Urinary tract infections (UTIs) have been reported

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1 o r i g i n a l c o m m u n i c a t i o n Antimicrobial Resistance in Escherichia coli Strains From Urinary Tract Infections Oladipo A. Aboderin, FMCPath; Abdul-Rasheed Abdu, MSc; Babatunde W. Odetoyin, MSc; Adebayo Lamikanra, PhD Background: An increase in resistance against many different drugs among urinary tract infection (UTI) E coli isolates has been observed in the last 2 decades. This study determined the trends of antimicrobial resistance in E coli to commonly used antibiotics. Methods: The study was conducted in Ile-Ife, southwest Nigeria. Patients with features suggestive of UTI were investigated for presence of significant bacteriuria. Urine isolates were identified. Antimicrobial susceptibility was evaluated in accordance with standard bacteriological methods. Results: Of 442 urine specimens, 158 (35.8%) yielded significant growth, including 41 (25.6%) with E coli. Among the E coli isolates, antimicrobial susceptibility varied in prevalence by agent in descending order as follows: nitrofurantoin (80%), ofloxacin (24%), ciprofloxacin (15%), nalidixic acid (10%), cotrimoxazole (5%), and amoxicillin/clavulanic acid (2%). No isolate was susceptible to amoxicillin, gentamicin, or tetracycline. All were also found to be resistant to at least 3 commonly used drugs. All 25 isolates tested for extendedspectrum ß-lactamase (ESBC) production were found to be presumptive ESBCs producers. Conclusion: The results demonstrate the continued susceptibility of E coli to nitrofurantoin and their widespread and increasing resistance to amoxicillin, gentamicin, cotrimoxazole, ciprofloxacin, ofloxacin, and tetracycline. Nitrofurantoin is a and, in this locale, perhaps the only rational drug for empiric treatment of uncomplicated UTI. There is a need for a comprehensive study of the involvement of ESBC-producing E coli in UTI in this environment. Keywords: urinary tract infection n bacteria n Nigeria J Natl Med Assoc. 2009;101: Author Affiliations: Department of Medical Microbiology and Parasitology, College of Health Sciences (Messers Aboderin and Odetoyin)and Department of Pharmaceutics, Faculty of Pharmacy (Dr Lamikanra), Obafemi Awolowo University, Ile-Ife, Osun-State, Nigeria; and Department of Medical Microbiology and Parasitology, College of Health Sciences, Niger Delta University, Wilberforce Island, Bayelsa-State, Nigeria (Mr Abdu). Corresponding Author: Adebayo Lamikanra, PhD, Department of Pharmaceutics, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Osun- State, Nigeria (alamikanra@yahoo.com). Introduction Urinary tract infections (UTIs) have been reported to affect up to 150 million individuals annually worldwide. 1 They are the most common bacterial infection in women and account for significant morbidity and health care costs. 2 In the United States, UTIs result in approximately 8 million physician visits and more than hospital admissions per year 3 with young, otherwise apparently healthy, sexually active women being at highest risk for community-acquired UTIs. The main risk factors that have been identified for this infection are sexual intercourse, spermicide-based contraception, and a history of UTIs. 4 Infections, including cystitis and pyelonephritis, are common both in community and hospital settings with Escherichia coli being the predominant pathogen. 5 There is increasing evidence that some E coli strains that cause UTIs and other extraintestinal infections are responsible for community-wide epidemics. 6-8 Up to 95% of UTI cases, including even some of those with severe symptoms, are treated without bacteriological investigation, with the empirically selected antibiotic treatment varying according to the patient s age, sex, and the infecting agent. 9 A short course of trimethoprim-sulfamethoxazole (cotrimoxazole) is the recommended empirical antimicrobial agent for treating acute uncomplicated bacterial cystitis in settings where the prevalence of resistance to cotrimoxazole in E coli is less than 20%. 2,3 However, the changing spectrum of microorganisms involved in UTIs and the emergence of acquired microbial resistance dictate the need for continuous surveillance to guide empirical therapy. 