Phenotypic and molecular characterization of CTX-M extended-spectrum beta-lactamase-producing Escherichia coli isolates in Shiraz , Iran

Introduction: The aim of this study was to detect the prevalence of the extended-spectrum beta-lactamase (ESBL)encoding CTX-M gene in Escherichia coli isolates. Methods: Phenotypic screening of 376 E. coli isolates for ESBL was conducted using disk diffusion. ESBL-producing isolates were tested using PCR and specifi c primers. The blaCTX-M cluster was identifi ed using the RFLP method, and its genotype was sequenced. Results: From 202 ESBL-producing E. coli, 185 (91.5%) possessed CTX-M genes. CTX-M-1 subtypes were found in 98% of the isolates. The blaCTX-M gene was identical to CTX-M-15. Conclusions: A high prevalence of CTX-M-1-producing E. coli apparently exists in Shiraz, Iran.

Escherichia coli, one of the most common intestinal commensals, causes various hospital-and community-acquired infections (1) .Specifi cally, the pathogen can cause intestinal and extra intestinal infections such as gastroenteritis, urinary tract infection, and meningitis (1) (2) .Escherichia coli strains utilize several mechanisms of antibiotic resistance, one of which is beta-lactamase production (3) .The extended-spectrum beta-lactamases (ESBLs) are a group of beta-lactamases with the ability to hydrolyze the extended-spectrum cephalosporin and monobactam antibiotics (4) .The extended-spectrum betalactamases were fi rst detected in Klebsiella spp., and then later identifi ed in E. coli and other species of Enterobacteriaceae (3) .These enzymes are divided into several main groups [e.g., sulphydryl variable (SHV), temoneira (TEM) and cefotaximases (CTX-M)] (5) (6) .Until the year 2000, SHV and TEM types were thought to be the most prominent ESBLs; however, since 2000, CTX-M types have emerged as new forms of ESBL that, unlike TEM and SHV, are more active against cefotaxime and ceftriaxone rather than ceftazidime (3) .
CTX-M-15-producing E. coli strains have now emerged in many countries and are a major cause of community-acquired infections, especially those that involve urinary tract infection (UTI) (4) .Furthermore, the E. coli ST131 and ST405 clones, which are part of virulence phylogenetic groups D and B2 respectively, are associated with the spread of CTX-M-15 (4) (8) .Because CTX-M-producing E. coli is widespread among various communities, hospitals, and environments, the treatment of patients exposed to this strain should be considered carefully in order to prevent the spread of CTX-M-producing antibiotic-resistant strains (3) (5) .Furthermore, because these strains are also multidrug-resistant, it is essential that epidemiological studies be conducted to enable identifi cation and treatment of the infections they cause (8) (9) .In the present study, our aim was to detect the prevalence of the CTX-M gene and its subtypes in clinical E. coli isolates collected from four hospitals in Shiraz, Southwest Iran.
This study was conducted from January to June, 2012, and involved analysis of 376 E. coli clinical samples from urine, stool, blood, wound, and sputum (Table 1), which were collected from Nemazee, Faghihi, Ali Asqhar, and Dastgheib hospitals affi liated with Shiraz University of Medical Sciences, Shiraz, Iran.Bacteria were identifi ed in samples by inoculating the clinical specimens into conventional culture media such as TSI, SIM, Simmons' Citrate Agar, MR/VP (Merck, Germany), and using the API 20E kit (bioMérieux, France).The bacterial isolates were then stored at -70°C in trypti case soy broth containing 20% glycerol.Fresh subcultures were used for testing.Antimicrobial susceptibility of the identifi ed isolates was determined by using the disk diffusion method according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) (10) .Eight antimicrobial agents were tested: ceftazidime, ceftriaxone, cefotaxime, cefepime, ceftizoxime, aztreonam, amikacin (30μg of each agent), and ciprofl oxacin (5μg) (Rosco, Denmark).Production of ESBL in the isolates was determined by using the combination disk method with antibiotic disks containing ceftazidime, cefotaxime, and cefepime per antibiotic) either alone or in combination with clavulanic acid (10μg) (Rosco, Denmark).The results were interpreted as previously described (10) .Quality control for the production of ESBL was assessed using E. coli ATCC 25922 (negative control) and Klebsiella pneumoniae ATCC 7881 (positive control).
Deoxyribonucleic acid was extracted from ESBL-positive isolates using an AccuPrep® Genomic DNA Extraction Kit (Bioneer, Korea) according to the manufacturer's instructions, and extracted DNA was stored at -20°C.
All 202 ESBL-positive isolates were screened for the presence of bla CTX-M by using polymerase chain reaction (PCR) with previously published primers and methods (11) .For PCR amplifi cations, 2µL of DNA was added to a 50-µL solution containing 1µL of deoxynucleotide triphosphates (dNTPs), 1.5µL Mgcl 2 , 2µL of each primer, and 0.3µL of Taq polymerase (CinnaGen, Iran) in 1x PCR buffer.An Eppendorf Thermocycler (Germany) was used for amplifi cation, with cycling parameters comprising an initial denaturation at 95°C for 10 min followed by 35 cycles of denaturation at 95°C for 30s, annealing at 51°C for 40s, amplifi cation elongation(extension) at 72°C for 30s, and a fi nal extension at 72°C for 3 min.
The amplifi ed CTX-M product (10μL) was directly subjected to digestion with 9U PST-I and 4U Pvu-II enzymes (Fermentas, Lithuania) in One-Phor-All Plus Buffer (10mMTris acetate; pH 7.5), 10mM magnesium acetate, and 50mM potassium acetate for 18h at 37°C.The restriction fragments of the PCR products were analyzed by electrophoresis in 3% agarose containing ethidium bromide (Imogene, Iran).Six PCR products were randomly selected and purifi ed using the AccuPrep® Gel Purifi cation Kit (Bioneer, Korea).They were subsequently sent to Bioneer Corp., Korea, for sequencing (GenBank accession numbers: KC465234/1, KC465233/1, and KC465232/1).The relationship between the presence of the bla CTX-M gene and antibiotic susceptibility was analyzed using non-parametric tests (chi-squared test), with analysis conducted in Statistical Package for the Social Sciences (SPSS) v18 software.
Of the 202 (53.7%)E. coli isolates with an ESBL phenotype, 185 (91.5%) strains carried bla CTX-M genes (Figure 1).The frequency of CTX-M genes in inpatients and outpatients was 122 (65.9%) and 63 (34.1%), respectively.In total, 182 (98.4%)CTX-M-producing isolates belonged to the CTX-M-1 group (Figure 2).A randomly selected bla CTX-M gene was sequenced and was shown to be identical to CTX-M-15.The authors declare that there is no confl ict of interest.Extended-spectrum beta-lactamases produced in E. coli account for the most common forms of antibiotic resistance against extended cephalosporins and monobactams (6) .

