann et al. 2017). Extraintestinal pathogenic E. coli

(ExPEC) have a complex phylogenetic structure, harbour diverse but specific/wide range

of virulence factors (VFs) with the potential to colonize highly specialized ecological

niches, such as the urogenital tract (Belanger et al., 2011, Kohler & Dobrindt, 2011), and

considerable plasticity of the genome. These strains not only cause uncomplicated UTIs,

extraintestinal pathogen, E. coli is the most common cause of community- acquired

urinary tract infections (UTIs) and the most common Gram-negative bacterium

associated with bloodstream infections (BSIs) in both developed and developing

countries. Community-onset UTIs or healthcare-associated BSIs caused by extraintestinal

pathogenic E coli (ExPEC) are not often recognized to occur as an outbreak or an

epidemic. However, EXPEC strains belonging to related lineages are distributed locally,

regionally, and globally (Riley. 2014)

The ExPEC group includes uropathogenic E. coli (UPEC), neonatal meningitis E. coli

(NMEC), sepsis-associated E. coli (SEPEC), and avian pathogenic E. coli (APEC), with

UTI being the most prevalent. The extraintestinal pathogenic E. coli (ExPEC) are a major

cause of UTI in pregnancy (Farshad et al., 2011).

2.7.2 Bacteriology

29
Escherichia coli is a Gram-negative, facultative anaerobic and nonsporulating bacterium,

bacilli (rod-shaped about 2.0µm long and 0.25-1.0µm in diameter, with a cell volume of

0.6-0.7µm3 (Yu et al., 2014), coliform bacterium of the genus Escherichia which

constitutes a major microflora of the lower intestines of warm-blooded organisms

(endotherms) (Tenaillon et al., 2010). It possesses adhesive fimbrae and a cell wall

consisting of the outer membrane made of lipopolysaccharides, a periplasmic space

having a peptidoglycan layer and a cytoplasmic membrane. The organism has a simple

cell structure with only one chromosomal DNA and plasmid and the ability to perform

complicated metabolism in order to maintain its cell growth and division and some of its

strains are piliated, also capable of transferring and accepting plasmid to and from other

bacteria. This unique characteristic enables E. coli to survive under bad conditions (Gray

et al., 2015). Escherichia coli is well adapted to its habitats, as it can grow in different

media, indicating media with glucose as its sole organic constituent (Kremling et al.,

2015). The wild-type E. coli has no growth factor requirements and is able to transform

glucose into molecular constituents of the cell. The bacterium is able to grow in the

presence or absence of oxygen. In the absence of oxygen, fermentation occurs, producing

mixed acids and gas (Shruthi et al., 2012). Escherichia coli responds to environmental

factors such as pH, temperature and chemicals. The presence or absence of chemicals can

be sensed by the bacterium, making it either swim towards or away from such chemicals

or gases (Murashko et al., 2017). The most commonly living microorganism of the

human gastrointestinal tract, and so is always found in faeces, making the bacterium a

sort indicator of faecal and water pollution and also the most common causative agent of

bacterial urinary tract infection and severe infections in many animals such as horses,

30
dogs, sheep, and humans (Bien et al., 2012; Tajbakhsh et al., 2016; Prince & Wildeboer,

2017). It is also found temporarily in environments such as vegetables and raw meat

(Samad et al., 2018; Luna-Guevara et al., 2019).

This common inhabitant of the gastrointestinal tract usually remains in a symbiotic

relationship with the host and plays a role in maintaining the homeostasis of the intestinal

tract.

Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in

their hosts, and are occasionally responsible for product recalls due to food contamination

(CDC, 2012). The harmless strains are part of the normal microbiota of the gut, and can

benefit their hosts by producing vitamin B12 and K. (Tsaku et al., 2017) (which helps

blood to clot) and preventing colonization of the intestine with pathogenic bacteria,

having a symbiotic relationship (in digestion and synthesis of certain vitamins). Strains of

E. coli, however, obtaining ability to colonize inside the urinary tract and to make

themselves safe from the host immune system, become uropathogenic E. coli.

Escherichia coli is expelled into the environment within fecal matter. The bacterium

grows massively in fresh fecal matter under aerobic conditions for 3 days, but its

numbers decline slowly afterwards.

Escherichia coli and other facultative anaerobes constitute about 0.1% of gut microbiota,

and fecal-oral transmission is the major route through which pathogenic strains of the

bacterium cause disease. Cells are able to survive outside the body for a limited amount

of time, which makes them potential indicator organisms to test environmental samples

for fecal contamination. A growing body of research, though, has examined

31
 environmentally persistent E. coli which can survive for and grow outside a host

(Montealegre et al., 2018).

It is classified into three groups: (i) commensal, (ii) intestinal pathogenic and (iii) extra-

intestinal pathogenic (Croxall, 2011). The ability of E. coli to colonize different

anatomical sites is due in part to genome plasticity and remodeling by acquisition or loss

of genetic material from which it acquired resistance or virulence factors. Therefore,

horizontal transfer is an important factor in the evolution and adaptation of E. coli to

different niches (Mellata, 2010). That is, due to its classification into different pathotypes

a common set of virulence factors and specific clinical manifestations, strains from a

single pathotype are not restricted to one phylogroup; such strains can share the same

genomic profile with other pathotypes (Didelot et al., 2012) and be distributed over the

entire span of the E. coli phylogenetic diversity (Vow et al., 2014), indicating a common

evolutionary origin and divergence into different pathotypes as a result of the

independent acquisition of specific virulence genes via multiple events of horizontal gene

transfer.

Therefore, E. coli represents one of the most sequenced microorganisms in public

databases, with draft and complete genome sequences available for strains isolated from

different hosts, disease associations, and geographic locations.

2.7.3 Uropathogenic Esherichia coli (UPEC)

Extraintestinal pathogenic E. coli (ExPEC) are a major cause of UTI in pregnancy

(Farshad et al., 2011). Among the ExPEC involved in UTIs, uropathogenic Escherichia

coli strains (UPEC) are the most common. Uropathogenic Escherichia coli (UPEC) is the

32
primary pathogen that account for about 80% of uncomplicated UTIs, 80%-95% of

community-acquired infections, and 50% of hospital-acquired infections (Foxman, 2014;

Flores-Mireles et al., 2015; Tabasi et al. 2016). UPEC also remains the most frequent

pathogen in complicated UTIs (Bartoletti et al. 2016). UPEC is a heterogeneous group of

extraintestinal pathogenic E.coli (ExPEC) that seem to originate from the gut (normal

microbiota). It colonizes the human intestine a few hours after birth and can be

introduced in many ways. In particular for females, the direction of wiping after

defecation (wiping back to front) can lead to fecal contamination of the urogenital

orifices. Many studies suggest that farm animals may be reservoirs of E. coli strains

carrying virulence genes responsible for UTI in humans. Comparison of the antimicrobial

resistance profiles and genetic virulence determinants in the Ecoli strains isolated from

UTI patients, farm animals or meat (particularly chicken) showed high similarity

(Jakobsen er al., 2010; Mellata et al., 2018).

Food-borne urinary tract infections (FUTI) include UTIs acquired from bacteria-

contaminated food (Nordstrom et al., 2013). ExPEC can survive in the alimentary tract

but do not cause diseases of the digestive system. However, ExPEC strains present in

other sites such as central nervous system, blood or the urinary tract may cause serious

illness. Adhering bacteria may be internalized into the uroepithelial facet cells, and then

they can enter the cytoplasm, replicate and form intracellular bacterial communities

(IBCs) being a source of quiescent intracellular reservoirs (QIRs) (Asadi Karam et al.,

2019). The host immune system may remove some of the IBCs by exfoliation of bladder

surface facet cells and excreting them with the urine but the remaining bacteria can grow

33
as a biofilm resistant to immune mechanisms and antibacterial agents (McLellan &

Hunstad, 2016).

Some of these bacteria escape from the biofilm, convert to motile form and disseminate

into the bladder lumen or even ascend into the kidneys, causing pyelonephritis (Flores-

Mireles et al., 2015). UPEC may also spread from the urinary tract to the bloodstream

causing bacteremia.

