Isolation, Characterization and Antibiotic Susceptibility Test Pattern of Uropathogens Isolated from Urine Samples

CONTENTS

CHAPTER ONE: INTRODUCTION
Background of the Study
1.1 Statement of the Problems
1.2 Justification
1.3 General Objectives

CHAPTER TWO: LITERATURE RVIEW
2.1. The Urinary System and its Infection
2.2. Etiology of Urinary Tract Infection
2.3. Types of Urinary Tract Infection
2.3.1. Lower and Upper Urinary Tract Infections
2.3.2 Complicated and Uncomplicated Urinary Tract Infections
2.4. Pathogenesis
2.5. Clinical Features
2.6. The Epidemiology of Urinary Tract Infection.
2.7. Risk Factors to Urinary Tract Infections
2.7.1. Anatomical and Physiological Factors
2.7.2. Age
2.7.3. Sex and Contraception
2.7.4. Pregnancy
2.7.5. Diabetes Mellitus
2.7.6. Urinary Catheterization
2.8. Virulence Factors
2.9. Laboratory Diagnosis of Urinary Tract Infection
2.10. Treatment and Prevention of Urinary Infections
2.10.1. Treatment
2.10.2. Prevention
2.11. Antibiotic Susceptibility

CHAPTER THREE: MATERIALS AND METHODS
3.1. Collection of Samples
3.2. Preparation of Culture Media
3.3. Nutrient Agar.
3.3.1. Blood Agar.
3.3.2. MacConkey Agar
3.4. Culturing of the sample
3.5. Biochemical Identification
3.5.1. Catalase Test
3.5.2. Oxidase Test
3.5.3 Indole Test
3.5.4. Citrate Utilization Test
3.5.5 Urease Test
3.5.6 Motility Test
3.5.7 Nitrate Reduction Test
3.6 Screening through Gram Staining
3.7 Antimicrobial Susceptibility
3.7.1 Susceptibility Test by Disc Agar Diffusion

CHAPTER FOUR

CHAPTER FIVE
Discussion
Conclusion
Recommendation

References

Appendix

CHAPTER ONE: INTRODUCTION

Background of the Study

Urinary tract infection (UTI) is a term applied to a variety of clinical conditions ranging in severity from asymptomatic which is carrier status in the urine to symptomatic acute infection of the kidney with resultant sepsis (Tanagho, and Mcaninch, 2004). UTI is defined also as the growth of a known bacterial pathogen of more than 103cfu/ml in association with a positive dipstick or urinalysis (Zorc and Levine, 2005). It is usually classified by the infection site: an infection of the lower urinary tract, urethra (urethritis) and urinary bladder (cystitis); an infection of the upper urinary tract, ureter and kidneys (pyelonephritis); and an infection of the renal pelvis (pyelitis) (Heffner and Gorelick, 2008).

Urinary tract infections also divided into complicated and uncomplicated infections. UTIs that occur in a normal genitourinary tract with no prior instrumentation are considered as uncomplicated. A patient is diagnosed with uncomplicated cystitis if his or her midstream urine specimen has between 103 to 105 colony forming units (cfu)/ml. the clinical symptoms of UTI usually include frequency, dysuria, pyuria, suprapubic tenderness, back pain, fever and urgency (Gupta el al., 2011).

Complicated UTIs are diagnosed in genitourinary tracts that have structural or functional abnormalities, such as indwelling urethral catheters, and are frequency asymptomatic (Gonzalez and Schaeffer, 1999; Stamm and Hooton, 1993). Recurrent infection by pathogens with increased resistance is a troubling consequence of complicated UTIs (Nicolle, 2005). However, other conditions such as, treatment with immunosuppressive drugs of old people over 65years, the presence of human immunodeficiency virus-infection and diabetes mellitus (DM) also predispose to an enhanced susceptibility for the development of a UTI with a complicated course that is more difficult to treat and often recur (Johnson et al., 1987 and Hoepelman et al., 1992). In community-acquired UTIs, women are significantly more likely to experience these infections during their life time than men where such cases are rare except in association with anatomic or functional abnormalities in the first year of life (Stamm, 2001 and Foxman, 2002).

Recurrent infection is a common problem and can affect women of all ages, particularly the elderly and pregnant women (Dwyer and O’Reilly, 2002). The source of UTI pathogens is generally considered to be the patient’s own flora. Infection is preceded by colonization of the vagina and periurethral area by uropathogens from the GI tract (Wagenlehener et al., 2005). The susceptibility of women to UTIs is due, in part, to the female anatomy in that a much shorter urethra allows pathogens’ easier access to the bladder. The structure of the female’s urethra and vagina makes it susceptible to trauma during sexual intercourse and hence making it easier for bacteria to be force into the bladder during intercourse and thereby resulting an increased bacterial count in the bladder (Kolawale et al., 2009). Bacteria present is fecal matter inoculate the periurethral area, and then the bladder, causing symptoms clinically termed cystitis and in more than 95% of cases it is monomicrobial (Yamamoto et al. 1997); Johnson et al. 1998 and Warren et al. (1999). Left untreated, bacteria ascend the ureters to the kidney and establish a secondary infection, acute pyelonephritis. At this juncture, there is risk of permanent renal scarring and bacteria can get access to the blood stream (Warren, 1996).

Urinary tract infections remains to be the most common infectious diseases diagnosed in outpatients (Gales et al.). it is estimated that 150 million people with UTI are diagnosed each year on a global basis, costing in excess of 6 billion dollars in direct health care expenditures (Gupta, 2011). UTIs are a serious health problem affecting millions of people each year. According to National Institution for Health, it is estimated from surveys of office practices hospital-based clinics and emergency departments that there are over 8 million cases of UTI annually in USA (NIH, 1999). The annual cost to the health care system of the United States attributable to community-acquired UTI alone is estimated to be approximately $ 1.6 billion (Foxman, 2002). Asymptomatic bacteriuria in Nigeria was reported as showing prevalence rates of 24 and 6% among rural and urban children, respectively (Okafor et al. 1993). UTIs are commonly encountered diseases in developing countries with an estimated annual accidence of at least 250 million cases (Nicolle, 2001 and Ronald, 2005).

Many different microorganisms can cause UTIs. It is most often due to bacteria (95%), but may also include fungal and viral infections (Amdekar et al, 2011). Ninety percent of UTI cases are caused by gram-negative bacteria while only 10% of the cases are caused by gram-positive bacteria including Enterococus, staphylococcus, and streptococcus agalactiae (Lazarevic et al. 1998). Members of the Enterobacteriaceae are the most common organisms isolated from uncomplicated UTI and most frequent uropathogen is Escherichia coli, accounting for 65% - 90% of urinary tract infections (Gupta and Schlager, 2001). Although Candida is an increasing nosocomial problem, isolation of yeast from urine does not necessarily always indicate infection (Fidel, 1999). In complicated urinary tract infection and hospitalized patients, organisms such as Enterococcus Faecalis and highly resistant Gram-negative rods including Pseudomonas spp. Are comparatively more common. The relative frequency of the pathogens varies depending upon age, sex catheterization, hospitalization and previous exposure of antimicrobial (Raz et al, 2000).

Antimicrobial resistance to the most commonly prescribed drugs used in the treatment of UTIs is an increasing problem world-wide and vary considerably in different areas of the world (Tenover and Hughes, 1996; Turnidge et al, 2002). The emergency of antibiotic resistance in the management of urinary tract infections is a serious public health issue, particularly in the developing world where, apart from high level of poverty, ignorance and poor hygienic practices, there is also a high prevalence of fake and spurious drugs of questionable quality in circulation. Their easy availability in the community without prescription and their low cost make them subject to abuse (Abubakar, 2009). With regards to resistance rates in Ethiopia, a report showed that high incidence of resistance to the commonly prescribed antibiotic agents was observed at Tikur Anbessa hospital (Wolday and Erge, 1997). Likewise, a study conducted in Gondar indicated that above 68% of the isolated pathogens showed resistance to two to nine antimicrobials and 15.7% were resistant to one antibiotic (Moges et al. 2002).

