Science ABSTRACT. Keywords: E. coli; extended-spectrum ß-lactamase; metallo-ßlactamase;

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1 Prevalence and Antibiotic Susceptibility Patterns of Extended-Spectrum ß-Lactamase and Metallo-ß-Lactamase Producing Uropathogenic Escherichia coli Isolates Hamed Ghadiri, MSc, 1 Hamid Vaez, MSc, 2* Kamal Razavi-Azarkhiavi, PhD, 3 Ramin Rezaee, PhD, 3 Mehdi Haji-Noormohammadi, MSc, 4 Ali Asghar Rahimi, PhD, 5 Vahid Vaez, DVM, 6 Enayatollah Kalantar, PhD 7 Lab Med Fall 2014;45: DOI: /LMHEP4VQHEY2POOK ABSTRACT Healthcare professionals worldwide have expressed concern over infections by extended-spectrum ß-lactamase (ESBL) and metallo-ßlactamase (MBL) producing bacteria. We evaluated the prevalence of ESBL- and MBL-producing Escherichia coli (E. coli) isolated from community-acquired urinary tract infections (UTIs) and their antibioticresistance profiles at 3 private laboratories in Tehran, Iran. E. coli isolates were mostly susceptible to meropenem (90.4%) and imipenem (90.0%), followed by amikacin (89.0%) and gentamicin (84.7%). Moreover, we detected that, of the E. coli isolates, 67 (22.3%) were ESBL producers and 21 (7.0%) of E. coli isolates were MBL positive via the imipenem-ethylenediaminetetraacetic acid (EDTA) combined disc test. This report is the first, to our knowledge, on the prevalence of MBL-producing uropathogenic E. coli (UPEC) strains in Iran. The antibiotic resistance of E. coli isolates revealed that 122 (40.7%) were multidrug resistant. The high number of antibiotic-resistant and ß-lactamase producing UPEC strains necessitates further attention and consideration, particularly MBL-producing strains. Keywords: E. coli; extended-spectrum ß-lactamase; metallo-ßlactamase; multidrug-resistant Urinary tract infections (UTIs) are common infectious diseases that may be symptomatic or asymptomatic. 1 Among the causative agents, bacteria are responsible for more than 95% of UTI cases. 2 Abbreviations UTIs, urinary tract infections; ESBLs, extended-spectrum ß-lactamases, MBL, metallo-ß-lactamase; EDTA, ethylenediaminetetraacetic acid; UPEC, uropathogenic E. coli; CLSI, Clinical and Laboratory Standards Institute; MDR, multidrug-resistant; PCR, polymerase chain reaction; KPCs, Klebsiella pneumoniae carbapenemases 1 Department of Microbiology, Faculty of Sciences, University of Tehran, Tehran, Iran, 2 Department of Microbiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran, 3 Department of Pharmacodynamics and Toxicology, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran, 4 Department of Microbiology, Islamic Azad University, Pharmaceutical Sciences Branch, Tehran, Iran, 5 Department of Biology, Faculty of Sciences, Imam Hosein University, Tehran, Iran, 6 Veterinary School, Islamic Azad University, Karaj, Iran, 7 Department of Pathobiology, School of Medicine, Alborz University of Medical Sciences, Karaj, Iran *To whom correspondence should be addressed. h_vaez@resident.mui.ac.ir Escherichia coli (E. coli) is the most common bacterial cause of UTIs. 2 The major concern in isolates of the Enterobacteriaceae family is antibiotic resistance, so it is necessary to continuously refine guidelines for empiric antibiotic therapy by monitoring the occurrence of resistance strains. 3,4 Extended-spectrum ß-lactamases (ESBLs) are a heterogeneous group of enzymes that cleave the ß-lactam ring and are responsible for the resistance of bacteria to ß-lactam antibiotics such as penicillins and cephalosporins. 5 Enterobacteriaceae family members, particularly E. coli and Klebsiella pneumoniae, are the most common producers of ESBL. 6 Carbapenems are commonly used to treat infections caused by ESBL-producing bacteria; however, carbapenemase enzymes such as metallo-ß-lactamase (MBL) have Fall 2014 Volume 45, Number 4 Lab Medicine 291

2 been reported 7 in Enterobacteriaceae such as E. coli. The MBL enzymes belong to the molecular class B family, or functional group 3 ß-lactamases, that can hydrolyze all ß- lactams except monobactam. 