The interaction between bacteria and epithelial cells is a multifactorial and complex

phenomenon which involves several adhesins produced according to the stage of

infection, while adherence to epithelial cells is essential for successful colonization and

establishment; the expression of other genes encoding toxins, siderophores,

lipopolysaccharide (LPS), capsule, and invasins determines the disease severity and the

strain's virulence. UPEC strains can cause acute infections and recurrent infections that

do not respond to common antimicrobial treatments.

During UTIs, UPEC pathogenesis includes: (a) UPEC colonization of the periurethral and

vaginal areas with colonization the urethra; (b) ascending into the bladder lumen and

growth as planktonic cells in urine; (c) adherence to the surface and interaction with the

bladder epithelium defense system; (d) biofilm formation; (e) invasion and replication by

forming bladder Intracellular Bacterial Communities (IBCs) where quiescent intracellular

reservoirs (QIRs) form and reside in the underlying urothelium; (f) kidney colonization

and host tissue damage with increased risk for bacteremia/septicemia.

Replication of bacteria in the IBC can easily reach as many as 10º bacteria per cell;

furthermore, bacteria in the IBC undergo morphological changes, flux out of the infected

34
cell, and go onto infect neighboring cells (Flores-Mireles et al., 2015, Spaulding &

Hultgren, 2016). The flushing of urine removes most of the invading bacteria, along with

UPEC-filled exfoliated bladder epithelium cells (BECs).

UPEC colonize the bladder using a variety of virulence factors that therefore play critical

roles in UTI pathogenesis. for instance, pathogenic strains of E. coli express adherence

factors which form pili or fimbriae of different types for their attachment in the sites

where they usually do not live, and that contributes to their ability to cause disease. These

include surface structural components and predominantly includes: adhesions (P.S and

type 1 fimbriae), lipopolysaccharide (LPS), polysaccharide capsule, invasins, flagella,

outer-membrane vesicles, pili, curli, non-pilus adhesins, outer membrane proteins

(OMPs), UPEC can impair host immune system by a variety of ways (Bien et al., 2012;

Olson & Hunstad, 2016), such as toxins and iron acquisition systems, and these are called

secreted virulence factors (for example, alpha- hemolysin, cytotoxic necrotizing factor 1,

autotransporter toxins), iron/heme-acquisition systems, and iron ion transport, secretion

systems, and TonB-dependent iron-uptake receptors, including siderophore receptors are

responsible, among others, which are usually encoded on pathogenicity islands (PAIs),

plasmids, and other mobile genetic elements (Tivendale et al., 2010; Köhler & Dobrindt,

2011) for adhesion to epithelial cells of intestines, kidneys, or lower urinary tract, and for

stimulating cytokine production by T cells.

Moreover, they are an important colonization factor in extraintestinal infections. A

characteristic feature of UPEC is the ability to multiply intracellularly (Baldy-Chudzik et

al., 2015). The loss of a portion of the genome involved in the production of type 1

35
 fimbriae and inactivation of genes encoding P fimbriae have led to the formation of

strains responsible for asymptomatic bacteriuria (ASB).

These strains can colonize the urinary tract without inducing inflammation. A potential

source of UPEC is host own intestinal flora, but the infection can also be transmitted

through the fecal-oral route or through sexual contact (Terlizzi et al., 2017). Fimbrial

adhesins such as PapG and CsgA are virulence factors that facilitate the attachment of E.

coli. In animal models, type 1 fimbriae aggrandize the chance survival of E. coli (Vizcara

et al., 2016). The production of these virulence factors by UPEC may cause an

inflammatory response which makes a possible pathway for UTI symptoms (Bien et al.,

2012). However, both the host and the uropathogenic E. coli strain has different roles in

the establishment and colonization process in the urinary tract (Parvez & Rahman, 2018).

2.8 Antimicrobial Treatment of Escherichia coli UTI

The treatment and control of UTIs are accomplished through therapy with different

classes of antibiotics namely B-lactams, fluoroquinolones, aminoglycosides,

sulfamethoxazole/trimethoprim, nitrofurantoin and cephalosporins, particularly the third

and fourth generation cephalosporins which have greater activity against gram-negative

bacteria, the main etiological agents of community acquired UTI (Lawal, 2012; Raji et

al., 2013; Reis et al., 2016).

Resistance developed against these classes of antimicrobial agents and others by bacteria

have limited their successful application to manage UTIs. The increasing therapeutic

failure observed in empirical treatment using antimicrobial agents has made it important

to identify the pattern of susceptibility and resistance of bacterial agents, through in vitro

36
 antimicrobial susceptibility testing, which can guide the therapeutic approach (Reis et al.,

2016).

2.9 Chemistry and Mechanism of Action of Antimicrobial Agents Tested

2.9.1 Ampicillin (AMP)

Chemistry: It has a molecular formula C16H19N3O4S. Ampicillin is a broad-spectrum,

semi-synthetic, beta-lactam penicillin antibiotic with bactericidal activity. It binds to and

inactivates penicillin-binding proteins (PBP) located on the inner membrane of the

bacterial cell wall. Inactivation of PBPs interferes with the cross-linkage of peptidoglycan

chains necessary for bacterial cell wall strength and rigidity (pubchem.ncbi.nlm.nih.gov).

Mechanism of Action: Ampicillin is in the penicillin group of beta-lactam antibiotics

and is part of aminopenicillin family. It able to penetrate Gram-positive and some Gram-

negative bacteria. It differs from penicillin G, or benzylpenicillin, only by the presence of

an amino group which present on both ampicillin and amoxillin that helps these

antibiotics pass through the pores of the outer membrane of Gram-negative bacteria

(Hauser, 2012; ASHSP, 2015). Ampicillin acts as an irreversible inhibitor of the enzyme

transpeptidase, which is needed by bacteria to make the cell wall. It inhibits the third and

final stage of bacterial cell wall synthesis in binary fission, which ultimately leads to cell

lysis (usually bacteriolytic) (Petri, 2011; ASHSP, 2015).

2.9.2 Trimethoprim/Sulfamethoxazole (TMP/SMX)

Chemistry: It is a dose combination consist of one-part Trimethoprim (Dihydrofolate

reductase inhibitor) to five parts Sulfamethoxazole (Sulfonamide antibiotic) (Hamilton,

2015).

37
 Mechanism of Action: Trimethoprim serve as a competitive inhibitor of dihydrofolate

reductase (DHFR), hence inhibiting the de novo synthesis of tetrahydrofolate, the

biologically active form of folate. Sulfamethoxazole, a sulfanilamide is a structural

analog of para-aminobenzoic acid (PABA). They compete with PABA to bind to

dihydropteroate synthetase and inhibit conversion of PABA and dihydropteroate

dihydrosphate to dihydrofolic acid, or dihydrofolate. Inhibiting the production of

dihydrofolate intermediate interferes with the normal bacterial synthesis of folic acid

(folate). Trimethoprim and sulfamethoxazole have a greater effect when given together

than given separately, because they inhibit successive steps in the folate synthesis

pathway (potentiated sulfonamide). They result in synergistic, bactericidal actions on

susceptible organisms (Boothe, 2015; www. Merckvetmanual.com. Retrieved 2015).

2.9.3 Cephalosporins

Chemistry: Several cephalosporins are associated with hypoprothrombinemia and a

disulfiram- like reaction with alcohol, due to the N-methylthiotetrazole side-chain of

these cephalosporins, which blocks the enzyme vitamin K epoxide reductase causing

hypothrombinemia and aldehyde dehydrogenase - causing alcohol intolerance (Stork,

2006).

Mechanism of Action: Cephalosporins are bactericidal and have the same mode of

action as other beta-lactam antibiotics (such as penicillins), but are less susceptible to

beta-lactamases. Cephalosporins distrupt the synthesis of the peptidoglycan layer forming

the bacterial cell wall. (structural integrity). The final transpeptidation step in the

synthesis of the peptidoglycan is facilitated by penicillin-binding proteins (PBP). PBPs

bind to the D-Ala-D-Ala at the end of muropeptides (peptidoglycan precursors) to

38
 crosslink the peptidoglycan. Beta-lactam antibiotics mimic the D-Ala-D-Ala site, thereby

irreversibly inhibiting PBP crosslinking of peptidoglycan

(https://en.m.wikipedia.org/wiki/Cephalosporins).