Treatment of UTIs cases is often started empirically based on information determined from the antimicrobial resistance pattern of the urinary pathogens (Wilson and Gaido, 2004). The estimation of local etiology and susceptibility profile could support the most effective empirical treatment (Farajnia et al. 2009). Therefore, investigating epidemiology of UTIs (prevalence and risk factors) and the antibiotic sensitivity of uropathogens is fundamental for caregivers and health planners to guide the necessary interventions.

In general, because of the continuous emergence of antibiotic resistance, regular monitoring of this phenomenon appears to be necessary to improve guidelines for empirical antibiotic therapy, which must consider the most probable microorganisms like E. coli, their susceptibility according to the characteristics of the population concerned, their side effects, and biological and economic consequences (Sotto et al, 2001).

In Ethiopia, even though there are published information concerning the etiology and resistance pattern of UTIs (Gedebou, 1988; Ringertz et al, 1990; Woldaye and Erge, 1997; Moges et al, 2002), there was no previous study and published information on UTI in the study area. This study was conducted between June and September, 2012 and is very much needed to define the problem of urinary tract infections; and to provide information that helps as a useful guide for controlling urinary tract infection around Shashemene. Therefore, this study was intended to assess the prevalence of bacteria uropathogens and their in vitro susceptibility patterns to commonly used antibiotic agents amongst outpatients with complaints of UTI is Shashemene Referral Hospital.

1.1 Statement of the Problems

Uropathogens such as E. coli is responsible for approximately 85% of community acquired infection, besides Proteus, Klebsiella and Pseudomonas (bacteriology Text Book). This pathogens cause’s varieties of urinary tract infection in both male and females.

1.2 Justification

The effect of uropathogen on an individuals or community at large cannot be over emphasized as it constitute majorly to the urinary tract infections and often referred to as community acquired infections. Some of these uropathogens are found to be susceptible while some are resistant. Therefore, need to study its susceptibility pattern is very significant.

1.3 General Objectives

The aim of this study is to characterize and how the susceptibility pattern uropathogens isolated from urine samples.

Specific Objectives are to:

The specific objectives of this study are:

- To determine the prevalence of bacteria uropathogens isolated from outpatient with complaints of UTI.
- To determine the susceptibility patterns of the isolates of uropathogens to the common used antibiotics.
- To identify the association of UTI with possible risk factors.

CHAPTER TWO: LITERATURE RVIEW

2.1. The Urinary System and its Infection

The urinary system is a system that maintains the volume and composition of body fluids within normal limits. It consists of the kidneys, which filter the blood, remove the end products of metabolism and excrete the waste in the urine; ureters carry the urine away from kidneys to the urinary bladder, which is temporary reservoir and urethra transports the urine from the urinary bladder to the outside (Tortora and Derroclspm. 2007) urinary tract infection (UTI) is a term applied to a variety of clinical conditions ranging from a systematic presence of bacteria, or fungi in the urine to severe infection of the organs of the system with resultant sepsis (Tanagho and Mcaninch, 2004). UTI is defined also as the growth of a known bacterial pathogen more than 10000 efu/ml tested with a positive dipstick or urinalysis (Zore and Levine, 2005). According to the National Institute for Health and Clinical Excellence (NIHCE) guidelines, urinary tract infection is defined by a combination of clinical features and the presence of bacteria and / or fungi in urine (NITCE, 2007)

2.2. Etiology of Urinary Tract Infection

Many different microorganisms can cause UTIs, but by far the most common agents are bacteria (95%), may include fungal and viral infection (Amdekar et al., 2011). Member of the Enterobacteriaceae are the most common organism isolated from uncomplicated UTI and the most frequent uropathogen is Escherichia coli, accounting for 65%-90% of urinary infection (Gupta and Schlager, 2001). Staphylococcus saprophyticus accounts for 5-15% of uncomplicated UTIs. Enterococcus and a variety of other primarily gram-negative aerobes account for other 5-10% of UTIs: Proteus mirabilis, Klebsiella species, and Pseudomonas species (Stamm and Hooton, 1993). More than 95% of UTIs are caused by a single bacterial species. Uncomplicated cystitis and pyelonephritis are due to Escherichia coli accounting for 80%, Proteus mirabilis 10%, and Klebsiella spp. Infections accounts 6% (Tanagho, 2004; Ghedira et al., 2004).

According to Tsao, after E. coli, Salmonella was the second most common pathogen (Tsao, 2002). Salmonella UTI do occur in healthy children and adolescents, especially in the presence of gastroenteritis (Buchta and Dunn, 2003). Salmonella typhimurium, S. Heidelberg, S. enteritidis, S. infantis, S. Newport and S. typhi are species most frequently isolated form urine and the pre-existence of stones, deformities or local tissue damage predisposed to the development of chronic Salmonella infection of the kidney (Hasham and Uehling, 1976). Salmonella has been postulated to enter the urinary tract either hematogenously or by direct invasion of the bladder via the urethra (Chebl et al. 2005).

Klebsiella terrigena and K. orinthinolytica have been transferred to the genus Raoultella and account for a smaller proportion of uncomplicated urinary tract infections (Janda and Abbott, 2006). Gram-positive cocci were isolated more frequently from a hospital setting and the most common were Enterococcus species (Wilson and Gaido, 2004). Leuconostoc isolates were earlier considered to be non pathogens however now gained important as opportunistic pathogens implicated in causing a small outbreak of nosocomial urinary tract infection (Cappelli et al., 1999). More commonly, Enterococci and Staphulococcus aureus cause infection in patients with renal stones or previous instrumentation or surgery. Although are among the dominant organisms colonizing the urethra and periurethra in males and females, they typically account for fewer than 10% of all UTIs in the United State. Most of those coagulase-negative staphylococcal UTIs are caused by the two species Staphylococcus epidermidis and S. saprophuyticus (Latham et al,. 1983).

According to the study conducted by Gernot et al., (2010), it was reported that Dermacoccus nishinomiyaensis (Microccocus nishinomiyaensis) is prevalent in urinary tract during microbial urethral stent colonization (MUSC). In similar studies by Riedl et al., (199) and Klis et al., (2009), urethral stents inserted during urinary tract infection (UTI) were more frequently colonized (59%) compared to those placed in sterile urine (26%). Female sex and continuous stenting were significant risk factors for MUSC. During manipulation or instrumentation, biofilm organism could be shed in to the urine and lead to uncomplicated UTI up to urosepsis.

In complicated UTIs, E. coli is recovered in only approximately one-third of cases (34.5%) Strepotococcal faecalis is recovered in 16%, Staphylococcus epidermidis in 13%, and proteus nurakukus ub 13%. Other bacteria involved in complicated UTIs include Pseudomonas sp. (5%), Staphylococcus aureus (4%), Enterobater sp (2.5%), Serratia sp. (1.5%), Staphyloccocus spl (1.5%), Acinetobacter sp (1.5%), Citrobater sp. (1.0%), providencia sp. (0.25%), Morganella morganii (0.255), and candida sp. (0.25%) (Naber, 1989). According to data of the National Nosocomial infection Surveillance (NNIS) study, during 1986-1989, Citrobacter accounted for 2% of total nosocomial infections (Schaberg et al., 1991).

Urinary tract infection may be caused by fungi and viruses. Fungi, such as candida, are the second most cause of nosocomial UTLs in children. It can be spread systemically and can be life threatening. The prevalence of UTIs due to Candida increased gradually by the duration of hospitalization, with a prevalence rate 27.2% (Parlak et al., 2007). Fungi infections are seen in infants and children who are on long-term antibiotics, patients who are immouno-compromised, or patients using invasive devices like IVs, grains and catheters. Viral UTI can be carsed by Adenoviruses types 11 and 21, polyoma virus BK, and herpes simplex viruses (Naber, 1989). The relative frequency of the pathogens varies depending upon age, sex, catheterization, and hospitalization (Sefton, 2000).

