ANTIBIOGRAM OF UROPATHOGENIC BACTERIA ISOLATED FROM PATIENTS IN SOME HOSPITALS IN BIRNIN KUDU, JIGAWA STATE

ABSTRACT

Urinary tract infection (UTI) is a common infection of human being and if untreated could lead to serious complications. This study was conducted to investigate the antibiotic susceptibility pattern of uropathogens from patients in two hospitals in Birnin kudu, Jigawa State, Nigeria. In this study, the antibiotic resistance profile and the plasmid profile of some multi-antibiotic resistant bacteria isolated from urine samples of patients from Birnin kudu community in North-west, Nigeria were analysed. Rapid diagnostic kit systems were used in identification of the isolated bacteria and agar disc diffusion technique was used for the determination of antibiotic susceptibility profiles of the isolated bacteria. Presence of β- lactamase was determined using standardized β-lactamase identification sticks while acridine orange was used for curing of multi-drug resistant isolates. The cultures of some multi- antibiotic resistant isolates irreversibly lost their antibiotic resistance with acridine orange treatment, which suggests that the resistant genes could be harboured in the plasmids. The result showed that 94.3% of the isolates were resistant to Ampicillin, Amoxycillin-clavulanic acid (71.5%), Ceftriaxone (35.4%), Cefuroxime (57.3), Cotrimoxazole (73.1%), Nitrofurantoin (24.6%), Chloranphenicol (36.9), Doxycycline (58.0%), Ciprofloxacin (60.0%) and Gentamicin (61.2%). Out of 36 isolates tested for presence of β- lactamse, 66.1% possessed β- lactamases. Plasmid profile studies revealed the presence of plasmid of size range 5184.8kb – 5673.9bp.

CHAPTER ONE

1.1 BACKGROUND OF THE STUDY

Urinary Tract Infection (UTI) is an infection that affects part of the urinary tract. When it affects the lower urinary tract, it is known as a simple cystitis (a bladder infection) but when it affects the upper urinary tract, it is known as pyelonephritis (a kidney infection). Lower urinary tract infection is characterized by burning sensation during urination with either frequent urination or urge to urinate or both and is often accompanied with significant pain (Nicolle, 2008). These symptoms may vary from mild to severe (Lane and Takhar, 2011) and in healthy women, can last an average of six days (Colgan and Willliams, 2011). Upper urinary tract infection is characterized by flank pain, fever, or nausea and vomiting in addition to classic symptoms of a lower urinary tract infection (Lane and Takhar, 2011). Rarely, the urine may appear bloody or contain visible pus. In children, the symptoms may be a fever. Infants may feed poorly, vomit, sleep more or show signs of jaundice. In older children, new onset urinary incontinence may occur.

Escherichia coli is the cause of 80-85% of urinary tract infections, with Staphylococcus saprophytic-us being the cause in 5-10% of the cases (Nicolle, 2008). Other bacterial causes include: Klebsiella, Proteus, Pseudomonas, and Enterobacter. These are less common and typically related to urinary catheterisation (Salvatore, 2011). Urinary tract infection due to Staphylococcus aureus occur secondary to blood-borne infections (Lane and Takhar, 2011).

Acute uncomplicated urinary tract infections (UTIs) are a common clinical syndrome that occurs in women with otherwise normal genitourinary tract (Hooton et al., 2000) with about 3% of the women in the United States visiting a Physician at least once each year have uncomplicated urinary tract infection (NCHS, 1979). Sexual intercourse is the cause of 75-95% of bladder infection in young, sexually active women. The risk of infection is related to frequency of sexual intercourse (Nicolle, 2008). Urinary tract infections is very frequent when women first get married, hence the term ―honeymoon cystitis‖ is often used. More women get urinary tract infections than men because women have a urethra that is much shorter and closer to the anus (Dielubanza and Schaeffer, 2011). As a woman‘s estrogen levels decreases with menopause, the risk of urinary tract infections increases due to the loss of protective vaginal flora. Other risk factors include diabetes (Nicolle, 2008) and having a large prostate (Lane and Takhar, 2011). The use of catheter increases the risk of urinary tract infections. The risk of contracting bacteriuria is 3-6% every day the catheter is used: antibiotics do not stop these infections.