2.9.4 Amoxicillin Clavulanic Acid

Chemistry: It has molecular formula C24H27KN4O10S. It is a semisynthetic beta-lactamase

inhibitor isolated from streptomyces. Clavulanic acid contains a beta-lactam ring and

binds strongly to beta-lactamase at or near its active site, thereby hindering enzymatic

activity; which protects other beta-lactam antibiotics from beta-lactamase catalysis,

thereby enhancing their antibacterial effects.

(https://pubchem.ncbi.nih.gov/compound/clavulanic-acid).

Mechanism of Action: Amoxicillin clavulanic acid is a beta-lactamase inhibitor often

used in conjunction with amoxicillin to broaden its spectrum further and combat resistant

amoxicillin and ampicillin. It has little to no antimicrobial activity of its own and instead

works by preventing bacterial destruction of beta-lactams. (Evans et al., 2021).

2.9.5 Gentamicin

Chemistry: Its molecular formula is C21H43N5O7 and belongs to aminoglycosides group.

It works by disrupting the ability of the bacteria to make proteins, which typically kills

the bacteria from performing vital functions needed for survival. Gentamicin is naturally

produced by the bacterium Micromonospora purpurea, (ASHSP, 2015). Gentamicin is

derived from the species. Micromonospora, the backbone for this antibiotic is the

aminocyclitol 2-deoxystreptamine (https://pubchem.ncbi.nlm.nih.gov/compound/2-

Deoxy streptamine; Dewick, 2009).

39
The six-carbon ring is substituted at the carbon positions 4 and 6 by the amino sugar

molecules cyclic purpurosamine and garosamine

(https://pubchem.ncbi.nlm.nih.gov/compound/Garosamine), respectively. (Kumar, et al.,

2008; Yu et al., 2017).

Mechanism of Action: Gentamicin is a bactericidal antibiotic that works by binding the

30S subunit of the bacterial ribosome, negatively impacting protein synthesis. The

primary mechanism of action is worked through ablating the ability of the ribosome to

discriminate on proper transfer RNA and messenger RNA interactions. Typically, if an

incorrect tRNA pairs with an mRNA codon at the aminoacyl site of the ribosome,

adenosines 1492 and 1493 are excluded from the interaction and retract, signaling the

ribosome to reject the aminoacylated tRNA. (Dao et al., 2018). However, when

gentamicin binds at helix 44 of the 16S rRNA, it forces the adenosines to maintain the

position they take when there is a correct, or cognate, match between aa-tRNA and

mRNA. (Wilson, 2014). This leads to the acceptance of incorrect aatRNAs, causing the

ribosome to synthesize proteins with wrong amino acids placed throughout (roughly

every 1 in 500). The non-functional, mistranslated proteins misfold and aggregate,

eventually leading to death of the bacterium. A secondary mechanism was based on

crystal structures of gentamicin in a secondary binding site at helix 69 of the 23S rRNA,

which interacts with helix 44 and proteins that recognize stop codons. At this secondary

site, gentamicin is believed to preclude interactions of the ribosome with ribosome

recycling factors, causing the two subunits of the ribosome to stay complexed even after

translation completes. This creates a pool of inactive ribosomes that can no longer re-

initiate and translate new proteins. (Borovinskaya et al., 2007).

40
2.9.6 Nitrofurantoin:

Chemistry: Nitrofurantoin, a chemotherapeutic compound of the nitrofuran family. It is

a synthetic antimicrobial derived from furan by the addition of a nitro group and a side

chain containing hydantoin. Nitrofurantoin is a weak acid and its solubility is affected by

PH (Munoz-Davila, 2014).

Mechanism of Action: It is a bacteriostatic medication. Its activity is based on inducing

lipid oxidation, as well as damage to bacterial cell walls, ribosomal proteins and DNA

strands (Glaser & Schaeffer, 2015). The nitrogroup coupled onto the heterocyclic furan

ring represents the specific active site of the drug and has to be activated by microbial

nitroreductases (Koulaouzidis et al., 2007).

2.9.7 Ciprofloxacin

Chemistry: Ciprofloxacin belong to the drug class of fluoroquinolone with molecular

formula C17H18FN3O3. Ciprofloxacin is 1-cyclopropyl-6-fluoro-1, 4-dihydro-4-oxo-7-(1-

piperazinyl)-3- quinolinecarboxylic acid. It is a faintly yellowish to light yellow

crystalline substance (FDA, 2009).

Mechanism of Action: Ciprofloxacin is a broad-spectrum antibiotic of the

fluoroquinolone class. It is active against some Gram-positive and many Gram-negative

bacteria. It inhibits a type II topoisomerase (DNA gyrase) and topoisomerase IV

(Pommier et al., 2010), necessary to separate bacterial DNA, thereby inhibiting cell

division.

2.10 Antimicrobial Resistance

41
Antimicrobial resistance is a serious public health problem resulting in increased

morbidity and mortality. In urinary tract infections, resistance rates against commonly

prescribed antibiotics are constantly rising. Nowadays, in many countries more than 20%

of uropathogens are resistant to Trimethoprim/Sulfamethoxazole (TMP/SMX) and

cephalosporins. This increasing resistance is also being observed for Flouroquinolones

with resistance rates rising up to 10%. Worldwide, Flouroquinolones are being used as

the most common antimicrobials for all UTIs, both complicated and uncomplicated.

Antibiotics are invariably used for the treatment of UTIs, though resistance to antibiotics

has been reported all over the world, particularly in developing countries. Treatment of

UTIs is a challenge due to the increasing level of antimicrobial resistance (Zeyaullah &

Vinod, 2015). There is an increased emergence of antimicrobial resistance in the

uropathogens, probably due to the empirical administration of anti-bacterial therapy, even

before the availability of the urine culture results, is a matter of concern worldwide

(Oladeinde et al., 2011). The prevalence of antimicrobial resistance in patients with UTI

is increasing and can vary according to geographical and regional location. Many factors

play a role in the emergence of resistance such as from poor utilization of antimicrobial

agents, transmission of resistant bacteria from patient to patient and from Health-care

workers (HCWs) to patients and vice versa, lack of guidelines for appropriate and

judicious use of antimicrobial agents and lack of easy-to-use auditing tools for restriction.

In addition, there is clear misuse of antimicrobial agents in the animal industry, and most

agents are the same agents used in humans.

All these factors, together, have led to the inevitable emergence and rise of resistance

(Aly & Balkhy, 2012). The emergence of antibiotic resistance is increased because of the

42
 random use of antibiotics and subsequent redundancy of antibiotics. The ineffectiveness

of antibiotics for treating UTIs is associated with antibiotic resistance of uropathogens,

therefore the range of antibiotics available to treat infections becomes a limit, well as

antibiotic use in pregnancy, will be harmful to the fetus. The risk of spontaneous

miscarriages due to the using of antibiotics during the period of pregnancy with the

infections of urinary tract. (Muanda et al., 2017). Multidrug resistant bacteria (MDR)

making treatment a difficult task and therefore increase threat to both mother and fetus

and will minimize chance of prescribing safe antibiotic (Nwadike et al., 2015). Bacteria

develop antimicrobial resistance through many mechanisms including mutations in

penicillin binding proteins, efflux mechanisms, alterations in outer membrane proteins

and the production of hydrolyzing enzymes such as extended spectrum ẞ lactamase

(ESBL) and carbapenemases (Zeyaullah & Vinod, 2015). These resistance mechanisms

may be encoded on transferable genes which facilitate the spread between bacteria of the

same species and between different species. Other resistance mechanisms may be due to

alterations in the chromosomal DNA (that is, bacterial genome by mutation or acquisition

by horizontal transfer of an extrachromosomal or chromosomal material which enables

the bacteria to withstand the harsh environment and multiply.

2.10.1 Antimicrobial resistance in Escherichia coli from urine

Urinary tract infections (UTIs) are associated with significant use of antibiotics that cause

implications for bacterial ecology and spread of resistance to antibiotics, especially when

it stems from the empirical antimicrobial treatment of recurrent UTIs.

43
 Antimicrobial resistance in UPEC and the spreading of MDR UPEC in recent decades is

a clinical problem, particularly in women with recurrent UTIs. The increasing frequency

of MDR UPEC, especially in developing countries, results in excessive use of broad-

spectrum antibiotics such as fluoroquinolones, cephalosporins, and aminoglycosides that

raise the cost of treatment and hospitalization (Bartoletti et al., 2016; Sanchez et al.,

2016). Antimicrobial resistance between UPEC is increasing in many countries.