2.3. Types of Urinary Tract Infection

2.3.1. Lower and Upper Urinary Tract Infections

Urinary tract infection is usually classified by the infection site:- Lower urinary tract infections include complications in the bladder and the organs below it. Cystitis is the most common lower urinary tract complication with inflammatory syndrome and infection of the bladder. It results from an irritation of the lower urinary tract mucosa (Roussey et al, 2008). Urethritis is the inflammation and infection of urethra; it is most often associated with sexually transmitted diseases. Upper UTIs include the kidneys which are the anatomical site of infection namely pyelonephritis, associated with inflammation of the renal parenchyma, calices and pelvis. This condition may be acute or chronic. (Wagenlehner et al, 2009)

2.3.2 Complicated and Uncomplicated Urinary Tract Infections

Urinary tract infections can also be divided into two categories: complicated and uncomplicated UTIs. Uncomplicated UTIs occur usually in healthy people, where the hosts have normal urinary tract and do not have systemic diseases subjecting them to bacterial infections. Complicated UTIs are infections occurring in individuals with predisposing lesions of the urinary tract that interferes with the normal flow of urine and urinary tract defenses. Common causes of predisposing lesions are birth defects, kidney stones, catheters, or obstruction (Masson et al, 2009). Complicated UTIs affect people with problematical urinary tract i.e. not functioning as it should due to anatomic or functional defects (Mobley and Warren, 1996). The second group affiliated with complicated UTIs is host generally susceptible to infections, for instance patient with immune suppression. UTI during pregnancy and Catheter associated urinary tract infections are also considered as complicated infections. Characteristics complicated UTIs are the spectrum of organism is broader and that antibiotic treatment may not have an effect (Gray and Moore, 2009).

2.4. Pathogenesis

Community-acquired UTIs usually result from a retrograde ascent of bacteria in the external urethral meatus and/or vaginal introitus to the bladder. However, infection may also occur via the blood or lymph. It is believed that the bacteria are usually transmitted to the urethra from the bowel, with female at greater risk due to their anatomy. The proximity of the urethra to the vagina and rectum allows fecal flora to colonize the periurethral area of woman. After gaining entry to the bladder, urepathogenic E. coli are able to attach to the bladder wall and form a biofilm that resist the body’s immune response. Women with recurrent UTIs have colonization of the vaginal and urethra areas with the urepathogen before the onset of infection (Salvatore et al, 2011). The relatively short urethra of women in comparison with men and the mechanical effect of sexual intercourse facilitate movement of bacteria into the bladder and explain the 50 times greater UTI rate in women than in men and the connection between UTIs and sexual activity. It thus, appears to be important in the pathogenesis of UTIs in younger women. In addition, use of spermicidal coated condoms dramatically alters the normal bacteria flora and has been associated with marked increases in vaginal colonization with E. coli and in the risk of UTI (Braunwald, et al, 2001).

Following invasion of superficial bladder epithelia cell, UPEC can replicate intracellularly and eventually reemerge from the infected host cells in a manner reminiscent of a lytic virus cycle. Upon exiting the superficial cell, UPEC can interact with and invade surrounding and underlying epithelia cells, leading to the establishment of a quiescent bacterial reservoir within the bladder tissue (Mulvey et al, 2001). The ability of uropathogenic E. coli to flux out of cells and colonize surrounding cells provides them a mechanism to subvert host defense mechanisms and persist in the bladder epithelium for weeks following the acute infection (Schilling et al, 2001).

Several studies have investigated bacteria adherence to uroepithelial cells. Strains of E. coli from women with pyelonephritis adhered to epithelial cells in much greater number than did strains of E. coli from those with asymptomatic bacteriuria or strains from feces (Svanborg and Jodal, 1979) Larger number of bacteria adhere to urothelium or buccal cells of women with recurrent UTIs than in women without UTIs (Svanborg and Jodal, 1979; Schaeffer, 1981). These later data indicate a genetic susceptibility to UTIs determined by adherence of bacteria to uropithelial cells. A genetic predisposition to UTIs is also suggested by findings of an increased risk of UTI and increased epithelia binding of E. coli in women who are non secretors of blood group antigens than in those who do secrete these antigens (Kinane et al, 19882; Lamberg et al 1986).

2.5. Clinical Features

The clinical features of UTIs are the result of a complex series of host pathogen interactions that can lead to bacterial invasion and persistence that ultimately can determine the course of the infectious disease. It may include both specific and nonspecific signs and symptoms. According to the infections site, the clinical features of UTIs can be classified as cystitis (Bladder), urethritis (urethra), pyelonephrities (Kidneys) (Schilling et al, 2001).

Cystitis involves inflammatory syndrome and infections of the bladder with signs and symptoms of dysuria, frequency, urgency and suprapubic tenderness (Gunther et al. 2001). The urine often becomes grossly cloudy and malodorous and when it is blood 30% of cases, it is termed as hermorrhagic cystitis. It can be caused by an infection or as a result of radiation, cancer chemotherapy, or immunosuppressive medication (Gupta et al, 1999). However, some women with cystitis have only 102 to 104 bacteria per milliliter of urine and in these instance bacteria cannot be seen in a Gram stained preparation. Physical examination generally reveal only tenderness of the supra - pubic area urethritis is an inflammation with/ without bacterial infection, causing symptoms similar to those of cystitis and it is most often associated with sexually transmitted disease such as Chlamydia trachnomatis and Neisseria gonorrhoeae (Braunwald et al.2001).

White blood cells and bacteria can be detected by examination of unspun urine in most cases. Pyuria is the presence of ≥ 104 leucocytes/ml in fresh urine. It is present in 96% of symptomatic women but in only 1% of women with no symptoms. Pyuria without bacteriuria may be associated with Chlamydia infections, but is equally common in the absence of obvious infections from vaginal leucorrhoea, or when UTIs fail to meet laboratory criteria for diagnosis (Stamm et al, 1982; Kunin et al, 1993). In nursing homes more than 50% of elderly women have pyuria (Kaye, 1995). Asymptomatic bacteriuria is defined as the presence of bacteria in two consecutive urine samples in the absence of typical symptoms. It appears in women of all ages but is more common in the elderly and women with diabetes (SIGN, 2006; NIHCE, 2007).

Pyelonephrities usually results from ascension of the bacteria to the kidney from the lower urinary tract and the condition may be acute or chronic. The more severe upper urinary tract disease acute pyelonephrities involves colonization of the kideys and represents as infection capable of progressing to bacteremia (Gunther et al, 2001). Symptoms of acute pyelonephrities generally develop rapidly over a few hours or a day and include fever, chills, nausea, vomiting, and diarrhea. Symptoms of cystitis may or may not be present. Besides fever, tachycardia and generalized muscle tenderness, physical examination reveals marked tenderness on deep pressure in one or both costo-vertebral angels or no deep abdominal palpation. In some patient, signs and symptoms of gram-negative sepsis predominate. Most patients have significant leukocytosis and bacteria detectable in Gram-stained unspun urine. Hematuria may be demonstrated during the acute phase of the diseases; If it persists after acute manifestation of infections have subsided, a stone, a tumor, or tuberculosis should be considered and the clinical manifestations associated with acute pyelonephritis are familiar chronic one that of (Braunwald et al, 2001).

2.6. The Epidemiology of Urinary Tract Infection.

Urinary tract infection (UTI) remains to be one of the most common infectious diseases diagnosed in outpatient (Gales et al, 2000). In 1997 these infections results in 7 million cases of physician office visits and 1 million cases of emergency room visits (Foxman, 2002). It is estimated that 150 million people with UTIs are diagnosed each year on a global basis, costing in excess of 6 billion dollars in direct health care expenditure (Gupta, 2011). UTIs are serious health problems affecting million of people each. In the USA, it is estimated from surveys of office practices, hospital-based clinic and emergency departments that there are over eight million cases of UTI annually (National Institutes of Health, 1999). The annual cost to the health care system of the system of the United States attributable to community – acquired UTI alone is estimated to be approximately $1.6 billion (Foxman, 2002). Asymptomatic bacteriuria in Nigeria was reported as the prevalence rates of 24 and 6% among rural and urban children, respectively (Okafor et al, 1993). UTIs are commonly encountered diseases by clinicians in developing countries with an estimated annual incident of at least 250 million (Nicolle, 2001 and Ronald, 2005). In Ethiopian the estimated number according to statistics by country for urinary tract infections is more than 2 million cases (Tessema et al, 2007).

In general, because of the continuous emergence of antibiotic resistance, urinary tract infections is a serious public health issues, particularly in the developing world where apart from high level of poverty, ignorance and poor hygienic practices, there is also a high prevalence of fake and spurious drugs of questionable quality in circulation (Abubakar, 2009). Therefore, investigating epidemiology of UTIs (prevalence and risk factors) and the antibiotic sensitivity of uropathogens is fundamental for caregivers and health planner to guide the expected interventions.