8 Because worldwide data indicate that ß-lactamase producing E. coli isolates are becoming more common, studies on their antibiotic resistance patterns are essential. Limited data are available on the resistance pattern of communityacquired uropathogenic E. coli (UPEC) strains in Iran, and this study investigated the prevalence of ESBL and MBLproducing E. coli isolates and their antibiotic resistance profiles against antibiotics that are commonly prescribed. Materials and Methods Subjects We conducted this study from January 2011 through December 2012 in 3 nonaffiliated pathobiology laboratories in the central part of Tehran, Iran. During the study, we evaluated data from 2500 outpatients whom we suspected had a UTI and whose specimens had been referred to our laboratories. We included UTI cases with significant E. coli positive culture results. We collected demographic (ie, age and sex) data from each patient. According to the registered data, all the cases were community acquired (without a history of hospitalization or antibiotic therapy 1 month before sampling). The study was approved by the Ethics Committee of Tehran University of Medical Sciences. Sample Collection and Bacterial Identification We collected midstream urine specimens, based on the recommendations of Girou et al. 9 The specimens were inoculated onto blood and MacConkey agar plates. Inoculated agar plates were incubated at 37 C for 24 hours. The presence of more than 10 5 colony-forming units per ml bacteria (single bacterial species) signified a UTI. We microbiologically identified isolated bacteria using standard biochemical identification methods. 10 Only 1 isolate per patient was included in the study. Antibiotic Susceptibility Testing We tested antibiotic susceptibility using the Kirby-Bauer disk diffusion method on Müller-Hinton agar plates in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. 11 The following antimicrobial agents were included: ampicillin (10 μg), ciprofloxacin (5 μg), cotrimoxazole (25 μg), gentamicin (10 μg), amikacin (30 μg), imipenem (10 μg), nitrofurantoin (300 μg), meropenem (10 μg), and ceftazidime (30 μg). These antibiotics are commonly used to treat UTIs. We used E. coli ATCC as the control strain and determined the current prevalence and phenotypes of multidrug-resistant (MDR) E. coli isolates in this region. We defined an MDR entity as having nonsusceptibility to at least 1 agent in 3 or more antimicrobial categories. 12 Detection of ESBL We subjected all E. coli isolates that were non-susceptible (inhibition zone 22) to ceftazidime to the ESBL production confirmatory test using CLSI guidelines. 13 We inoculated Müller Hinton agar plates with 0.5 McFarland suspension of each isolate to form a lawn culture. Separate antibiotic discs containing cefotaxime (30 μg) and ceftazidime (30 μg) with and without clavulanic acid (10 μg) were placed over the lawn culture. The results were interpreted as specified by the CLSI. 13 We used E. coli ATCC as an ESBL-negative reference strain and Klebsiella pneumoniae ATCC as an ESBL-positive reference strain. Detection of MBL E. coli isolates that were resistant to imipenem (10 μg) and/or meropenem (10 μg) were tested phenotypically for MBL production using the imipenem-edta combined disc synergy test, as previously described by Galani and Rekatsina. 14 Briefly, we placed 2 imipenem (10 μg) discs on the surface of a Müller-Hinton agar plate (inoculated with 0.5 McFarland suspension of each isolate) and added 10 µl of EDTA solution (0.5 M) to 1 of them. We used the EDTA disk by itself as the negative control. Plates were incubated for 16 to 18 hours at 37 C. We considered a difference of 7 mm or greater between the inhibition-zone diameter of the imipenem-edta disk and that of the imipenem-only disk to constitute MBL positivity. Statistical Analysis Data entry and statistical analysis were performed using SPSS software, version 13 (SPSS Inc., Chicago, IL). Comparisons were made using the Pearson ξ-square and Fisher exact tests. A P value of less than.05 indicated statistical significance. Results Of the 2500 nonrepetitive urine specimens, 352 specimens (14.1%) showed significant bacteriuria with monomicrobial origin. We obtained 300 isolates of E. coli from 352 specimens that had tested positive for UTIs, 292 Lab Medicine Fall 2014 Volume 45, Number 4

3 Table 1. Prevalence and Antibiotic Resistance Profiles of ESBL and MBL-Producing E. coli Isolated From Community-Acquired UTIs. Antibiotics ESBL Positive, no. (% within ESBL) MBL Positive, no. (% within MBL) Total Resistance, no. (%) Imipenem 7 (10.4%) 21 (100%) 30 (10%) Meropenem 7 (10.4%) 20 (95.2%) 29 (9.6%) Gentamicin 16 (23.9%) 9 (42.9%) 46 (15.3%) Ciprofloxacin 27 (40.3%) 21 (100%) 108 (36%) Ampicillin 67 (100%) 21 (100%) 285 (95%) Cotrimoxazole 27 (40.3%) 20 (95.2%) 96 (32%) Nitrofurantoin 22 (32.8%) 21 (100%) 83 (27.7%) Ceftazidime 67 (100%) 20 (95.2%) 184 (61.3%) Amikacin 12 (17.9 %) 9 (42.9%) 33 (11%) Total 67 (22.3%) 21 (7%) 300 (100%) ESBL: Extended-spectrum ß-lactamases, MBL: Metallo ß-lactamases and confirmed the E. coli isolates according to their morphologic and biochemical characteristics. The age of the 300 patients in