2.10.1.1 Resistance to Nitrofurantoin

Nitrofurantoin is recommended for the treatment of uncomplicated cystitis as first-line

empiric therapy. At present, the resistance of UPEC to nitrofurantoin is very low,

favoring its use as a first-line antibacterial agent and retains a high level of antibiotic

activity against urinary E.coli as the drug of choice for the treatment of uncomplicated

cystitis, although it should not be used for the treatment of pyelonephritis since its

concentration in the renal parenchyma is too low. Additional characteristics, as high

efficiency, cost-effectiveness, and weak adverse environmental impact suggest that

nitrofurantoin should be the first-choice treatment in uncomplicated UTI in women (Kot,

2019).

2.10.1.2 Resistance to trimethoprim-sulfamethoxazole

Resistance to trimethoprim-sulfamethoxazole (TMP-SMZ) is another important and

widely used first-line antimicrobial in the treatment of uncomplicated cystitis. Yamaji et

al. (2018) showed on comparative study in USA that frequencies of TMP-SMZ resistance

in UPEC isolates obtained from outpatients with UTI symptoms in 1999-2000 and in

2016-2017did not significantly change (resistance slightly increased from 16.9% to

44
 17.1%). However, increasing resistance to this drug has recently been observed in many

countries. A majority of studies show resistance at or above the accepted level of 20%,

and thus TMP-SMZ should not be used in empiric treatment (Bartoletti et al., 2016).

High resistance rate to TMP-SMZ (24.5%) among E. coli isolated from urine in 2012-

2015 was reported in Switzerland (Erb et al., 2018). The considerable geographic and

age-related differences in resistance of UPEC to TMP-SMZ were observed, and the

highest resistance was poted in certain regions of Europe in young women and in India,

Brazil, Iran, Ethiopia, Mongola and Jordan (Cunha et al., 2016; Munkhdelger et al.,

2017; RegasaDadi et al., 2018; Shakhatreh et al. 2018; Prasada et al., 2019). The activity

of TMP-SMZ against UPEC in other regions of the world was significantly lower as the

frequencies of TMP-SMZ resistant UPEC isolates ranged from 72.7% in Mexico to 82%

in Pakistan (Ali et al. 2016; Ramirez-Castillo et al, 2018). These results indicate that in

many countries TMP-SMZ should not be used in empiric UTI therapy due to the high

frequency of UPEC resistant to this antimicrobial.

2.10.1.3 Resistance to amoxicillin-clavulanic acid

Amoxicillin-clavulanic acid was recommended as first-line therapy for mild and

moderate pyelonephritis or complicated UTI or as an alternative empiric therapy for

uncomplicated cystitis Cheung et al., 2017). Resistance rates for amoxicillin-clavulanic

acid vary regionally. These results demonstrate that the levels of UPEC resistance to

amoxicillin-clavulanic acid varied between geographical regions or patient populations.

Therefore, the empiric regimens for uncomplicated and complicated UTIs should be

guided by the local susceptibility of E.coli. However, definitive regimens should be

developed according to the susceptibility results of UPEC, when available.

45
2.10.1.4 Resistance to fluoroquinolones

Quinolone antimicrobials are widely used extensively in clinical medicine/applications

(Kim & Hooper, 2014). An important addition of a fluorine substituent at position C6

added to potency and became the common feature of the fluoroquinolone class with the

introductions of porfloxacin in 1986 and ciprofloxacin in 1987 that showed substantially

greater potency against Gram-negative bacteria. Because of their potency, spectrum of

activity, oral bioavailability, and generally good safety profile, fluoroquinolones were

used widely for a range of clinical indications worldwide and are the only current class of

agents that directly inhibit bacterial DNA synthesis. Quinolones dually target DNA

gyrase and topoisomerase IV binding to specific domains and conformations so as to

block DNA strand passage catalysis and stabilize DNA - enzyme complexes that block

the DNA replication apparatus and generate double breaks in DNA that underlie their

bactericidal activity. Resistance has emerged with clinical use of these agents and is

common in some bacterial pathogens. Mechanisms of resistance include mutational

alterations in drug target affinity and efflux pump expression and acquisition of

resistance- conferring plasmid- encoded genes. Resistance mutations in one or both of the

two drug target enzymes are commonly in a localized domain of the GyrA and ParC

subunits of gyrase and topoisomerase IV, respectively, and reduce drug binding to the

enzyme-DNA complex. Other resistance mutations occur in regulatory genes that control

the expression of native efflux pumps in the bacterial membrane(s). These pumps have

broad substrate profiles that include other antimicrobials as well as quinolones. Mutations

of both types can accumulate with selection pressure and produce highly resistant strains.

46
Resistance genes acquired on plasmids confer low-level resistance that promotes the

selection of mutational high-level resistance.

Plasmid-encoded resistance is because of Qnr proteins that protect the target enzymes

from quinolone action, a mutant aminoglycoside-modifying enzyme that also modifies

certain quinolones, and mobile efflux pumps. Plasmids with these mechanisms often

encode additional antimicrobial resistances and can transfer multidrug resistance that

includes quinolones (Hooper & Jacoby, 2016).

Fluoroquinolones (FQs) are recommended for empirical oral antimicrobial therapy in

uncomplicated pyelonephritis (Bonkat et al., 2017) and they are frequently used for the

treatment of UTIs (Drekonja et al., 2013; Yamasaki et al., 2015; Walker et al., 2016).

The increasing emergence of E. coli resistant to FQs was reported worldwide, and it has

emerged probably due to the excessive use of these antibiotics. The resistance of UPEC

to FQs was reported from different countries and the level of resistance is significant. The

results of Prasada et al. (2019) revealed a high rate of fluoroquinolone resistance in

UPEC in India (>60%). The results of the meta-analysis on resistance to ciprofloxacin in

the community and hospital-acquired UTIs showed that UPEC resistance to ciprofloxacin

was higher in the hospital when compared to the community setting (Fasugba et al.,

2015). In Europe, resistance to FQs was reported in 22% of strains, and the prevalence of

fluoroquinolone-resistant UPEC strains was about 31% among hospitalized patients in

the United States (Asadi Karam et al., 2019). In many parts of the world, >20% of E. coli

isolated from patients with community-acquired uncomplicated UTI and >50% of E. coli

isolated from complicated UTI showed resistance to FQs (Talan et al., 2016). In Poland,

resistance to FQs was observed in about 30% of UPEC (Michno et al., 2018). MDR

47
 UPEC demonstrated much higher rates of resistance to FQs, ranging from 49% to 72%

(Walker et al., 2016).

Among FQs, ciprofloxacin is the most commonly prescribed for UTIs because it is

available in eral and intravenous preparations. In the group of UPEC strains isolated in

2013-2014 from cutpatients in Brasilia, 18.8% were resistant to ciprofloxacin, and

resistance to ciprofloxacin was associated with multidrug resistance (Moreira da Silva et

al., 2017). In the United States in the period from 2013 to 2014, 12.1% of E.coli isolates

from patients with acute uncomplicated and complicated pyelonephritis were resistant to

this antibiotic (Talan et al., 2016). A similar percentage of UPEC resistant to

ciprofloxacin was noted in other research conducted in the United States in 2016-2017

for isolates from the urine samples from outpatients with symptoms of UTI (Yamaji et

al., 2018).. Resistance to ciprofloxacin was significantly higher in developing countries

(Ethiopia 85.5%, Nepal 64.6%, Pakistan 60.8%, Mongolia 58.1%, Jordan 55.5%) (Ali et

al., 2016; Khatri et al., 2017; Munkhdelger et al., 2017; RegasaDadi et al., 2018;

Shakhatreh et al., 2018) than in developed countries (USA 5.1%, Germany 10.5%,

Switzerland-17.4%, France-24.8%) (Erb et al., 2018; Hitzenbichler et al., 2018; Yamaji

et al., 2018). The widespread use of fluoroquinolones in the outpatients is the cause of the

continuous increase in resistance to this drug. Therefore, ciprofloxacin should be avoided

in first-line treatment of UTIs and be used only in more severe infections or as an

alternative when the recommended agents cannot be used. Restrictions of ciprofloxacin

usage should be enhanced especially in developing countries, where no regulations

concerning the use of this antibiotic currently apply (Asadi Karam et al., 2019).