10 This being the case, the World Health Organization and the European Commission have recognized the importance of studying the emergence and determinants of acquired antimicrobial resistance and the need to devise appropriate strategies for their control. 11 Over the last 2 decades, the proportion of community-acquired strains with resistance to first-line agents such as ampicillin, nitrofurantoin, and cotrimoxazole have been shown to be on the increase More recently, resistance to fluoroquinolones such as ciprofloxacin and levofloxacin has been on the increase too, 16,17 thus further complicating the man JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION VOL. 101, NO. 12, DECEMBER 2009

2 agement of these infections. Recent studies indicate that UTIs caused by extendedspectrum ß-lactamase (ESBC)-producing E coli constitute an emerging problem in outpatient settings in various parts of the world. 18,19 Possible community acquisition of ESBCs-producing E coli was first reported in 1998, when a nalidixic acid-resistant uropathogenic E coli producing an ESBC was isolated from an elderly patient who did not have a recent history of hospitalization in Ireland. 20 Since then, ESBC-producing E coli have been increasingly reported in different communities It is imperative that the prevalence of antimicrobial resistance in organisms associated with UTI be the subject of regular study and that these studies be performed at different geographic locations. 28 Since the most recent such work from Ile-Ife (a semiurban community in southwest Nigeria) was published in 1988, 29 we sought to define the current trends of antimicrobial resistance in our region. Moreover, since ESBC-producing strains have emerged in other parts of the world in the intervening period, we evaluated ESBC production in some of the strains isolated. Materials and Methods Patients All the patients recruited presented at the various departments (outpatient clinics and wards) of the Ife State Hospital unit of the Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC) between September 2004 and April They all complained of symptoms associated with UTI and were thus referred to the medical microbiology laboratory for investigation. Sociodemographic information, including age, sex, and status regarding hospitalization was obtained from each patient. Mid-stream urine specimens were collected from each of the patients into sterile bottles (Sterilin, England) and immediately processed appropriately. Bacterial Strains Urine microscopy, isolation and identification of organisms were carried out as part of the routine procedures in the diagnostic microbiology laboratory of OAU- THC, and cultures with a colony count of 10 5 colonyforming unit (CFU/mL) were considered significant for inclusion. Briefly, a semiquantitative technique was performed by inoculating the urine specimens on a welldried cystine-lactose-electrolyte-deficient medium. The plates were incubated overnight aerobically at 37ºC. The identities of all the isolates with significant growth ( 10 5 CFU/mL) were determined by employing standard microbiological techniques. 30 Isolates that were gram negative, lactose positive, indole positive, and citrate negative were defined as E coli and were stored in tryptone soya agar. Antimicrobial Susceptibility Testing Susceptibility to 9 antimicrobial agents was tested by the disc diffusion technique according to the guidelines by the Clinical and Laboratory Standards Institute (CLSI, formerly National Committee for Clinical Laboratory Standards). 31 The antibiotic discs used were amoxicillin, 25 μg; amoxicillin/clavulanic acid, 30 μg; ciprofloxacin, 5 μg; cotrimoxazole, 25 μg; gentamicin, 10 μg; nalidixic acid, 10 μg; nitrofurantoin, 300 μg; ofloxacin, 5 μg; and tetracycline, 30 μg (Remel, Lenexa, Kansas). The discs were placed on to the surface of the agar by an autodispenser (Remel) that is capable of placing 8 discs equidistantly close to the periphery of the plate, leaving the center for the ninth disc. Inhibition zone diameters were measured to the nearest millimeter, and isolates were classified as susceptible, intermediate, or resistant according to CLSI-specified interpretive criteria. 