2.7. Risk Factors to Urinary Tract Infections

2.7.1. Anatomical and Physiological Factors

Among several factors contributing to the risk of acquiring UTIs, anatomical and physiological factors are predisposing to UTI. Urinary tract abnormalities affecting the flow of urine and emptying of the bladder increase the risk of UTI. Urine voiding disorders such as those associated with prolapsed, multiple sclerosis, bladder cancer or bladder stones increase the risk (Scholes et al, 2000). Women with any urinary tract abnormality are more prone to the female anatomy in that a much shorter urethra allows pathogens easier access to the bladder (Neumann et al, 2005). Constipation increases the residue after micturiation, causes functional obstruction and affects the flow of urine (Dohil et al, 1994; Blethyn et al 1995; Leoning-Baucke, 1997). Male circumcision reduces the risk of UTI substantially (Singh-Grewal et al, 2005). The reported incidence of UTI in infancy is greater in non circumcised boys than in circumcised boys (Schoen et al, 2000).

2.7.2. Age

UTI affects people in varying incidences depending on age group. The incidence of UTI is highest during the first year of life and peaking again during adolescence. Approximately 3% of prepubertal girls and 1% prepubertal boys are diagnosed with a UTI. (4). Bacteriuria is more common at the extremes of life. (Heffner and Gorelick, 2008). In women after the menopause vaginal prolapsed changes in virginal flora and urinary incontinence contributes to the increased susceptibility to UTIs (Perrotta et al, 2008).

2.7.3. Sex and Contraception

Gender is an important factor in UTI. Women are much more susceptible than men to community –acquired UTIs except in association with anatomic or functional abnormalities in the first year of life (Stamm, 2001 and Foxman, 2002). It is thought that bacteria are forced into the bladder during intercourse and an increase in bacteria count in the bladder after intercourse predisposing to UTI (Buckley, et al, 1978). A prospective cohort study showed that among sexually active young women the incidence of symptomatic urinary tract infection is high, and the risk is strong associated with recent sexual intercourse, recent use of a diaphragm with spermicide and a history of recurrent urinary tract infections (Hooton et al, 1996). Adding a spermicide coating further increase (7.4 – 11.5 times) the risk of UTI, and the highest risk occurs with the use of Nonoxyno1-9 (Handley et al, 2002). Spermicide alters normal vaginal flora and facilitates colonization with pathogens (Fihn et al, 1985). Additionally, in a case- control study spermicide use of nonoxyno1-9, male condoms particularly nonxyno1-9 coated condom, poor personal hygiene, and a new sex partner during the last year frequency of sexual intercourse contributes to the increased susceptibility to UTIs (Scholes et al, 2000; Handley et al, 2002).

2.7.4. Pregnancy

During pregnancy, the female urinary tract undergoes profound physiologic changes that facilitates the development of cystitis and acute pyelonephritis. Physiologic bladder changes, including decreased smooth muscle tone, increased capacity and incomplete emptying, predispose pregnant women to vesicoureteral reflux and ascending pyelonephritis. Symptomatic bacteriuria occurs in a rate well above the normal annual incidence of UTI (Vazquez and Villar, 2003). Women with bacteriuria in the first trimester develop upper urinary tract infection in the second or third trimester (Hill et al, 2005; SIGN, 2006; Sharma and Thapa, 2007).

2.7.5. Diabetes Mellitus

Patient with diabetes have a higher risk for UTI. Lower tract infections are commoner in diabetes and ascent of infection is easier, probably due to deficiency of local immune mechanisms and increased adherence of bacteria to the linings of the urinary system (Geerlings et al, 2002; Meiland et al, 2002). UTIs are more severe in diabetic women and can cause renal and perirenal damage (Baron et al, 1994).

2.7.6. Urinary Catheterization

Urinary catheterization is an indwelling catheter (urethral or suprapublic) to be used either temporarily r permanently. Urethral catheter is one that possess into the bladder through the urethra and suprapublic catheter go through the lower abdomen directly into the bladder. They share many of urinary catheter associated problems. They are quick and easy to put in and very reliable at draining the bladder in the short term. They are therefore ideal for managing the bladder in the initial phases of spinal shock (Barbara, 2001).

Many complications can arise depending on the insertion techniques, gender, state of the patient’s health and duration of catheterization. Studies show that bacteria initiate infection by entering the bladder via migration from the catheter-urethral-meatus interface along the external surface of the catheter (Sabbuba, et al, 2002). The most important risk factor for the development of catheter associated bacteriuria is the duration of catheterization. Most of the catheterized patients are bacteriuric by the end of 30 days, which is the dividing line between short term and long term catheterization (Warren, 2001). In short term catheterization most cases of bacteriuria are asymptomatic. Whereas, long term catheterization is indicated by complications such as symptomatic UTIs which cause fever, bacteria, acute upper UTI, obstruction, urinary tract stones, chronic upper UTI, and with prolonged us, loss of bladder capacity due to contraction of the bladder, urethra damage and bladder cancer (Warren, 2001; John et al, 1987; Jackson, 2006).

2.8. Virulence Factors

All uropathogens are equipped with a variety of virulence factors. Bacterial virulence factors marked influence the likelihood that a given strain, once introduced into the bladder, will cause UTI. The best characterized are those from E. coli (Oelschlaeger et al, 20002). However, not all strains of E. coli are equally capable of infecting urinary tract. Most E. coli strain that cause symptomatic UTIs is non catheterized patients belongs to a small number of specific O, K, and H serogroups. These urepathogens clones have accumulated a number of virulence genes that are often closely linked on the bacterial chromosome in “virulence island” (Braundwald et al, 2001). Uropathogenic Escherichia coli (UPEC) isolate are a genetically heterogeneous group that consist of several virulence factors associated with adherence colonization and persistence of the bacteria in the urinary tract (Yamamoto, 2007). Adherence properties of some organisms prevent the normal washout for of these organisms by bladder empting and mucosal host defense mechanism (Kamada et al, 2005).

Virulence factors associated with UPEC include adhesions (P fimbriae, tyoe 1fimbriae, S and FIC fimbriae, afimbrial adhesin), toxins like hemolysin and acrobactin (a siderophore for scavenging iron) produce increased K1 capsular antigen that protect bacteria from phagocytosis, and released endotoxins which are resistant to the bacterial actions of human serum (Yamamoto, 2007). Type 1 fimbriae are hair – like projections that entend from the surface of E. coli and other genera of the enterobacteriaceae (Gunther et al, 2001). These fimbriae bind mannose containing oligosaccharides via the Fimbriae tip protein and are required for colonization of the urinary tract by uropathogenic E. coli. Besides their primary function as adhesion loecules, several other additional functions can now be attributed to these orgenelles. Once attachment to the urothelium occurs, bacterial virulence factors other than adhesion come into play. They may also function as invasions, promote biofilm formation and transmit signal to epithelia cells resulting in inflammation (Oelschaeger et al, 2002).

Recently, it was found that there is new urovirulence factor gene, which was more frequently found in UPEC strains. Tis gene encoding a protein designated UPS (uropathogenic-specific protein) (Yamamoto, 2007; Kurazono et al, 2000). Nearly all E. coli strains causing acute pyelonephritis and most of those causing acute cystitis are uropathogenicc. In contrast, infections I protein with structural or functional abnormalities of the urinary tract are generally caused by bacterial strain that lack these uropathogenic properties; the implication is that these properties are not needed for infection of the damaged urinary tract (Braunwald et al, 2001).

2.9. Laboratory Diagnosis of Urinary Tract Infection

The diagnosis of UTs begins with the screening of patient clinically suspected of having urinary tract infection due to their claiming with problems suggestive of UTIs by a physician. Determination of the number and types of bacteria in the urine is an extremely important diagnostic procedure (Braunwald et al, 2001). Although the diagnosis is primarily based on symptoms, individual symptoms are rarely diagnostic, but in combination their accuracy is greater. Signs and examination findings are generally unhelpful in uncomplicated cystitis, but have more values where upper UTIs is possible. Thus, only patient who had pyuria and significant bacateriuira obtained from appropriate urine samples (a clan – catch midstream) are included in the microbiological analysis (Kahlmeter 2003; Wazait et al, 2003). Bacteriuria is regarded as significant when the enrine contain 105 organisms or more per ml (108/liter) in pure culture (Cheesbrough, 2001). This is true usually in symptomatic patients, two consecutive urine specimens should be examined bacteriologically before therapy is instituted, and ≥105 bacteria of a single specimen per milliliter should be demonstrable in both specimens. Samples of urine from the ureters or renal pelvis may contain < 105 bacteria per milliliter and yet indicated infection (Braunwald et al, 2001; Cheesbrough, 2001).