2.10.1.5 Resistance to Other Antibiotics

48
 Some UPEC isolates can be resistant to ampicillin and first-generation oral

cephalosporins (Moya-Dionisio et al., 2016). The resistance to cefuroxime (second

generation cephalosporin) in Belgium, Germany, and Spain was 5.5%, 12.8%, and

16.6%, respectively (Kresken et al., 2016).

Escherichia coli isolates from the urine samples from hospitalized patients in England

were found to be resistant (13.8-21.3%) to third-generation cephalosporins

(cefotaxime/ceftazidime) (Abernethy et al., 2017). In Mexico, the resistance rates to

antibiotics belonging to aminoglycosides were 28.2%, 19.1%, 10%, and 5.5% for

gentamicin, tobramycin, amikacin, and netilmicin, respectively (Ramírez-Castillo et al.,

2018). However, a recent study from Saudi Arabia about the presence of carbapenem-

resistant uropathogenic E. coli clones in community acquired UTIs reported the

occurrence of carbapenemases and 5 (the New Delhi metallo-lactamase), and

carbapenemases of the OXA-181 type in these strains (Abd El Ghany et al., 2018). The

emergence of carbapenem-resistant uropathogenic E. coli isolates makes treatment of

these infections increasingly challenging.

2.11 Mechanisms of uropathogenic Escherichia coli resistance to antibiotics

Resistance to β-lactams is related to the production of different types of β-lactamase

enzymes. Among the genes often located on plasmids are those coding multiple types of

β-lactamases (bla genes) (Adamus-Białek et al., 2018). β-lactamases hydrolyze the amide

bond of the four- membered β-lactam ring of β-lactam antibiotics (penicillin,

cephalosporin, monobactams, and carbapenems). ESBL are enzymes that confer

resistance to β-lactam antibiotics (all penicillins, cephalosporins, and monobactams),

except for carbapenems, cephamycins, and β-lactamase inhibitors. ESBL are the

49
predominant source of Enterobacteriaceae resistance to 3rd and 4th- generation

cephalosporins and they developed as a result of mutations in the genes coding for

ancestral enzymes blareM-1, blaTEM-2, and blasSHV-1. Three classes of β-lactamases

including TEM and SHV, and since 2000 a new group of ESBL, CTX-M

(cefotaximases), were observed among ESBL produced by UPEC (Ojdana et al., 2014;

Shahbazi et al., 2018).

The CTX-M enzymes are active against cefotaxime and ceftriaxone and less active

against ceftazidime (Bhat et al., 2012) UPEC producing ESBL are particularly often

detected in developing countries (Iran - 37.1%, Nepal -38.9%, Pakistan-40%, and Jordan

- about 50%) (Ali et al., 2016; Parajuli et al, 2017; Shakhatreh et al., 2018). Prasada et al.

(2019) revealed that in India the percentage of ESBL-producing UPEC increased from

45.2 to 59.6% over 5 years (2013-2017). The frequency of ESBL producing E coli

isolates is different in various parts of the world and sometimes even in various hospitals

within the country.

In addition to resistance to B-lactam antibiotics, ESBL producing E.coli isolates are also

resistant to other antimicrobial agents, such as aminoglycosides, tetracycline, and

trimethoprim/sulfamethoxazole (Rezai et al., 2015). Shahbazi et al. (2018) has found that

higher number of ESBL-producing UPEC isolates were resistant to aminoglycosides and

quinolones when compared to the UPEC strains that not produce ESBL. Carbapenems

(imipenem and meropenem) represent the best option for the treatment of UTIs caused by

ESBL-producing strains (Idil et al., 2016). Cephalosporins, penicillins, and monobactams

should be used with ẞ- lactamase inhibitors (Bartoletti et al., 2016).

50
Quinolones and fluoroquinolones are extensively used worldwide in the treatment of

UTIs because of their efficacy against a broad spectrum of gram-positive and, in

particular, gram-negative bacteria, lesser side effects and convenient oral dosages

(Redgrave et al., 2014, Aldred, et al., 2014, Röderova et al., 2016, Yeh et al., 2017) and

their common use led to increased resistance in UPEC. For years, quinolone resistance

was thought to occur through chromosomal mechanisms including mutations in DNA

gyrase and topoisomerase genes on quinolone resistance determining regions (QRDRs),

active export of the drugs via efflux pumps and also decreased permeability related to

porin loss (Ruiz et al., 2012).

The mechanism of fluoroquinolone action is based on binding to and impeding the

activity of topoisomerase II (DNA gyrase) and topoisomerase IV (parC and parE).

DNA gyrase is encoded by the gyrA and gyrB genes (PourahmadJaktaji&Mohiti, 2010).

The resistance of E. coli to quinolones frequently results from a mutation in the gyrA and

gyrB genes that catalyze DNA supercoiling. The point mutations in gyrA protein N-

terminal sequence (amino acids 67 (Ala-67) to 106 (Gln-106)) strongly correlate with

phenotypic resistance to quinolones and fluoroquinolones, and this sequence is named a

quinolone resistance-determining region (QRDR); carriage of the plasmid-mediated

quinolone resistance (PMQR) genes; and reduced accumulation of drugs or chemicals

due to active efflux pump activity (Yeh et al., 2017,Osei & Amoako, 2017).

Change in genes encoding the targeted enzymes. DNA gyrase and topoisomerase IV

are the two targeted enzymes of quinolones. The quinolone resistance associated with

chromosomal mutations occurs due to errors in the replication of the genes encoding the

GyrA subunits of DNA gyrase and ParC of topoisomerase IV (Hooper and Jacoby, 2015,

51
2016). Mutation in single amino acids in either one of these two enzymes weakens the

interaction between the quinolones and enzymes, reducing quinolone susceptibility.

These mutations have been reported to be primarily located on the amino terminal

domains of GyrA or ParC of the enzymes. The most common mutated amino acids are

the serine residue and an acidic residue (glutamic acid or aspartic acid) four amino acids

away. Mutations of Ser83 and Asp87 are the most common resistance mutations in GyrA

of E. coli, with similar mutations in other species at the equivalent positions. (Pham et al.,

2019) This domain of the enzymes has been shown to be responsible for anchoring the

water-metal ion bridge, which is termed as the quinolone resistance determining region

(QRDR). Mutations at this QRDR disrupt the water-metal ion bridge, thereby reducing

drug affinity for the enzyme-DNA complex. Mutation at the serine accounts for more

than 90% of the mutant pool, followed by the mutations at the acidic residue (Redgrave

et al., 2014). Mutations at the serine residue on GyrA and ParC appear to have little

effect the catalytic activity of the enzymes.

However, the mutations at the acidic residue were reported to significantly reduce the

catalytic activity from 5 to 10-fold (Aldred et al., 2014). Presumably this explains why

mutation occurs more often at the serine residue, as it does not impact enzyme activity. It

is notable that the serine residue is highly conserved across bacterial species despite its

minimal contribution to the activity of the enzymes.

Based on a study on nybomycin on Streptomyces spp, it was proposed that this conserved

serine residue is responsible for protection against 'natural' antibiotics rather than

synthetic antibiotics (Hiramatsu et al., 2012). Mutations in the amino acids of the GyrB

and ParE domains also cause quinolone resistance; however, they are less frequent than

52
the mutations located on the GyrA and ParC. It was reported that the QRDRs of the

GyrB/ParE are distant from the QRDRs of the GyrA/ParC. However, the structure also

showed that the conformation of these two QRDRs is homologous to each other. Other

structural studies on cocrystals of the quinolones and these domains have shown that the

mechanism of resistance in these domains is similar to that in the GyrA/ParC domains via

mutations of the QRDRs by charge interactions to decrease the drug affinity. The degree

of resistance caused by mutation of a single amino acid in the enzymes varies among

bacterial species and quinolones. Different bacterial strains have different primary targets

for quinolones, thereby having different relative sensitivities to a given quinolone.

Resistant clinical isolates indicate that a single target-site gene mutation on either of the

two enzymes results in an 8-16-fold increase in resistance (Pham et al., 2019).