our cohort ranged from 17 years to 65 years, with a mean of 43.2 years; 58 (19.3%) were male and 242 (80.7%) were female. We determined the antibiotic resistance rates of E. coli isolates from male and female patients with UTIs for the 9 antibiotics that we evaluated in this study. Comparison of the antibiotic resistance rate between the isolates from male and female patients revealed no significant difference (P >.05). E. coli isolates were mostly susceptible to meropenem (90.4%) and imipenem (90.0%), followed by amikacin (89.0%) and gentamicin (84.7%). The highest percentage of resistant strains were observed against ampicillin (285 isolates [95.0%]) and ceftazidime (184 isolates [61.3%]) (Table 1). Of 300 E. coli isolates, 67 (22.3%) were ESBL producers and 7.0% were MBL positive. Moreover, coexistence of ESBL and MBL was observed in 5 (1.7%) of the isolates. Among ESBL-producing E. coli isolates, 40.3%, 40.3%, and 32.8% were resistant to cotrimoxazole, ciprofloxacin, and nitrofurantoin, respectively. Imipenem and meropenem demonstrated the strongest activity against ESBLproducing isolates: only 10.4% of ESBL-producing isolates of E. coli were resistant to these antibiotics. In this study, 70.0% of the imipenem-resistant E. coli isolates were MBL producing; among them, we observed the lowest overall level of resistance in amikacin and gentamicin (Table 1). The results of our evaluation of the antibiotic resistance pattern of 300 E. coli isolates indicated that 122 (40.7%) of the isolates were MDR. Also, 13.9% (17/122) of MDR isolates were resistant to all 9 antibiotics tested. Discussion Indiscriminate use of antibiotics has resulted in the emergence of ß-lactamase producing strains that are a therapeutic challenge healthcare. 15 Considering the high rate of UTIs and the antibiotic resistance caused by overuse of antibiotics, this study aimed to assess the resistance of E. coli to commonly prescribed antibiotics and the prevalence of MBL and ESBL production by UPEC strains. The E. coli isolates in this study were highly resistant to different antibiotics such as penicillins, cephalosporins, and quinolones. These data correspond with data presented in a previous community-acquired UTI study. 16 Although sex-dependent antibiotic resistance was established in a previous investigation, 17 we observed no significant difference in antibiotic resistance between the males and females (P >.05). In our study, E. coli strains isolated from patients with UTIs were most often resistant to ampicillin (95.0%) (Table 1). Surveys 18,19 performed in the United States, Europe, Africa, and Asia indicated that most UPEC isolates are resistant to this antibiotic. High resistance to ampicillin and its ineffectiveness in treating UTI in Iranian children and adults have also been reported. 20,21 In the present study, resistance of isolates to nitrofurantoin was 27.7%, which is higher than the percentages reported in Canada (4%) and the United States (1.6%) and lower than that from India (80%) A report from central Iran 25 contained similar data. Fall 2014 Volume 45, Number 4 Lab Medicine 293

4 Resistance to ciprofloxacin in our study was 36.0%, which is comparable to the data published by Kashef et al 25 concerning Iranian community-acquired UTI isolates. Along with the data reported from different parts of Iran in recent years, our results revealed that resistance to ciprofloxacin has increased and that it is more pronounced in the central regions of the country than in the western and northwestern regions The regional dissimilarity in antibiotic resistance patterns and the rate of ß-lactamase production may be partly explained by local practices of antibiotic use. 28 Compared with ampicillin, multiple mechanisms through which ciprofloxacin exhibits its antimicrobial effects enable it to retain potent activity on UPEC. 29 In our study, 61.3% of E. coli isolates were resistant to ceftazidime. This result contradicts those of other studies on community acquired UTI in Europe, 30 Canada, 22 and the United States. 23 According to our data, aminoglycosides such as gentamicin and amikacin (with resistance rates of 15.3% and 11.0%, respectively) had a considerable effect on the isolates. These aminoglycoside resistance rates are higher than in an earlier experiment by some of us 31 on nosocomial E. coli strains and data reported from other parts of the world. 22,23 Although 40.7% of the isolates in our study were MDR, the rate of MDR UTI isolates reported from other parts of Iran ranged between 10.9% and 65%. 