Dipstick screening test and microscopy are the major components of urinalysis which are useful for rapid screening of UTI. Urine dipstick consist of chemically treated paper which displays different color indicating the presence of leukocyte esterase, nitrites, blood, and protein when dipped into urine smaple (Dulczak and Kirk, 2005). Leukocyte esterase test are based on the hydrolysis of ester substrate by protein with esteriolytic activity. These proteins react with ester substrates to produce alcohols & acids that then react with other chemicals to produce a color change that is proportional to the esterase in the specimen.

Microscope of urine from symptomatic patient can be of great diagnosis value (Braunwald et al 2001; Cheesbrough, 2001; Wilson and Gaido, 2004). Bacteriuria can be detected microscopically using Grain staining of uncentrifuged specimens or direct observation of bacteria in urine specimen. Grain staining of uncentrifuged urine specimens is a simple method (Wilson and Gaido, 2004), Detecting bacteria in uncentrifuged (fresh) urine indicates urinary infection, i.e. bacteriuria is excess of 104/ml (Cheesbrough, 2001). However, bacteria can not usually be detected microscopically in infections with lower colony count (102 to 104). The detection of bacteri by urinary microscopy thus constitutes firm evidence or infection, but the absence of microscopically detectable bacteria does not exluded the diagnosis (Braunwald et al, 2001.)

Bacteriuria can be detected chemically when bacteria produce nitrite from nitrate. The biochemical reaction that is detected by the nitrite test is associated with members of the family Enterobacteriaceae (Wilson and Gaido, 2004). Urinary pathogens, e.g. E. coli Proteus, and Klebsiella species, are able to reduce the nitrate normally present in urine in sufficient concentration. When first morning urine is tested, about 80% to 90% of UTI caused by nitrate reducing pathogens can be detected. The test is negative when the infection is caused by pathogens that do not reduce nitrate such as Enterococcus faecalis, Pseudomonas and Staphylococcus species or when as previously mentioned the bacteria are too few in the urine (Cheesbrough, 2001). Another limitation to the test is that it is requires testing a specimen of the first urine produced in the morning as > 4 hours is required for bacteria to convert nitrate to nitrite as level that are reliable detectable (Wilson and Gaido, 2004). Occassionaly the nitrite test is negative because nitrite is lacking in the urine due to person being on diet lacking vegetables (Cheesbrough, 2001).

Urine culture is the best and capable method in the diagnosis of UTI to identify the causative organism and to make an accurate decision about the best treatment is urine culture (NIHCE, 2007). It is done through plating a loop full of urine on a culture media and should be incubated overnight at 370C in an incubator before being read thus identifying the uropathogens and counting the number of bacterial colonies that grow on the culture palte by colony forming unit per millimeter CFU/ml. the presence of 105 colony forming units (cfu) of bacteria per ml in a clean catch or midstream urine specimen is considered diagnostic of UTIs (Wilson and Gaido, 2004).

Routine urine culture should be plated using calibrated loops for the semi quantitative method. This method has the advantage of providing information regarding the number of CFU/ml, as well as providing isolated colonies for identification and susceptibility testing. The types of median used for routine cultures should be limited to blood agar and MacConkey agar (Wilson and Gaido 2004). For urine specimens obtained from outpatients, it is not necessary to routinely inoculate a medium that is selective for gram-positive bacteria (Haryniewicz et al, 2001; Wilson and Gaido, 2004).

Although many authorities have recommended that urine culture and antimicrobial susceptibility testing be preformed for any patient with a suspected UTI, it may be more practical and cost effective to manage women who have symptoms characteristics of acute uncomplicated cystitis without an initial culture. A positive result for pyuria and/ or bacteriuria provides enough evidence of infection to indicate that urine culture and susceptibility testing can be omitted and the patient treated empirically. Urine should be cultured, however, when a woman’s symptoms and urine examination findings leave the diagnosis of cystitis in questions. Pre-therapy culture and susceptibility testing are also essential in the management of all patients with suspect upper tract infections and of those with complicating factors, as in these situations any of a variety of pathogens may be involve and antibiotic therapy is best tailored to the individual organism (Braunwald, et al, 2001). Susceptibility testing is also essential in the management of patients with a history of recurring UTI (Cheesbrough, 2001). Thus, each laboratory should have guidelines by which pathogens are tested for antimicrobial susceptibility. These guidelines should be developed and antimicrobial susceptibility test should be performed and reported according to the most recent version of the NCCLS guidelines (NCCSLS, 2003).

2.10. Treatment and Prevention of Urinary Infections

2.10.1. Treatment

The development of new diagnostic tests and the introduction of new antimicrobial agent have allowed physicians to appropriately tailor specific treatment for each patient (Tanagho and Mcaninch, 2004). Identifying whether patients have cystitis or pyelonephritis and complicated or uncomplicated UTI is important for treatment. Except in acute uncomplicated cystitis in women, a quantitative urine culture, a gram stain, or an alternative rapid diagnostic test should be performed to confirm infection before treatment is began. When culture results become available, antimicrobial sensitivity testing should be used to direct therapy (Braunwald, et al, 2001). Considering specific patient factors, such as severity of symptoms, allergic history, results of recent microbiological test (if available), risk factors for resistance and accessibility of medical care, treatment of UTI with the appropriate antibiotic are chosen since it can minimize mortality, morbidity and any real damage from acute UTI (Gupta et al, 2001).

Majority of patients respond to oral antibiotic, but in some cases intravenous antibiotics must be used, these cases are seriously ill or septic patients, children less than 1 month of age, and in the case of vomiting. The fact that the spectrum of organisms causing acute UTI (E. coli accounts for 75% to 90%) and the susceptibility patterns of these organisms have been predictable, empiric therapy with short – course TMP-SMX has been a standard management approach for uncomplicated cystitis or TMP alone for patients with sulfa allergies (Nicolle, 2002). Three-day antibiotic regimens have emerged as the preferred management because they are often as efficacious as 7- to-10-day courses for acute uncomplicated UTI, produce fewer side effect, and thus are associated with better patient compliance (Warren et al, 1999). A 3-days course of trimethoprim-sulfamethoxazole is more effective and less expensive than 3-day courses of oral nitrofurantoin, amoxicillin, or cefadroxil in treatment of acute uncomplicated UTI (Hooton et al, 1995). However, antibiotic resistance is now becoming a major factor not only in nosocomial UTIs, but also in uncomplicated community-acquired UTIs (Gupta, 2003).

According to the recent infectious diseases society of America Clinical Management Guidelines for UTI, fluoroquinolones can be used as first-line therapy for uncomplicated UTI in areas where resistance is likely to be of concern due to its high bacterioloic and clinical cure rates, as well as low rates of resistance, among most common uropathogens, Fluoroquinolones are indicated for the management of acute uncomplicated UTIs, as well as complicated and pyelonephritis, in adults due to its braod spectrum of coverage, low rates of resistance and good safety profiles (Schaeffer, 2002).

Therapeutic drug concentrations with extended- release ciprofloxacin are established immediately after dose administration and maintained throughout the 24-hour dosage interval, permitting convenient once daily treatment. Clinical trials results confirm that extended-release ciprofloxacin is as safety used and effective as the conventional, immediate-release formulation of ciprofloxacin in patients with uncomplicated UTIs complicated UTIs acute uncomplicated pyelonephritis. These findings support the use of extended-release ciprofloxacin as a well-tolerate, effective and convenient therapy for UTIs, which may improve patients’ adherence to therapy and thereby, reduce the risk of infection recurrence and emergence of antimicrobial resistance (Blondeau, 2004).