31 Intermediate and resistant strains were further grouped together in the resistant group for analysis. E coli K12 C600 was used as the control strain. Extended-Spectrum ß-Lactamase Tests ESBC production was detected by disk diffusion technique as described by CLSI. 32 In brief, the test plates were inoculated as for a standard disk diffusion test. Disks containing expanded-spectrum cephalosporins (ceftazidime, 30 μg; cefotaxime, 30μg) were then applied. After overnight incubation at 37ºC, the presumptive production of ESBCs was detected by the Table 1. Pathogens Recovered From 158 Urine Samples During the Period September 2004 to April 2005 Organism Category Specific Organism (Any Species) No. of Isolates (% of 160) Gram-negative bacilli 121 (75%) Klebsiella spp 49 (30.6) Escherichia coli 41 (25.6) Pseudomonas aeruginosa 18 (11.3) Proteus spp 13 (8.1) Gram-positive cocci Staphylococcus aureus 27 (16.9) Yeast Candida spp 6 (3.8) Other Mixed growth 6 (3.8) JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION VOL. 101, NO. 12, DECEMBER

3 presence of inhibition zones 22 mm and 27 mm for ceftazidime and cefotaxime, respectively. Data Handling All data were entered into a Microsoft Excel database (Microsoft Corp, Redmond, Washington). Data obtained were analyzed using descriptive statistics. Figure 1. Antimicrobial Resistance Patterns of Urinary Tract Infection E Coli Abbreviations: AMX, amoxicillin; AUG, amoxicillin/clavulanic acid; CIP, ciprofloxacin; COT, cotrimoxazole; GEN, gentamicin; NAL, nalidixic acid; NIT, nitrofurantoin; OFL, ofloxacin; TET, tetracycline. Table 2. Antibiotic Resistance Patterns of 41 E coli Isolates Resistance Pattern No. of Isolates (% of 41) Resistance to 4 antibiotics Amx Cot Gen Tet 1 (2.4%) Amx Aug Gen Tet 1 (2.4) Resistance to 5 antibiotics Amx Aug Cot Gen Tet 2 (4.9) Resistance to 6 antibiotics Amx Aug Cot Gen Nal Tet 2 (4.9) Resistance to 7 antibiotics Amx Aug Cip Cot Gen Nal Tet Amx Aug Cip Gen Nal Ofl Tet 5 (12.2) 1 (2.4) Resistance to 8 antibiotics Amx Aug Cip Cot Gen Nal Ofl Tet 23 (56.1) Resistance to 9 antibiotics Amx Aug Cip Cot Gen Nal Nit Ofl Tet 6 (14.6) Abbreviations: AMX, amoxicillin; AUG, amoxicillin/clavulanic acid; CIP, ciprofloxacin; COT, cotrimoxazole; GEN, gentamicin; NAL, nalidixic acid; NIT, nitrofurantoin; OFL, ofloxacin; TET, tetracycline JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION VOL. 101, NO. 12, DECEMBER 2009

4 Results During the study period (September 2004 through April 2005), a total of 442 urine specimens were investigated by microscopy, culture, and (if culture positive) susceptibility testing to 9 antimicrobial agents. Nearly twothirds (ie, 284 [64.2%]) of the specimens were culture negative, whereas 158 (35.8%) yielded significant growth of 1 or more organisms, for a total of 160 isolates. Table 1 shows the pathogens recovered. Of the 158 positive cultures, 152 (96%) yielded the growth of a single organism, whereas 6 yielded 2 or more different pathogens. Of the 160 isolates, more than 60% were Enterobacteriaceae, with Klebsiella spp being the most commonly isolated organism (49 [30.6%]). E coli (41 [25.6%]), whereas Proteus spp and other gram-negative bacteria (Pseudomonas spp) accounted for 13 (8.1%) and 18 (11.3%), respectively. Only 27 (16.9%) of the isolates were gram positive, and all of these were identified as Staphylococcus aureus. Candida albicans was isolated from 6 individuals. Of the 41 patients from whom E coli was isolated, 23 were male and 18 were female. The mean age of all the patients was 47.7 years (range, 1-80 years). Twenty-two (10 males, 12 females) were hospitalized, while 19 (13 males, 6 females) were outpatients. Of the hospitalized patients, 10 of 23 (43.5%) men and 12 of 18 (66.7%) women were identified in the study. As illustrated in Figure 1, the E coli isolates exhibited a high prevalence of resistance to the 9 antibiotics tested except nitrofurantoin, to which 20% were resistant. Overall, 8 patterns of antibiotic resistance were observed among the E coli isolates, and further analysis revealed that 56% of them exhibited coresistance to as many as 8 of the 9 drugs used in the study. The predominant resistance pattern is that which involved 8 antibiotics namely, amoxicillin, amoxicillin/clavulanic acid, ciprofloxacin, cotrimoxazole, gentamicin, nalidixic acid, ofloxacin, and tetracycline. Other