32,33 One cause of the prevalence of MDR microorganisms is the production of ß-lactamases such as ESBL. According to results of the phenotypic confirmation test, 22.3% of our E. coli isolates were ESBL producing. We speculate that other resistance mechanisms that are not detectable by this method, such as presence of clavulanic acid resistant ESBLs, can contribute to ceftazidime resistance. 3 A comparison of our results and those from studies on E. coli isolates from hospitals in the central part of Tehran indicate a higher prevalence of ESBL-positive isolates in hospital-acquired infections. 34,35 The prevalence of ESBLproducing UPEC strains in Iran is greater than in Europe (5.5%), South America (5% 10%), and North America (<5%) but lower than in India (34.4%) and Pakistan (57.4%). 24,39 Among the oral antibiotics we evaluated in this study, ciprofloxacin and nitrofurantoin showed the strongest activity against ESBL-positive E. coli strains. Considering the average resistance to these agents, their administration should be preceded by an antibiogram test. Moreover, injected antibiotics such as meropenem, imipenem, amikacin, and gentamicin had a substantial effect on ESBL-positive isolates. The high susceptibility of ESBLproducing strains to carbapenems should not lead to their excessive use, which could in turn result in more serious challenges to the healthcare system. Previous studies have reported no resistance in UPEC to imipenem in Iran; however, our study and a 2013 report from Alborz province in central Iran 40 are the first in this field to demonstrate the occurrence of UPEC resistance to imipenem. MBL production is one of the most important mechanisms for the resistance of microorganisms to carbapenems such as imipenem and meropenem. 41 Although the combined disc synergy test, according to previous studies, 14,42 is a sensitive method for detection of MBL-producing isolates, it may not detect some MBL-producing strains. Use of this phenotypic method is one of our study limitations. The MBL inhibitors, such as EDTA, act nonspecifically and affect other structures and processes, increasing the risk of false-positive results. 43 Thus, molecular techniques (polymerase chain reaction [PCR] or DNA hybridization methods) are necessary to confirm the results of this phenotypic method. Two reports from different countries 44,45 revealed an increase in carbapenem resistance due to the acquisition of MBL genes such as bla IMP and bla VIM. To our knowledge, all investigations concerning MBL-producing species in Iran have been limited to Pseudomonas and Klebsiella species; this is the first report on the prevalence of MBL-producing UPEC strains. Among the E. coli isolates in this study, 7.0% were MBL producers, which should be regarded as a threat to public health. A study, performed as part of the SENTRY antimicrobial surveillance program in 83 medical centers in the United States, Europe, and Latin America, revealed that the rate of MBL-producing E. coli and Klebsiella strains were 0% and 0.6%, respectively. 46 The prevalence of MBL-producing E. coli strains in our study was higher than that reported from other parts of the world and was equal to that reported from India In this study, 70.0% of the imipenem-resistant E. coli isolates were MBL producing. We suggest that this finding can be associated with other resistance mechanisms, such as K. pneumoniae carbapenemases (KPCs) or AmpC-type ß-lactamase production. 50,51 Although isolates that harbor KPCs are now rarely being reported from Iran, detection of KPC can be considered for future studies. 52, Lab Medicine Fall 2014 Volume 45, Number 4

5 Conclusion The rate of antibiotic-resistant and ß-lactamase producing UPEC strains in the central region of Iran is high; this is of great concern, especially regarding MBL-producing strains. These results should be used to set stricter criteria for the use of antibiotics. Regular monitoring of resistance patterns and further clinical study is required to develop more effective therapies for conditions that commonly warrant antibiotic treatment. Acknowledgments We thank the patients, physicians, nurses, and laboratory staff of Ehsan medical center for their cooperation. LM References 1. Pezzlo M. Detection of urinary tract infections by rapid methods. Clin Microbiol Rev. 1988;1: Bonadio M, Meini M, Spitaleri P, Gigli C. Current microbiological and clinical aspects of urinary tract infections. Eur Urol. 2001;40: Bush K. Alarming ß-lactamase-mediated resistance in multidrugresistant Enterobacteriaceae. Curr Opin Microbiol. 2010;13: Taneja N, Rao P, Arora J, Dogra A. 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