A study conducted on nitrofurantoin indicated that it use does not share cross-resistance with more commonly prescribed antimicrobials and its more widespread use is justified from a public health perspective as a fluroquinolone-sparing agent. Beta-lactams and fosfomycin should be considered second-line agents foe empirical treatment of cystitis(Hooton, 2003).

Treatment of asymptomatic bacteriuria offers no benefit for most healthy, adult, non pregnant women since it can be frequently resolved without treatment and has no long-term medical consequences (Abrutyn et al, 1994). In addition, there are special consideration in the management of UTI among selected populations, including postmenopausal and pregnant women, and women with frequent recurrent UTIs (Nicolle, 2003). In pregnancy, acute cystitis can be managed within 7 days of treatment with amoxicillin, nitrofurantoin, or a cephalosporin. After treatment, a culture should be performed to ensure cure, and culture should be repeated monthly thereafter until cdelivery. Acute pyelonephritis in pregnant should be managed with hospitalization and parenteral antibiotic therapy, generally with cephalosporin or extended-spectrum penicillin. Continous low-dose prophylaxis with nitrofurantoin should be given to women who have recurrent infections during pregnancy (Braunwald et. al, 2001).

In general, factors to be considered in the selection of appropriate antimicrobial therapy include pharmacokinetics, spectrum of activity of the antimicrobial agent, resistance prevalence of the community, potential for adverse effects, and duration of therapy. Ideal antimicrobial agents for UTI management have primary excretion routes through the urinary tract to achieve high urinary drug levels (Nicolle, 2003).

2.10.2. Prevention

Educating people to raise awareness about the risk factors and pathogens of UTI are predominantly important in the prevention of the disease. Before resorting to antimicrobial, the progress from colonization to UTI can usually be prevented by simple measure like: sufficient fluid intake for diluting urine, good personal hygiene, complete emptying of the bladder during voiding to clear bacteria in the bladder, less use of spermicides or diaphragm by modifying birth control method and restrictive catheter use are important to prevent UTIs (Gupta et al, 1999). In some instances, urine acidifiers such as citrus juice. Berries of the Vaccinium family (bilberries, lingonberries and cranberries) and their extracts may decrease symptomatic UTIs and bacteriuria for women with recurrent infections. Chemicals in all of these fruits appear to reduce bacterial adherence to the limings of the urinary system and inhibit bacteria in urine (Ronald, 1996; Jackson, 2006; Jepson and Craig, 2008).

The worldwide increasing problem of resistant uropathogens call for additional non-antimicrobial strategies, both for the treatment and for the prevention of UTIs. There are some preliminary data on antimicrobial agents for preventing UTIs. An anti E. coli vaccine which provides exhibitions of adherence of uropathogen to uroepithelial cells has been tried in women with recurrent UTIs (Zakri et al, 2008). Vaginal probiotics containing lactobacillus have been tested to restore normal vaginal flora to reduce recurrent UTIs (Reid, 2002). The ingestion of fermented milk products containing probiotics has also been said to reduce the risk of UTI (Kontiokari, et al., 2003).

Prophylaxis should be initiated only after bacteriuria has been eradicated with a full-dose treatment procedure. All pregnant women should be screened for bacteriuria in the first trimester and should be treated if bacteriuria is demonstrated. Prophylactic treatment should be used after sexual intercourse to prevent episodes of symptomatic infection in women UTIs are temporally related to intercourse. Other patients for whom prophylaxis appear to have some merit include men with chronic prostates, patients underlining prostatesctomy, both during the operation and in the postoperative period; and pregnant women with symptomatic bacteriuria (braunwald, et al, 2001).

2.11. Antibiotic Susceptibility

Antimicrobial agents are the main therapeutic tools used against human and animal bacterial infections. Antimicrobial resistance among urinary tract isolates has recently been reported with an increased frequency all over the world (Turnidge et al, 2002). The emergence of antibiotic resistance in the management of urinary tract infections is a serious public health issue, particularly in the developing world where apart from high level of poverty, ignorance and poor hygienic practices, there is also a high prevalence of fake and spurious drugs of questionable quality in circulation, their easy availability in the community without prescription and their low cost which make them subject to abuse (Abubakar, 2009). Resistance rates to the most common prescribed drugs used in the treatment of UTIs vary considerably in different areas world-wide depending on antibiotic consuming habit and laws in that region (Tanagho and Mcaninch, 2004).

Within the community setting, due to repeated or prolonged use exposure of uropathogens to antibiotics is a strong risk factors for resistance (Hillier et al, 2007; SIGN, 2006). Repeated use of antibiotics can damages periurethral flora, allowing uropathogens to colonize and subsequently to infect the urinary tract, leaving clinicians with very few choices of drugs for the treatment of UTI. Moreover, this condition enables bacteria to exchanges their genetic material through horizontal gene transfer resulting resistant genes that confers resistance to a particular antibiotic (Tessma et al, 2007; Tanagho and Mcaninch, 2004). The estimation of local etiology and susceptibility profile could support the most effective empirical treatment and laboratory analysis is only recommended in certain circumstances (Farajnia et al, 2009).

Several studies in Netherlands on the antibiotic susceptibility patterns of uropathogens showed an increased resistance to widely used agent like trimethoprim and amoxicillin (Goettsch et al, 2000). Rate of multi-drug resistance was recorded in some studies in both community and hospital isolates with highest degree among enterococcus spp. Proteus spp. P. aeruginosa, Klebsiella spp., and S. aureus (Aiyegoro et al, 2007; Abubakar, 2009). In previous studies done in Ethiopia by Getenet and Wondewosen, 2011, it has been reported that E. coli and Klebsiella spp. Showed high percentage of resistance to ampicillin and amoxicillin (100%), and low resistance to ciprofloxacin (14.3%).the high prevalence of antimicrobial resistance among UTI E. coli isolates emphasizes the necessity to review empirical therapies (Smaoui et al, 2004).

CHAPTER THREE: MATERIALS AND METHODS

3.1. Collection of Samples

Mid stream urine samples were collected aseptically from and cultured on different media like Nutrient agar, MacConkey agar etc. and incubated for 24 hrs at 370C. Identification of isolated microorganisms was done by Gram staining. A total of 60 urine samples of outpatients (GOPD) suspected to be having urinary tract infection were collected from university of Ilorin Teaching Hospital.

3.2. Preparation of Culture Media

The culture used are Nutrient agar, MacCkonkey agar among others and are prepared according to the manufacturer specifications and also base on the user depression i.e. amount required.

3.3. Nutrient Agar.

28g of powdered nutrient agar was weighed on weighing balance and was carefully dissolved in 1L of distilled water in a conical flask and stirring rod was used to stir the mixture so as to dissolve completely. Then it was autoclaved at 1kg/cm3 pressure and 1210 for 15 minutes.

3.3.1. Blood Agar.

28g of powdered blood agar base was weighed using balance and was dissolved in 1L of distilled water in a conical flask and was stirred with stirring rod until it dissolved completely. Then the mixture was autocalved 1kg/cm3 pressure for 15 minutes. After 15 minutes of autoclaved, the mixture was allowed to cooled to about 600c and then 5mls of sterile blood sample was poured into the mixture and shaked well until it mixed completely.

3.3.2. MacConkey Agar

48.5g of MacConkey agar was weighed on a weighing balance and carefully dissolved in 1L of distilled water stirred with stirring rod and allowed to dissolved for 10 minutes. Then, it was autoclaved at 1kg/cm3 pressure at 1210c for 15 minutes.

3.4. Culturing of the sample

The samples were cultured on blood agar, MacConkey agar and blood agar respectively by making a smear on a sterile media plates labeled correctly and incubated for 24 hours at 370c in an incubator. The number of colonies in the impression area was counted and if over 25 colonies were present, the original urine sample was known to have contain greater than 105 hu/ml organism per milliliter, indicating significant bacteriuria “Kass concept of significant bacteriuria (Collee et al, 1996).

3.5. Biochemical Identification

3.5.1. Catalase Test

Both on tube and slide catalase test of isolates was performed according to MacFaddin (MacFaddin, 2000) using 3-6% hydrogen peroxide (H2O2). About 2 to 3 drops of hydrogen peroxide were introduced and observed for effervecense. i.e. gas formation.

3.5.2. Oxidase Test

Oxidase test of isolats was performed by filter paper method according Snell JJS et al. 1999 using 1% N, N, N’, N’ –tetramethy1-pphenylenediamine dihydrochloride solution.