A single target-site muation in both DNA gyrase and topoisomerase TV results in

increasing the levels of resistance. Sequential mutations in both target enzymes in clinical

isolates increases the resistance up to 100-fold (Hooper & Jacoby, 2016). Some bacteria,

such as Mycobacterium nberculosis. Treponema pallidum, and Helicobacter pylori, have

only the DNA gyrase enzyme.

Other genomic mutations. As DNA gyrase and topoisomerase are cytoplasmic

enzymes, quinolones must pass through the bacterial envelope to exert their functions.

Therefore, quinolone activity is also affected by their ability to penetrate the cellular

barrier and the effectiveness of efflux pumps at removing the antibiotic from the

cytoplasm. Quinolones are known to enter bacterial cells by using both porin- and lipid-

mediated pathways.

53
Therefore, resistance can occur via mutations that reduce drug accumulation by under-

expression of porins, by over-expression of efflux pumps, or by modifications of the

lipopolysaccharide (LPS) structures. Many quinolone-resistant strains do not have

mutations in the enzymatic target QRDR and were less susceptible to unrelated

compounds, such as cyclohexane, salicylate, and tetracycline, proving that resistance is

associated with broad spectrum efflux activity. The multiple antibiotic resistance (mar)

gene is known to cause tolerance to a variety of compounds. (Pham et al., 2019).

Mutation of this gene leads to both overexpression of the acrAB efflux pump and reduced

expression of OmpF (outer membrane protein F) porin. MarA, a positive regulator of

acrAB transcription, can be induced either by mutation of the mppA gene or by exposure

to salicylate and tetracycline. Thereby, salicylate and tetracycline may stimulate

quinolone resistance. Moreover, MarA prevents translation of OmpF and activates the

expression of OmpX, which is a porin expression down-regulator, thus reducing the

expression of a variety of porins, such as OmpC, OmpD, OmpF, LamB, and Tsx.

(Correia et al., 2017; Dalhoff, 2012).

Another gene that contributes to the resistance against quinolones and other antibacterial

agents is the nfxB gene, which confers alterations in expression of functional Ompf at the

cell surface Ompf, thereby decreasing quinolone entry. In addition, modifications of

OmpA, a B-barrel protein associated with the integrity of the cell envelope or acting as a

porin, depending on the species, may lead to reduced quinolone susceptibility, as can

changes in SoxRS regulons resulting from bacterial adaption to superoxide stress (Pham

et al., 2019).

54
Quinolone resistance associated with efflux pumps include modification of the major

facilitator superfamily (MFS) of Gram-positive bacteria or the resistance-nodulation-

division (RND) family, multiple antibiotic and toxin extrusion (MATE), and ATP-

binding cassette (ABC) of Gram-negative bacteria. (Correia et al., 2017). Mutations of

efflux systems can alter their specificity for quinolones or cause upregulation.

Acquisition of resistance plasmids. Plasmid-mediated quinolone resistance, which has

been reported in clinical and environmental isolates around the world, appears to be

spreading (Zhang et al., 2012; Jacoby et al., 2014). Plasmid-mediated quinolone

resistance was first reported in 1998 in the United States in a multi-resistant urinary

Klebsiella pneumoniae that could transfer low level resistance to nalidixic acid,

ciprofloxacin, and other quinolones to a variety of gram- negative recipients (Akova et

al., 2015). However, PMQR is now being reported in other gram- negative as well as

Gram-positive bacteria (Jacoby et al., 2014). The transmission of these resistance

plasmids is through horizontal transfer from bacteria to bacteria as well as vertical

transfer from generation to generation. To date, at least three types of PMQR

determinants gene families are involved namely: gur family, acc(6')-1b-cr and active

efflux pump have been described in members of Enterobactericeae species, including the

pentapeptide-repeat family Qnr proteins (QnrA, QnrB, QnrS, QnrC, and QnrD) protect

DNA gyrase and topoisomerase IV from quinolone inhibition, the modified

acetyltransferase aac(6)-lb-cr, and the efflux pumps QepA and OqxAB (Andres et al.,

2013). They reduce the bacterial susceptibility to quinolones and mediate the selection of

mutants promoting treatment failure. Most of these determinants could be associated with

resistance integrons which may be embeded in elements related to horizontal gene

55
transfer (HGT). Resistance integrons (or mobile integrons) are elements that contain

genetic determinants of the components of a system for site-specific recombination that

recognizes and captures resistance genes in mobile cassettes (Di Conza and Gutkind,

2010).

Class 1, 2, and 3 integrons are widely associated with resistance determinants in human

clinical isolates. The first plasmid-encoded protein is Qnr, a pentapeptide repeat family

protein (Pham er 2019), These proteins are folded into a right-hand quadrilateral β-helix

shape and dimerize to format rod-like structure with a size, shape, and electrostatic

surface mimicking that of β-form DNA (Vetting et al., 2011).

More than 100 variants have been discovered in clinical isolates, which are classified into

6 subfamilies (qnrA, qnrB, qnrC, qnrD, qnrS, and qnrVC). The qnr gene has been

reported to originate from the chromosomes of many aquatic bacteria; with qnrA

originally from Shewanella algar, qnrB from Citrobacter spp, qnrC, qnrS, and qnrVC

from Vibrio spp, and qnrD and qnrE from Enterobacter spp. These Qnr proteins compete

with DNA binding to the enzymes, thereby inhibiting the quinolone from entering the

cleavage complexes and reducing the number of double-stranded breaks on the

chromosomes, resulting in reduced quinolone toxicity to the chromosomes (Pham et al.,

2019).

Investigation of mutations in codons 83 and 106 of the gyrA gene in UPEC isolates in

Iran presented the significant relationship between mutations in the gyrA gene and

quinolone and fluoroquinolone resistance pattern of UPEC isolates (Shenagari et al.,

2018).

56
Other genes responsible for the resistance to quinolones and fluoroquinolones are the qnr

genes (qnrA, qnrB, and qnrC), being the most important PMQR (plasmid-mediated

quinolone resistance) genes that induce antibiotic resistance by inhibition of binding of

quinolones to DNA gyrase and topoisomerases (Shahbazi et al., 2018). The second

plasmid-mediated mechanism involves acetylation of quinolones with an appropriate

amino nitrogen target (such as ciprofloxacin and norfloxacin) by aac(6')-1b-cr gene

encoding a variant of the common aminoglycoside acetyltransferase, AAC(6')-Ib-er (Ruiz

et al., 2012). Two unique mutations distinguish this variant enzyme from other ACC(6')-

Ib enzymes, and leads to specific targeting of quinolones with an amine on the

piperazinyl ring skeleton, such as ciprofloxacin, norfloxacin, and enoxacin. The enzyme

acetylated the unsubstituted nitrogen of the R7 piperazine ring. thereby decreasing the

quinolone activity. Both mutations are necessary for this specific enzyme action, with the

Trp102Arg mutation positioning the Asp179Tyr tyrosine aromatic ring for optimal

interaction with the quinolone, anchoring it in place.

Other mechanisms of E. coli resistance to quinolones and fluoroquinolones are related to

the presence of efflux pumps produced by plasmid genes qepA and oqxAB for pumps

QepA and OqxAB respectively (Jacoby et al., 2014) and decreased uptake of the

antibiotics due to changes in the outer membrane porin proteins (Asadi Karam et al.,

2019). OqxAB is a multidrug-resistant efflux pump encoded by conjugative plasmid

pOLA52 found in E. coli strains isolated from swine manure. It was recently detected in

human clinical isolates of E. coli and K. pneumoniae. Bacteria with this oqxAB gene

were 8- to 16-fold less susceptible to nalidixic acid and ciprofloxacin, respectively. This

efflux pump not only mediates low-level quinolone resistance but also helps bacteria to

57
survive under low concentration of quinolones, thus facilitating the subsequent

development of higher-level resistance (Pham et al., 2019). Another novel plasmid-

mediated quinolone eflux pump is QepA, which is encoded by PHPA plasmid found in

clinical isolates of E. coli from Japan, It is an efflux putip of the major facilitator family

that decreases susceptibility to hydrophilic quinolones (Aldred et al., 2013). These

multidrug-resistant efflux pump encoded genes do not directly cause high levels of

resistance to quinolones, but can facilitate the development of mutations to topoisomerase

enzymes by allowing the bacteria to adapt to low concentrations in quinolones inside the

bacteria. Abdelhamid and Abozahra (2017) showed that the increased expression of the

efflux pump-coding genes acrA and mdfA was related to the growing resistance to

levofloxacin, which confirms that efflux pump systems contribute to fluoroquinolone

resistance in urinary E. coli isolates.