The pathogens causing UTI are consistent across the globe. The pathogenesis of urinary tract infection involves ascending infection with coliform bacteria colonizing the perineum in susceptible women (80–90% Escherichia coli, 5–10% Staphylococcus saprophytic-us with the remainder caused by Proteus and other Gram negative rods) (Zalmanovici et al., 2010). While not generally considered a cause of significant mortality, UTI do represent an important cause of morbidity if left untreated. This is more likely to be the case where access to or availability of timely and appropriate medical intervention is limited due to inadequate numbers of health care providers. UTI can be particularly dangerous in pregnant women in whom it has been shown that up to 50% of those with asymptomatic bacteriuria (ABU) go on to develop pyelonephritis. In addition, these women experience higher rates of intrauterine growth restriction and low birth-weight infants. The presence of a UTI has also been shown to increase the risk of preterm labor, preterm birth, pregnancy-induced hypertension, preeclampsia, amnionitis and anemia (Delzell and Lefevre, 2000).

The antimicrobial misuse in clinical medicine has led, in part, to an increase in microbial resistance; the consequent spread of bacterial resistant strains is a serious public health problem. Urinary tract infection is one of the most common diseases of the community and also of the hospital setting. In 1997, it was reported that 29% of healthy adults outside the hospital environment are colonized by methicillin-resistant Staphylococcus aureus (MRSA). By 2008, the figure had increased to 74% (EARSS, 2008). This type of colonization is caused by strains of Staphylococcus aureus different from those found in the hospital environment and are often referred to as community-associated MRSA (CA-MRSA). Some studies have shown that CA-MRSA has high potential to become endemic in the community and that this will have a significant impact on the control of MRSA in hospital (Klutymans, 2006; Tang and Stratton, 2010).

Beta-lactamases are enzymes produced by bacteria that provide multi-resistance to β-lactam antibiotics such as penicillins, cephamycins, and carbapenems, although carbapenems are relatively resistant to beta-lactamase. Beta-lactamase provides antibiotic resistance by breaking the antibiotics’ structure. These antibiotics (β-lactam antibiotics) all have a common element in their molecular structure: a four-atom ring known as a β-lactam. The lactamase enzyme breaks the β-lactam ring by hydrolysis thereby opening and deactivating the molecule’s antibacterial properties. Beta-lactamases produced by Gram-negative organisms are usually secreted, especially when antibiotics are present in the environment (Neu, 1969).

Classification

Group 1

CEPHALOSPORINASE: Group 1 are cephalosporinases not inhibited by clavulanic acid, belonging to the molecular class C

Group 2

Group 2 are penicillinases, cephalosporinases, or both inhibited by clavulanic acid, corresponding to the molecular classes A and D reflecting the original TEM and SHV genes. Additionally, many class A TEM β-lactamases are inhibited by β-lactamase inhibitor protein (BLIP).

Group 3

METALLOENZYME, Molecular Class B

Group 3 are the zinc-based or metallo β-lactamases, corresponding to the molecular class B, which are the only enzymes acting by the metal ion zinc, as discussed above. Metallo B-lactamases are able to hydrolyse penicillins, cephalosporins, and carbapenems.

Group 4

PENICILLINASE, No Molecular Class

Group 4 are penicillinases that are not inhibited by clavulanic acid, and they do not yet have a corresponding molecular class. This group has been omitted from the current scheme, as when it was described originally it included enzymes not classifiable into other groups. It has been concluded that the enzymes that made up this classification in 1995 would be included in one of the other groups once more information becomes available on them and that it was not informative to have a separate group for them (Bush & Jacoby, 2010).