CONFLICT OF INTEREST
Cefotaximases is now identifi ed as a predominant ESBL that is increasingly disseminated among clones of E.coli, and it creates problems for treating infections caused by this bacterium (5) .Although strains that produce the CTX-M enzyme are often detected in hospital infections, the acquisition of these strains from community settings has increased substantially over recent years (2) (7) .The increased prevalence of such infections may be due to the indiscriminate use of extended-spectrum cephalosporins and other beta-lactams.Moreover, transmission and prevalence of the bla CTX-M gene could be attributed to several causes, which include the use of contaminated medical equipment, prolonged hospitalization of patients, low quality hospital hygiene and infection control systems, transmission through animal products, contact with repositories of CTX-M-producing bacteria, dissemination via the hands of medical personnel, recombination of the bla CTX-M gene with insertion sequences and transposons, and the spread of other bacteria such as Kluyvera spp.and Klebsiella spp (3) (8) .
Our results suggest that CTX-M type beta-lactamases are widespread in Shiraz, Iran.In previous studies, CTX-M production was significantly associated with resistance to fluoroquinolones and amino glycosides, and decreased susceptibility to cefotaxime and ceftriaxone led to a high prevalence of multi-drug resistant strains (3) (4) .In our study, 125 (67.5%) of the 185 identifi ed CTX-M-producing isolates were resistant to ciprofl oxacin (p <0.001).
Our results indicated that E. coli isolates weremore susceptible to ceftazidime, ceftizoxime, and cefepime than they were to cefotaxime and ceftriaxone: a fi nding that is consistent with other reports (7) .Cefotaxime and ceftriaxone are the third generation cephalosporins most commonly used for treating nosocomial and community acquired infections in Shiraz hospitals.Furthermore, 13% and 48.1% of the isolates from this study were resistant to amikacin and ciprofl oxacin, respectively.Therefore, our results suggest that ESBL-producing strains in Shiraz have become strongly resistant to antibiotics such as ciprofl oxacin.Nevertheless, amikacin remains an effective drug that could still be prescribed to treat infections caused by E. coli.The prevalence of ESBL-producing bacteria, according to the reports from other countries, differs in various geographical regions (12) .
In our study, the prevalence of ESBLs was 53.7%, which is consistent with the results of other reports in Iran (e.g., Mirzaee et al.) (1) .indicated that 135 of 250 strains were ESBL-producing) (1) .However, 91.5% of ESBL-producing strains in our study contained the bla CTX-M gene, which is higher than the rates reported by other investigators in Iran (1) (13) (14) .Our analysis of samples using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method indicated that more than 98% of isolates contained CTX-M-1subtypes from the CTX-M group; this is in agreement with reports of the predominant subgroup in most other countries and other areas of Iran (1) (14) (15) .The restriction patterns of the CTX-M-1 subtypes were similar, with restriction bands observed at 267bp, 156bp, and 120bp.According to Shahcheraghi et al. (15) and Sultan Dallal et al. (14) , CTX-M-1 is the predominant gene among Iranian isolates; after sequencing, 98% of randomly selected samples were found to be similar to CTX-M-15 reported from most countries (4) .CTX-M-15 was found in three sequenced isolates and it may be the most common group 1 type in the Shiraz geographic region.This CTX-M type is also relatively common among European isolates and has been responsible for community outbreaks of multi-drug resistant E. coli infections in the United Kingdom and elsewhere (5) (9) .
In conclusion, this study demonstrated signifi cant differences in susceptibility to various antibiotics among CTX-M-producing E. coli isolated in Shiraz, Iran, and a high prevalence of bla CTX-M-1 beta-lactamase was observed in these isolates.Therefore, similar studies are necessary to fully determine the frequency of the bla CTX-M gene and to identify the role of broad-spectrum cephalosporins in the treatment of infections caused by multidrug resistant CTX-M-producing E. coli.