Nitrofurantoin is recommended for the treatment of uncomplicated cystitis and, currently,

the resistance of UPEC to nitrofurantoin is very low. Resistance to nitrofurantoin did not

evolve as fast as to other drugs because of this antimicrobial act at multiple targets in the

bacterial cell (Veeraraghavan& Shakti, 2015), identified mutations conferring resistance

to nitrofurantoin and found that the mutation frequency is approximately 10-7/cell in E.

coli. The mutations in the A and nfsB genes that encode oxygen-insensitive

nitroreductases were responsible for nitrofurantoin resistance. It was also found that the

growth of bacterial cells in the presence of nitrofurantoin at therapeutic concentrations

was greatly reduced in nitrofurantoinresistant mutants. It may indicate that resistant

mutants in the presence of nitrofurantoin were probably unable to establish an infection.

58
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Materials

3.1.1 Glass wares and Equipment

The glass wares used were: Petri dishes, conical flash, beaker, test tube, microscope slide,

Eppendorf tube, Bijou bottle, sterile container, wire loop, sterile swab stick, forceps,

disposable hand glove, laboratory coat, aluminum foil, graduated cylinder, staining rack.

The equipment used include: Bunsen burner, autoclave (Yamato, USA), incubator (HME

Global, Model No.DNP-9022A, England), Panasonic (NN-ST765S) Microwave oven,

Refrigerator/Freezer(Model PRN 1313 HCA, BEKO, Germany), Sorvall MC 12V

Microcentrifuge(Model 5417R: Touch plate Super Mixer, CAT. No. 1291, Lab-line

Instrument Inc., USA), Electronic weighing balance (Model QT600), Nanodrop 1000

Spectrophotometer (Thermo Fisher Scientific, USA), Microscope (WildHerbrug M11,

Switzerland), Applied Biosystems PCR GeneAmp9700 (Scientific Support Inc, USA),

Piko Thermal Cycler (Thermo Scientific, USA), Vortex machine (XH-B) (Celtech Inc,

USA), UV Transilluminator (302nm; MD-25) (Wealtec Corp, USA). Block heater HB-2

(Wealtec Corp, USA), Agarose gel unit (HE 33: Hoefer San Francisco, CA, USA).

3.1.2 Media

59
 Media used for this study include: MacConkey Agar (MCA: HiMedia Laboratories Pvt.

Ltd., Mumbai, India), Eosin Methylene Blue Agar (EMB: Oxoid Ltd, Basingstoke,

United Kingdom), Mueller-Hinton Agar (MHA: Oxoid Ltd, Basingstoke, United

Kingdom), Luria-Bertani Broth (LBB: Oxoid Ltd, Basingstoke, United Kingdom),

Nutrient Agar (NA: Oxoid Ltd, Basingstoke, UK), Agarose gel (Gibco Life

Technologies, Paisley, United Kingdom), Methyl Red-Voges Proskauer (MR-VP) media

(Biotec Laboratories, Ltd., Ipswich, United Kingdom), Peptone Water (HiMedia

Laboratories Pvt. Ltd., India). All media were prepared according to the manufacturer's

instructions.

3.1.3 Chemicals and Reagents

Chemicals and reagents used include: sodium chloride (NaCl: BDH Chemical Ltd,

England), Kovac's Indole Reagent (HiMedia Laboratories Pvt. Ltd., Mumbai, India),

Barium chloride dihydrate (BaCl2.2H2O: BDH Chemical Ltd, England), sulfuric acid

(H₂SO₄: BDH Chemical Ltd, England), Crystal Violet and Safranin (HiMedia

Laboratories Pvt. Ltd., India), Lugol's lodine (HiMedia Laboratories Pvt. Ltd., India),

Acetone-Alcohol (HiMedia Laboratories Pvt. Ltd., India), Neutral Red (HiMedia

Laboratories Pvt. Ltd., India), Water, Ethidium bromide dye (Sigma-Aldrich Chemical

Ltd., England) and Molecular marker (1-kb DNA ladder, Gibco/BRL).

3.1.4 Antibiotic discs

The antibiotic discs used were all from Oxoid Ltd., Basingstoke, United Kingdom, and

include: Ampicillin (10 µg), Sulphamethoxazole/Trimethoprim (25 µg), Cefotaxime (30

µg), Ciprofloxacin (5 µg), Amoxicillin/Clavulanic acid (30 µg), Gentamicin (10 µg),

60
 Ceftazidime (30 µg), Nitrofurantoin (300 µg), Streptomycin (10 µg), and Ceftriaxone (30

µg).

3.2 Methods

3.2.1 Study Locations

The sampling locations for the study were Nyanya General Hospital (NGH), which is a

government hospital and Pan Raf Hospital (PRH) Nyanya, Abuja, private owned hospital.

These hospitals, located in sub-urban Abuja Municipal, and they served resident of

Nyanya and Maraba in Nasarawa state and environs due to their proximity to each other.

Both are referral centre and cater for all infectious disease cases such as gastroenteritis,

cholera, TB, Malaria, etc.

3.2.2 Ethical Consent

Ethical approval was obtained from the Federal Capital Territory Health Research Ethics

Committee, Abuja. Samples were collected only from the healthy pregnant women

attending antenatal care and willing to participate in the research.

3.2.3 Sample Population, Inclusion and Exclusion Criteria

Pregnant women in five age groups (in years) namely: 16-20, 21-25, 26-30, 31-35 and

36-40, were selected for this study. Only pregnant women attending antenatal clinic (out-

patient) in the selected hospitals during the period of the study, with (symptomatic) or

without (asymptomatic) visible signs, of urinary tract infections and not on antibiotics

61
 were enrolled. Pregnant women that are not registered in the antenatal clinic (out-patient)

or those registered but on antibiotics were excluded from the study.

3.2.4 Determination of Sample Size

The sample size was determined, using the formula below as earlier described by (Naing

et al., 2006):

2
z pq
N= 2
d

Where: N= desired sample size (when the population >10,000); 2 standard normal

deviate, usually set at 1.96, which usually correspond to 95% confidence level; p= 0.05 in

the target population, set at 14.5% (0.145) (Nkene et al., 2019); d tolerated margin of

error at confidence interval with degree of accuracy of d (0.05). The designed effect of 1

was used.

q = 1-p

P = prevalence of Escherichia coli (0.145) as local prevalence

N = the minimum sample size = 190

Therefore, the sample size was 252

3.2.5 Sample Collection

62
 A total of 252 early morning mid-stream urine samples of Pregnant women were

collected between September 2019 -December, 2019 using sterile container and

transported using ice pack to the Microbiology Laboratory, Nasarawa State University,

Keffi for analysis.

3.2.6 Isolation and Identification of Escherichia coli

Escherichia coli were isolated from the urine samples of the pregnant women using

streak plate method. The urine sample was streaked on MacConkey agar (Oxoid Ltd.,

Basingstoke, UK) plate and incubated at 37°C for 24 h. Pinkish colonies that grew on

MacConkey agar were further streaked on Eosin Methylene Blue (EMB: Oxoid Ltd.,

Basingstoke, UK) agar and incubated at 37°C for 24 h. Greenish metallic sheen colonies

that grew on the EMB agar were selected as presumptive E. coli.

The presumptive E. coli was identified by microscopy (Gram staining) and the minimal

biochemical tests for E. coli identification called 'IMVIC' (Indole test, Methyl red,

Voges- Proskaeur and Citrate) as earlier described Egheieye et al, (2018). The suspected

E. coli isolates (Gram negative, rod shape, indole positive, methyl red positive, citrate

negative and Voges- Proskauer negative) and further identified using KB003H125™

identification kit following the manufacturer's instructions.

3.2.7 Identification and Confirmation of Escherichia coli

3.2.7.1 Gram Staining

63
 Gram staining was carried out as described by Cheesbrough (2006). Briefly, a thin smear

of colony of the presumptive E. coli (test organism) was made on a clean glass slide

(microscope slide) with sterile wire loop and emulsified in a drop of distilled water until a

thin homogenous film was obtained. It was allowed to air dry and then heat fixed by

passing glass slide through the flame 3 times. The fixed smear was then covered with 3

drops of crystal violet for 60 sec and rinsed with clean water, then covered with Lugol's

lodine for 60 sec and rinsed with clean water gently. It was decolorized with 95% acetone

and rinsed immediately with clean water, the smear was covered with safranin for 30 sec

and rinsed with clean water before it was wiped clean and kept in draining rack to air-dry.