resistance patterns are illustrated in Table 2. All the isolates were resistant to at least 3 different classes of antibiotics. We carried out presumptive ESBC detection tests on 25 isolates, and all were found to be presumptive ESBC producers. DISCUSSION This study demonstrates that E coli strains that are simultaneously resistant to several antimicrobial agents normally used in the empirical treatment of UTI are a prominent cause of UTI in Ile-Ife. Reports of other studies have shown that the vast majority of uncomplicated UTIs within the study environment and other environments are caused by E coli. 29,33-35 In contrast, in the present study, Klebsiella spp outnumbered E coli for overall prevalence, and other pathogens encountered. The recovery rate of 25.6% E coli in our study is in contrast to previous reports that E coli is considered a major cause of UTIs, accounting for 75% to 90% of UTIs as earlier observed in the Central African Republic (55.6%), 33 Madagascar (67.2%), 34 and Turkey (73.6%). 35 However, in agreement with the low recovery rate of E coli from UTIs, the present result closely mirrors what was observed in the earlier study 29 and 36% observed by Kariuki et al 36 in Nairobi, Kenya. Reasons that might have been attributed to the observed variance may include (1) differences in the study design and patient selection, and (2) differing environmental conditions in various study centers. Antimicrobial resistance is recognized as an increasing global problem. 37 In this study, all of the E coli isolates were resistant to amoxicillin, gentamicin, and tetracycline, while 95% were resistant to cotrimoxazole. To our knowledge, this will be the highest report so far for these agents. The prevalence of antibiotic resistances in our study environment is considerably higher than what was reported within the same location in 1988 by Lamikanra and Ndep. 29 Their observed resistance prevalences were 85.9%, 72.6%, 68.7%, 21.1%, 18.7%, and 1.6% to ampicillin, tetracycline, streptomycin, gentamicin, cephalotin, and nalidixic acid, respectively. Though higher proportions of resistant strains have been reported in various parts of the developing world, 34,38,39 none has reported resistance rates higher than what is now reported for these agents and confirms the observation by Okeke, Fayinka, and Lamikanra 28 that the prevalence of antibiotic resistances within the study environment was increasing with time. The extensive multidrug antibiotic resistance patterns to amoxicillin, amoxicillinclavulanic acid, fluoroquinolones, and trimethoprimsulfamethoxazole observed among E coli clinical isolates in these studies, and the continuous increase in prevalence of infections caused by antibiotic-resistant E coli makes the effective empirical treatment of UTIs difficult in these environments. 9,40 The prevalence of cotrimoxazole resistance among urinary E coli varies considerably among different geographical locations, with current estimates from developed countries being 20% to 40%. 4,5 In North America, cotrimoxazole resistance ranges from 18% to 25%, 40 having increased from 7% to 9% in to 17% to 18% in The 1998 SENTRY surveillance program, reporting on isolates of E coli collected from 26 US centers, found the overall prevalence of cotrimoxazole to be 23.3%. 2 In developing countries, especially those in Africa, rates of resistance are higher, being 67%, 68.1%, 80%, and 85% in Sudan, 38 Senegal, 39 Madagascar, 34 and the Central African Republic, 33 respectively. In like manner, between 30% and 45% E coli isolates in more developed countries are resistant to amoxicillin. 5 This is in contrast to higher rates from the developing countries being 46% to 80%. 34,36,39-40 The extensive use and easy availability of antimicrobial drugs over the counter has been a contributing factor for JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION VOL. 101, NO. 12, DECEMBER