Molecular classification

The molecular classification of β-lactamases is based on the nucleotide and amino acid sequences in these enzymes. To date, four classes are recognised (A-D), correlating with the functional classification. Classes A, C, and D act by a serine-based mechanism, whereas class B or metallo-β-lactamases need zinc for their action.

Resistance in Gram-negative bacteria

Among Gram-negative bacteria, the emergence of resistance to expanded-spectrum cephalosporins has been a major concern. It first appeared in a limited number of bacterial species (C. cloacae, C. freundii, S. marcescens, and P. aeruginosa) that could mutate to hyperproduce their chromosomal class C β-lactamase. A few years later, resistance appeared in bacterial species not naturally producing AmpC enzymes (K. pneumoniae, Salmonella spp., P. mirabilis) due to the production of TEM- or SHV-type ESBLs. Characteristically, such resistance has included oxyimino- (for example cefotaxime, ceftriaxone, and ceftazidime, as well as the oxyimino-monobactam aztreonam), but not 7-alpha-methoxy-cephalosporins (cefoxitin and cefotetan); has been blocked by inhibitors such as clavulanate, sulbactam, or tazobactam, and did not involve carbapenems and temocillin. Chromosomal-mediated AmpC β-lactamases represent a new threat, since they confer resistance to 7-alpha-methoxy-cephalosporins (cephamycins) such as cefoxitin or cefotetan are not affected by commercially available β-lactamase inhibitors, and can, in strains with loss of outer membrane porins, provide resistance to carbapenems (Philippon et al., 2002).

1.2 STATEMENT OF THE RESEARCH PROBLEM

Urinary tract infection is one of the common infections of the community and also of the hospital settings, resulting in high rate of morbidity and high economic costs associated with its treatment (Arjunan et al., 2010; Rahman et al., 2009; Hryniewicsz et al., 2001).

Urinary tract infection (UTI) is the second most common infectious presentation in community medical practice. Worldwide, about 150 million people are diagnosed with UTI each year costing the global economy in excess of six (6) billion dollars (Gonzalez and Schaeffer, 1999). In the United States, UTI accounts for 8.3 million out-patient visits and one million hospitalizations annually (CDC, 2004).

UTIs are common in general practice, accounting for 1-3% of all consultations. Almost half of all women report at least one UTI sometime during their lifetime, and after an initial UTI, 20% to 30% of women experience a recurrence (Foxman, 2003).

Some studies carried out have shown that uropathogens such as Escherichia coli (46.4-74.2%), Klebsiella spp (6-13.45%), Proteus spp (4.7-11.9%) and Enterococcus spp (5.3-9.54%) represent the main causes of urinary tract infection (Rahman et al.,2009; Akram et al., 2007; Laupland et al., 2007). Escherichia coli has been indicated as the most frequent uropathogens involved in the community–acquired urinary tract infection (Francesco et al.,2007; Laupland et al.,2007) due to the fact that it belongs to the normal flora of the human intestine and therefore easily colonizing the urinary tract. Some strains of Escherichia coli isolated from sexually active patients matched with faecal isolates from their partners, which indicate that the urinary tract infection can be sexually transmitted (Wiles et al., 2008).

The prevalence of UTIs in Benin City was reported as 14.58% (Orhue, 2004). This prevalence level is relatively low compared to the 22% reported for Ibadan (Okesola and Oni, 2009) and much lower compared with figure reported in other parts of the country. For example 35.5% was reported for Jos (Ebie et al., 2001) and in Lagos; the figure is 38.6% (Akinyemi et al., 1997). Much higher incidences were reported in some towns, 60% in Lafia (Kolawole et al., 2009), 67.2% in Yola, Adamawa State (El-Mahmood et al., 2009) and 77.9% in Enugu (Mbata, 2007).

Antibiotic 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% (Schito et al., 2009; De Backer et al., 2008).