It was viewed first with 40X objective and then with oil immersion under the microscope

by the use of 100X objective lens. Gram- positive organisms retain the dark blue colour

inferred by the iodine/crystal violet complex, while Gram-negative organisms appear red,

maintaining the colour of the secondary dye. The organism retained the colour, safranin

which indicates a Gram-negative organism. Therefore, Escherichia coli was

presumptively indentified as the organism smeared.

3.2.7.2 Biochemical Identification Tests

Biochemical identification tests that carried out were include Indole, Methyl red, Voges-

Proskauer and Citrate (IMViC) as described in the Bacteriological Analytical Manual

(BAM, 2007). For the indole test, pure suspected isolates from EMB plates were

inoculated in 5ml of peptone water for 24h, prepared according to the manufacturer's

instruction. Three drops of Kovacs' indole reagent were added and shaken gently.

Positive result developed a red colour on the reagent layers above the broth within one

minute while a negative action remains its normal colour.

64
 For the Methyl red test, the isolated organisms were grown in ml of methyl red broth and

incubated for 48 h at 37°C. After incubation 1 ml of the broth was transferred into a test

tube and two drops of methyl red were added which increases the formation of a red

colour and that signified a positive methyl red test in which the result was recorded.

For the Voges-Proskauer Test, 2ml of sterile glucose phosphate peptone water were

inoculated with the test organism and incubated at 37°C for 48h. A very small amount of

creatine was added and mixed together, also 3 ml of the sodium hydroxide reagent were

added and shake very well and the bottle cap were removed, and leave for 1h at room

temperature. Positive result developed a pink-red colour while a negative action remains

its normal colour.

For the Citrate Test, the isolates were inoculated into simmon's citrate agar slant in a

Bijou bottle and incubated for 72h. The Simmon's citrate agar has a characteristic green

colour before inoculation. A change of colour from green to deep blue colour after

incubation due to the utilization of the citrate indicated a positive reaction.

3.2.7.3 Confirmation of Escherichia coli using Commercial Kit

The AP120E system (Analytical Profile Index) (BioMerieux™, USA), a commercial kit

designed for the identification of Enterobacteriaceae and other non-fastidious Gram-

negative bacteria was used to confirm the suspected isolates. The test strips consist of

twenty mini-test tubes that enable species identification to be made on the basis of the

test organism's ability to perform a series of biochemical reactions.

Briefly, suspensions of isolates were made in sterile distilled water, inoculated into the

wells in the strips and the strips inoculated aerobically for 24h at 37°C After 24h, the

65
 colour reactions were observed and read using the computerized APlweb™ software

(Biomerieux) and the species of the test isolates ascertained.

3.2.8 Antimicrobial Susceptibility Testing

The antimicrobial susceptibility testing of the bacterial isolates was carried out as earlier

described by (CLSI, 2015). Briefly, three distinct pure colonies of E. coli isolated from

urine samples of patients were inoculated into 5 ml sterile 0.85% (w/v) NaCl (normal

saline). Thereafter, the turbidity of the bacteria suspension was adjusted to the turbidity

equivalent to 0.5 McFarland's standard. The McFarland's standard was prepared as

follows; 0.5ml of 1.172% (w/v) BaCl2.2H2O was added into 99.5ml of 1% (w/v) H2SO4.

A sterile swab stick was soaked in the standardized bacteria suspension and streaked on

Mueller- Hinton agar (MHA) plates, and the antibiotic discs were aseptically placed at

the center of the plates and allowed to stand for 1 h for pre-diffusion. The plates were

placed in an incubator (Model 12-140E, Quincy Lab Inc.) at 37°C for 24h.

The diameter zone of inhibition in millimeter was measured and the result of the

susceptibility was interpreted in accordance with the susceptibility break point earlier

described by CLSI (CLSI, 2015).

32.9 Evaluation of Multiple Antibiotics Resistance (MAR) index

The Multiple Antibiotics Resistance (MAR) indices for the resistant isolates were

calculated as earlier described by Ngwai et al. (2014) as follows:

MAR Index=No . of antibiotics isolateis resistant ¿ ¿

No .of antibioticstested

66
3.2.10 Classification of Categories of Antimicrobial Resistance in the Isolates

Antimicrobial resistance in the isolates were classified into: multidrug resistance (MDR:

non susceptible to ≥1 agent in ≥3 antimicrobial categories); extensive drug resistance

(XDR: non- susceptible to ≥1 agent in all but ≤2 antimicrobial categories); pan drug

resistance (PDR: non- susceptible to all antimicrobial listed) (Magiorakos et al., 2012).

3.2.11 Molecular Detection of Plasmid-mediated Quinolone Resistance Genes

3.2.11.1 DNA Extraction

Five confirmed ciprofloxacin-resistant Escherichia coli (CREC) isolated from this study

were used.

The isolates were maintained on nutrient agar slants at 4°C Refrigerator/Freezer, and sub-

cultured on MacConkey agar at 37°C for 24h to obtain pure colonies before use in

experiments.

The DNA of the isolates was extracted using boiling method as described by Abimiku et

al. (2016). Briefly, following purification on MacConkey agar, three pure colonies of the

ciprofloxacin resistant isolates, was inoculated into 5ml of sterile LB broth in a Bijou

bottle and incubated at 37°C for 24h. Exactly 200 µl of the LB culture was then

transferred into Eppendorf tube and centrifuged in a microcentrifuge to isolate bacterial

DNA.

The bacterial cells were harvested by centrifugation at 3200rpm in a micro-centrifuge for

2min at room temperature and the supernatant was discarded. The harvested cells were

67
 re-suspended in 1ml of sterile normal saline and the micro-centrifuge tubes were placed

in the vortex for 5sec. Centrifugation was carried out at 3200rpm for 1 min and the

supernatant was discarded. Exactly 0.5 ml of sterile normal saline was added to the

pellets and the tubes were vortexed for 5sec after which they were heated in the block

heater at 90°C for 10 min. immediately after heating, rapid cooling was done by

transferring the tubes into the freezer for 10min. Cell debris was removed after

centrifugation was done at 3200rpm for 1min and 300µl of the supernatant was

transferred into a sterile 2ml Eppendorf tube as DNA and stored at-10°C until use.

3.2.11.2 Amplification of Target Genes

The DNA amplification of target PMQR genes in the ciprofloxacin-resistant E. coli

(CREC) as given in Table 4.9 as earlier described by Nkene et al. (2020). Briefly, the

target genes for fluoroquinolone resistance namely: oqxA, oqxB, qnrB and qnrS.

Amplification reactions was carried out in a thermocycler (Eppendorf master cycler, MA)

under the following conditions: initial denaturation at 94°C for 5min, followed by 32

cycles of amplification at 94°C for 45sec each, annealing at 53°C for 45sec, and 72°C for

60sec with final extension at 72°C for 5min.

The PCR conditions for aac-(6')-Ib-cr gene was carried out as follows: initial

denaturation at 95°C for 5 min, followed by 32 cycles of amplification at 95°C for 20sec,

annealing at 59°C for 40sec, and initial extension at 70°C for 30sec, with final extension

at 72°C for 5min. The PCR performed on total volume of 50µL containing 100ng

genomic DNA of resistance E. coli isolates, 200mM each of dNTP, 1xPCR buffer

(20mM Tris-HCl, pH 8.4), 50mM KCI, 1.5mM MgCl2, 0.5mM of each primer, and 1.5U

68
 of Taqpolymerase. Amplified samples (10µL) were separated on 1.0% agarose gel in

TBE buffer.

The gel was stained with ethidium bromide 0.5mg/mL.. The amplified bands were

visualized under ultraviolet light and photographed. Reaction mixtures without a DNA

template served as negative controls.

3.2.12 Statistical Analysis

The data obtained in this study was analyzed using chi-square by use of Smith statistical

Package (SSP) version (2.80) and the significance was determined at 95% confidence

interval.

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