3.5.3 Indole Test

Indole test was done by adding few drops of Kovac’s reagent to culture media according to (Cheesbrough, 1985).

3.5.4. Citrate Utilization Test

Citrate utilizayion test was performed according to the standard method of simmons J, 1990 using, 0.1gm% ammonium di-hydrogen phosphate, 0.5gm% NaCI 0.02 gm% Mg% MgS04, 0.008 gm% Bromothymol blue, 0.1 gm% di-postassium Phosphat, 0.2 gm% Sodium citrate and 1.5 gm% bacteriological agar.

3.5.5 Urease Test

Urease test for bacterial isolates was done according to the method Chakraborty SP et al. 2011b. Media were prepared with urea 2 gm %, Agar 1.5 gm%, Nacl 0.5gm%, KH2PO4 0.2 gm% and phenol red 0.0012 gm% in slant position.

3.5.6 Motility Test

Motility test of isolates was performed according to Cheesbrough, 1985. Briefly, semi solid agar was prepared using beef extract-0.3%, pancreatic digest of casein-1.0%, Nacl 0.5% and agar-0.4%.

3.5.7 Nitrate Reduction Test

Nitrate reduction tests were done according to Snell “Snell EE and Wright LD, 1941”. In brief, Nitrate broth was prepared (casein Peptone 0.5 gm%, Beef Extract 0.3 gm%, Potassium Nitrate 0.1 gm%, Galactose 0.5 gm% and Disodium Phosphate 0.25gm%) with Durham tubes (gas collector) in it. 0.8gm% sulphanilic acid (dissolved in 5M acetic acid) and 0.6 gm% alpha naphathole (dissolve in 5M acetic acid) and Zinc dust were simultaneously added to those cultures.

3.6 Screening through Gram Staining

Gram staining of isolates was performed according to standard method (Duguid JP, 1999). Briefly, on a glass slide bacterial smear was prepared from broth culture and heated gently to fix. The slide was flooded with 0.5% crystal violet and left for 30 sec. then it was titled and poured sufficient 1% Lugol’s iodine to wash away the excess stain. The slide was covered with fresh iodine and allowed to act for 30 sec. the slide was titled and washed off the iodine with 95-100% ethanol until colour ceases to run out of the smear. The slide was rinsed with water and 0.1% safranin was poured on it and left to act for 2 min. the slide was washed with water and blotted to dry and observed under microscope.

3.7 Antimicrobial Susceptibility

The susceptibility of the isolated organism against different antibiotics was checked by desk diffusion method on Mueller Hinton agar. The antibiotics which are commonly prescribed by doctors in UTI’s were selected for the test. The antibiotics used were ceftazidine, norfloxacin, ciprofloxacin, nitrofurantoin, amikacin and gentamycin. Inoculums was prepared by using isolated bacterial colonies. The well isolated bacterial colonies were inoculated in the nutrient broth. After than an incubation was given at 370C till turbidity reached equal to 0.5 Mc Farland barium sulphate standard solution (Devi, et al., 2011). The degree of inhibition shown by different antibiotics was measured by using a transparent plastic ruler and was compared with the ranges published by the manufacturer, Hi-media.

3.7.1 Susceptibility Test by Disc Agar Diffusion

Antimicrobial susceptibility was determined by the Kirby – Bauer disk diffusion method (Bauer AW et al, 1966) and according to the method of Chakraborty SP et al. 2011b. The tested bacterium was from an overnight culture (inoculated from a single colony) and freshly grown for 4 hour at approximately 106 CFU/ml. with this culture, a bacterial lawn was prepared on Mueller-Hinton agar. Filter paper disc of 6-mm size were used to observed antibiotic susceptibility patterns against 14 antibiotics (amount of antibiotics per disc in microgram (µg); Gantamycin (10 µg) Norfloxacillin (10 µg), Ciprofloxacin (10 µg), Amikacin (30 µg). antibiotic discs were obtained commercially from Himedia. The diameter of zone of bacterial growth inhibition surrounding the disc (including the disc) was measured and compared with the standard for each drug. This gave a profile of drug susceptibility vis-à-vis antibiotic resistance.

CHAPTER FOUR

Urinary Tract Infection, commonly known as UTI, affects as many 50% women, at least once during their lifetime. All individuals are susceptible to urinary Tract Infection (UTI); however the prevalence of infection differs with age, sex and certain predisposing factors. UTI was found to be prevalent in the female among the age group of 20 – 29 with (33.3%) but less common between 50 – 59 with (8.3%). In the present study, a total of 60 samples were taken from people of different age groups the following pathogenic bacteria were isolated, E. coli, Klebsiella, Enterobacter, Citrobacter, Pseudomonas, Staphylococcus, Proteus, Micrococcus, Streptococcus and Bacillus were isolated, shown in the appendix, most common uropathogens accounting for 40% of UTIs. Klebsiella (12%), Enterobacter (15%), Citrobacter (8%), Pseudomonas (12%), Staphylococcus (10%), are responsible for most of the rest shown in Table 2. From the present study it can be concluded that nearly 80% of UTI are caused by gram negative bacilli as shown in table no 1 which mostly includes the members of Enterobacteriaceae family. A few samples also contain gram positive bacteria. Table 3 showed susceptibility pattern of the isolates. E. coli were found to be susceptible to 8% of Amikacin, while 75% were resistant to Ceftazidime, Norfloxacin and Nitrofuratocin. In allAmikacin found to be effective against all the tested bacteria showing 100% active against Klebsiella, Enterobacter, Staphylococcus and Citrobacter. Enterobacter was found to be susceptible to four of the tasted antibiotics while Pseudomonas aerogenes was resistant to them.

Table 1: Frequency of UTI in Male and Female of different age group

Abbildung in dieser Leseprobe nicht enthalten

Total Sample = 300 47 (78.3%) 113(21.6%)

Table 2: Prevalence of Uropathogens Isolated from suspected UTI Patients.

Abbildung in dieser Leseprobe nicht enthalten

Total Isolate 97 17 (17.5%) 80 (82%)

Table 3: Antibiotics Susceptible Pattern of Isolated Uropatheogens %

Abbildung in dieser Leseprobe nicht enthalten

CA: Ceftazidime NF:Norfloxacin CF: Ciprofloxacine Nx: Nitrofuratoin AK: amikacin G: Gentamicin

CHAPTER FIVE

Discussion

Urinary tract infection (UTI) remains a worldwide therapeutic problem, not only as a nosocomial disease but also as a community-acquired infection (Bacheller et al, 1997; Hoberman et al, 1997; Orett et al, 1999; Gupta et al, 2001). Early diagnosis and prompt antimicrobial treatment are require to minimize these complications (CQISUTI-1999). Escherichia coli (38% and 2%) are the commonest cause of UTI in our study with both female and male followed by Enterobacter species (15.4%) only in female, Klebsiella sp (12%) and Pseudomonas species (12%) respectively. These findings are similar to studies (Mohammed and Khadri, 2011).

The study found Escherichia coli, Pseudomonas and Klebsiella species among the top 5 pathogens (Shariti et al, 1996). The majority of the bacteria were isolated from female 80 (82.3%), while the remaining 17 (17.5%) were from male. The sex distribution of parents in our study is consistent with other reported studied. In our study, the incidence of UTI was high among the female (54.34%) than males (45.66%). Factors such as short urethra and its closeness to the anus as well as sexual activity have been reported to influence the higher prevalence of UTI in females (Adedeji et al, 2009). The female to male ratio among age groups of less than 20 years and age group of more than 50 years was equal and higher for age group of 20 to 40 years. This may be due to increase sexual activity at this age range which predisposes the female to the possibility of contracting UTI. This result is in agreement with other reports which showed that UTI were more common in females than males during adolescences and adulthood (Adedeji et al, 2009; Gales et al 2000; Kebira et al, 2009; Kolawole et al, 2009; orret et al, 1998; Tambekar et al, 2006; Theodore, 2007).