Worldwide, Flouroquinolones are being used as the most common antimicrobials for all UTIs, both complicated and uncomplicated. Raul, (2007) explored risk factors for developing Quinolone resistance in uropathogens in the community. UTI is the most common bacterial infection seen in the community, and E. coli is responsible for about 70% to 80% of all the uropathogens. Quinolones are one of the most widely used antibiotics in the community for the treatment of UTI, and it is this unfortunate excessive use of the agent that has led to a considerable and worrying increase in the rate of E. coli resistant

isolates in many countries. The magnitude of the problem worldwide is becoming very apparent. In as much as the major risk factor associated with the development of these microorganisms is overuse, physicians must practice antibiotic stewardship and avoid the empiric use of quinolones when other antimicrobials may be adequate (Raul, 2007).

Fluoroquinolone resistance is increasing and it is associated with multi-drug resistance. The indiscriminate use of Fluoroquinolones as empirical treatment for the UTIs will facilitate the emergence of resistance to this class of compounds and promote the emergence of multi-drug resistant strains and it should be discouraged as it will undermine the efficacy of Fluoroquinolones to treat more serious infections (James et al., 2006).

The etiology and resistance pattern of community-acquired uropathogens has not been extensively studied in Birnin Kudu community.

It is therefore, important to study susceptibility of some common uropathogens (Escherichia coli, Klebsiella, Citrobacter, Serratia and Staphylococcus spp) and the reason for significant variation in antibiotic resistance through molecular characterization of these organisms.

1.3 JUSTIFICATION

Antimicrobial misuse has led to an increased microbial resistance and consequent spread of bacterial resistant strains in both community and hospital settings. Unfortunately there are few publications about the main uropathogens implicated in community-acquired urinary tract infection and their antimicrobial resistance pattern, when compared with nosocomial urinary tract infections.

The cost of microscopy, culture and susceptibility testing is more than the antibiotic treatment itself. These factors have complicated empiric treatment of UTI as data on prevalence of uropathogens and antimicrobial susceptibility are not readily available particularly in developing countries.

Considering the fact that as with many community-acquired infections, resistance rates to antimicrobials commonly used in treatment of UTI is increasing and susceptibility of microorganisms shows significant geographical variations (Gupta et al., 1999). Therefore, studies to increase knowledge on etiologic agents of UTIs and their resistance patterns to antibiotics at the local levels are very important as we know there is growing problem of drug resistance which means there is an urgent need for continuous surveillance of antibiotic susceptibility of uropathogens. Appropriate knowledge of local antimicrobial resistance trends is of utmost importance in order to step up evidence based recommendations in empiric antibiotic treatment of UTI.

There are also public health issues as UTI is related to quality of life. Lower urinary tract symptoms which accompany UTI such as urinary urgency, frequency, painful urination, hesitancy, and the sense of incomplete bladder emptying have negative impact on the quality of life (Liao et al., 2009). In addition to a reduction in quality of life for women who are symptomatic, in countries with limited health care resources, unnecessary UTIs might be expected to cause a drain on the already struggling health care apparatus.

1.4 AIM

To determine the antibiogram of uropathogenic bacteria isolated from patients in some hospitals in Birnin kudu, Jigawa State, Nigeria

1.5 SPECIFIC OBJECTIVES

The specific objectives of this work are to:

a. Characterize bacterial uropathogens from patients that are diagnosed with UTI using Rapid identification test kits.

b. Determine the antibiotics susceptibility profiles of the isolated bacterial uropathogens using the agar diffusion technique

c. Explore the presence of β- lactamase in the isolated bacteria

d. Determine if the resistance to antimicrobial agents is plasmid mediated using the Gel electrophoresis method.

1.6 HYPOTHESIS

NULL HYPOTHESIS (HO)

• Uropathogens isolated from UTI patients are generally susceptible to commonly used antibiotics.

ALTERNATE HYPOTHESIS (HA)

• Uropathogens isolated from UTI patients in FMC and GHC, Birnin kudu are not susceptible to commonly used antibiotics

1.7 RESEARCH LIMITATION

• Collection of isolates was limited to UTI patients that attended the hospitals from August, 2014 to January, 2015.

• Only isolates that were multi-drug resistant underwent further molecular studies to determine the R-plasmid profiles.