5 the high selection pressure for resistant bacteria. 28,29,41 Reports of antimicrobial resistance trends in uropathogenic E coli isolates from other countries raise the specter of wider resistance to fluoroquinolones. Resistance to the fluoroquinolones (ofloxacin and ciprofloxacin) in this study was strikingly prevalent, at 76% and 85%, respectively. Many studies worldwide have reported a sharp increase in ciprofloxacin resistance among E coli isolates from UTIs. For example, a study in China revealed that the prevalence of ciprofloxacin resistance increased steadily from 46.6% to 59.4%. 9 In Senegal, 39 a significant increase of resistance to quinolones was observed between 2004 and 2006, with current estimates being 23.9% to nalidixic acid, 15.5% to ciprofloxacin, and 16.4% to norfloxacin. In a recent study conducted in Mexico, Arredondo-Garcia and Amabile-Cuevas 42 reported that 44% (11.4% intermediate) of the organisms isolated were resistant to ciprofloxacin. However, in sharp contrast to this, ciprofloxacin remains very active in the United States and Canada, where the resistance prevalence has been reported as 2% 43 and 4%, 4 respectively. The high prevalence of resistance to the fluoroquinolones documented in the present study may be ascribed to high selective pressure prevalent in an environment in which antibiotics are freely available without restriction. 44 Furthermore, Davidson et al 45 had suggested that chloroquine might be an added selective pressure for quinolones resistance. We think this claim could be relevant in our situation, as chloroquine was the cheapest and most widely used antimalarial drug in our environment. In addition, the observed resistant pattern results may also be attributable to a previous hypothesis that fluoroquinolone-resistant E coli encountered in humans are likely transmitted from livestock, rather than having originated in humans by conversion of susceptible human intestinal E coli to resistant E coli under selective pressure from medical fluoroquinolone use These enormous differences may be the result of administering antimicrobials in poultry production. 49 Unlike what has been observed in other parts of the world 11,35,39 and indeed what was observed in an earlier study in this locality in which only 21% 29 of the organisms tested were found to be resistant to gentamicin, all the isolates obtained in the course of the present study were found to be resistant to this agent. This is not in agreement with the reports observed in different parts of the world. 11,35,39 This observation suggests that there is widespread use in this environment of gentamicin, an injectable antibiotic, and further suggests that conditions conducive to the spread of ESBC-producing organisms are present within the study environment, since resistance to gentamicin in E coli has been associated with strains that have the capacity to produce ESBC. 11,35 Because of the resistance of these isolates to several antibiotics, the need to perform an ESBC screening test was acknowledged, and all 25 isolates screened for ESBC production demonstrated the ability to produce ESBCs. Although, this is the first report from our community, community-associated ESBC-producing uropathogenic E coli have previously been reported in other parts of the world. 19,26,27,42 In the present study, nitrofurantoin was the only drug that was found to be active against a majority of the E coli isolates, the prevalence of resistance being only 20%. It is thus, the only one of the study drugs that can be regarded as being useful for the empirical treatment of UTI in this environment. However, it is worth noting that this figure is still considerably higher than what has been reported by Sire et al (10.1%), 39 Arredondo-Garcia, and Amabile-Cuevas (7.4%). 42 As the large majority of E coli isolates obtained in this study are resistant to cotrimoxazole and fluoroquinolones, we suggest that nitrofurantoin be an important alternative oral agent for the treatment of uncomplicated UTIs in Ile-Ife as earlier reported by Odetoyin et al. 50 The primary factor responsible for the development and spread of bacterial resistance has been identified as the injudicious use of antimicrobial agents. 51 Thus, strategies to improve prescription practices that use surveillance data to rationally guide more judicious antibiotic use warrant consideration in this locality. References 1. Stamm WE. The epidemiology of urinary tract infections: Risks factors reconsidered. Inter Sci Conf Antimicrob Agents Chemother. 1999;39: Karlowsky JA, Kelly LJ, Thornsberry C, et al. 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