The frequency of UTI is greater in women as compared to men and our results were similar to these reports (Schaeffer et al, 2001). This might be owing to anatomic and physical factors (Kumar et al., 2006; Khan et al., 2004). Also, the uropathogens identified in our study are similar to those of many other studies conducted in different countries either in the region or internationally (Astal et al, 2002), however different results have been reported. The similarities and differences in the types and distribution of uropathogens may result from different environmental conditions and host factors, practices such as health care and education programmes, socioeconomic standard and hygiene practices in each country (Mansour Amin et al., 2009).

The Enterobacteriaceae family were the common microorganisms isolated of urinary tract infection in present study accounting 71% of total isolated bacteria and amongst them E. coli (60%) was the most predominant bacteria. This result is consistent with reports from other studies (Abubakar, 2009; Daza et al., 2001; Dimitrov et al 2004; Guneberg, 1980; Orret et al; 1998; Omigie et al., 2009) but differs from the reports of Ehinmidu (2003) and Aboderin et al (2009) which recorded P. aeruginosa and Klebsiella spp, respectively as the predominant bacteria.

In our study, Pseudomonas aeruginosa was found to be the most common Multi Drug Resistance (MDR) uro-pathogens follows by Citrobacter Koseri and E. coli. MDR was defined as resistance to 3 or >3 classes of the antimicrobial (≥3drugs). Similar data is presented in other reports. In our study the most active antibiotic against all isolates was amikacin. In constrast to other isolates high resistance was found against ceftazidine, nitrofurantoin, and gentamicin. Although the prevelance of pathogens in different parts of the world is somewhat similar, antimicrobial resistance patterns reported from different regions are significant and antimicrobial resistance increases.

Multi-drug resistant P. aeruginosa, Klebsiella spp, S. aureus and Enterobacter spp strains have been widely reported in some studies (Abubakar, 2009; Gales et al, 2000). High prevalence of multi antibiotic resistance strains is a possible indication that very large population of bacterial isolates has been exposed to several antibiotics.

Conclusion

UTI is the most common problem throughout the world and in the study area as well. In addition, bacterial resistance to commonly used antibiotic agents is widespread phenomenon all over the world. The importance of this studies lies in it’s contribute on in assessing the prevalence of bacterial uropathogens and their in vitro susceptibility pattern to common used antibiotic agents amongst outpatients in University Ilorin Teaching Hospital.

On the basis of patient study undertaken on UTI it has been found E. coli is the major group of microorganisms found in female and males after which comes Enterococcus, Klebsiella, Proteus, Pseudomonas etc. From the present study it can be concluded that nearly 80% of UTIs are caused by gram negative bacilli which mostly includes the members of enterobacteriaceae.

Recommendation

Based on the result of this study, the following recommendations are made:

- Since this study involved only out patients, further studies should be conducted on inpatients in order to find the prevalence of other uropathogenes in the study area.
- Conducting antimicrobial susceptibility tests before prescribing antimicrobials for the treatment of UTI is advisable.
- Drugs for which isolates have developed resistance should be avoided in the study area.
- Establishing good management of drugs in order to prevent their easy available in the community without prescription and drugs of questionable quality in circulation.
- Creating awareness about the potential risk factors of UTI is important to reduce the risk of the disease.

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Appendix

Table 1

Colony morphology, Gram staining and microscopic appearance of the positive samples.

S/n Sample No Colony Colony Gram Staining Morphology on morphology on MacConkey agar nutrient agar.

Abbildung in dieser Leseprobe nicht enthalten

S/n Sample No Colony Colony Gram Staining

Morphology on morphology on

MacConkey agar nutrient agar.

Abbildung in dieser Leseprobe nicht enthalten

S/n Sample No Colony Colony Gram Staining Morphology on morphology on MacConkey agar nutrient agar.

Abbildung in dieser Leseprobe nicht enthalten

S/n Sample No Colony Colony Gram Staining Morphology on morphology on MacConkey agar nutrient agar.

Abbildung in dieser Leseprobe nicht enthalten

S/n Sample No Colony Colony Gram Staining Morphology on morphology on MacConkey agar nutrient agar

Abbildung in dieser Leseprobe nicht enthalten

S/n Sample No Colony Colony Gram Staining Morphology on morphology on MacConkey agar nutrient agar

Abbildung in dieser Leseprobe nicht enthalten

S/n Sample No Colony Colony Gram Staining Morphology on morphology on MacConkey agar nutrient agar

Abbildung in dieser Leseprobe nicht enthalten

LFC: Lactose Fermenting Colonies

NLFC: Non Lactose Fermenting Colonies

Table 2

Table showing biochemical results of sample no. Al-A9

The Greek Alphabelt

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Table 3

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Table 4

Table showing biochemical results of sample no. A37-A60

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Table 5

Table showing different types of bacteria isolated from different urine samples

S/n sample Age (years) Gender Name of the bacteria isolates

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S/n sample Age (years) Gender Name of the bacteria isolates

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S/n sample Age (years) Gender Name of the bacteria isolates

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S/n sample Age (years) Gender Name of the bacteria isolates

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S/n sample Age (years) Gender Name of the bacteria isolates

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Table 6

Table showing the result of antibiotic sensitivity test of isolated

Sample Ceftazidime Norfloxacin Ciprofloxacin Nitrofuratoin amikacin Gentamicin

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Sample Ceftazidime Norfloxacin Ciprofloxacin Nitrofuratoin Amikacin Gentamicin

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Frequently asked questions about "Urinary Tract Infections: A Language Preview"

What is a urinary tract infection (UTI)?

A urinary tract infection (UTI) is a term applied to a variety of clinical conditions ranging in severity from asymptomatic bacteriuria to symptomatic acute infection of the kidney with resultant sepsis. It involves the growth of a known bacterial pathogen of more than 10³ cfu/ml in association with a positive dipstick or urinalysis.

How are UTIs classified?

UTIs are usually classified by the infection site (lower or upper urinary tract) or as complicated or uncomplicated infections.

What are the common causes (etiology) of UTIs?

The most common agents causing UTIs are bacteria (95%), but fungal and viral infections may also be involved. Escherichia coli is the most frequent uropathogen, accounting for 65%-90% of urinary infections.

What are the types of urinary tract infections?

UTIs can be classified into lower and upper urinary tract infections, as well as complicated and uncomplicated UTIs.

What are the risk factors for developing UTIs?

Risk factors for UTIs include anatomical and physiological factors, age, sex, contraception, pregnancy, diabetes mellitus, and urinary catheterization.

What are the common clinical features of UTIs?

Clinical features of UTIs depend on the infection site but may include dysuria, frequency, urgency, suprapubic tenderness, fever, chills, nausea, vomiting, and diarrhea.

How is a UTI diagnosed in a laboratory?

Laboratory diagnosis of a UTI involves urinalysis (dipstick screening and microscopy) and urine culture to identify the causative organism and determine its antibiotic susceptibility.

What are the treatment and prevention strategies for UTIs?

Treatment strategies involve the use of appropriate antibiotics. Prevention strategies include sufficient fluid intake, good personal hygiene, complete emptying of the bladder, modified birth control methods, and restricted catheter use. Some evidence suggests urine acidifiers and cranberry extracts may reduce UTI recurrence.

What is antibiotic susceptibility and why is it important?

Antibiotic susceptibility refers to the sensitivity of uropathogens to various antimicrobial agents. Monitoring antibiotic susceptibility is crucial due to the increasing problem of antimicrobial resistance, which can complicate UTI treatment.

What sample types were used for this study?

Mid-stream urine samples collected aseptically from outpatients suspected of having urinary tract infections were used.

Which culture media was used for this study?

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The culture media used were Nutrient agar, MacConkey agar, and Blood agar.

What biochemical tests were used for this study?

Catalase test, Oxidase test, Indole test, Citrate Utilization Test, Urease Test, Motility Test, and Nitrate Reduction Test.

How was antimicrobial susceptibility tested?

Antimicrobial susceptibility was tested through the Kirby-Bauer disk diffusion method.

What Uropathogens were most frequently isolated from the tested samples?

The most common Uropathogens isolated were *Escherichia coli*, followed by Klebsiella, Enterobacter, Citrobacter, Pseudomonas, Staphylococcus, Proteus, Micrococcus, Streptococcus and Bacillus.

What antibiotics was the most frequently isolated *E. coli* susceptible and resistant to in the study area?

The study found *E. coli* susceptible to amikacin while resistant to ceftazidime, norfloxacin and nitrofuratoin.