Antibiotic Susceptibility Profile, ESBL Production and blaCTX-M1, blaSHV and blaTEM Types Among Escherichia coli Blood Isolates

International Journal of Enteric Pathogens Int J Enteric Pathog. 2017 February;5(1):9-12 http://enterpathog.abzums.ac.ir Original Article doi 10.15171/ijep.2017.03 Antibiotic Susceptibility Profile, ESBL Production and blaCTX-M1, blaSHV and blaTEM Types Among Escherichia coli Blood Isolates Farshad Nojoomi1, Abdolmajid Ghasemian1,2*, Majid Eslami2, Sepideh Khodaparast2 Microbiology Department, Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran 1 2 *Corresponding Author: Abdolmajid Ghasemian; Tel: +(98)9394514860; Fax: 021-82884555; Email: Published Online October 17, 2016 Keywords: Blood specimens, Escherichia coli, ESBL betalactamase, Multidrug resistance Abstract Background: Plasmid and chromosomal extended-spectrum beta-lactamases (ESBLs) have been increasingly spread everywhere and blaCTX-M1 is one predominant beta-lactamase. Objectives: This study was fulfilled to determine the production of ESBL and prevalence of blaCTX-M1, blaSHV, and blaTEM among Escherichia coli blood isolates in Tehran. Patients and Methods: Twenty-three isolates were adopted to be studied during 2015-2016. The antibiotic susceptibility testing was performed using Kirby–Bauer method. The combined disk method was used for the detection of phenotypic ESBL production. The most effective antibiotics were piperacillin, amikacin, and ofloxacin. The minimum inhibitory concentration (MIC) of ceftazidime was determined using micro-broth dilution method. Polymerase chain reaction (PCR) was used for detecting the blaCTX-M1, blaSHV, and blaTEM genes. Results: In the broth dilution test, 19 (82%) isolates showed MIC ≥1, and 18 (78.3%) isolates were ceftazidime resistant. In the combined disk test, 19 (82%) isolates were ESBL producers. The results of the MIC and ceftazidime resistance were the same for ESBL selection. The results of MIC, in fact confirmed the disk diffusion in determining the phenotypic ESBL production. The frequency of blaCTX-M1, blaSHV, and blaTEM genes among blood ESBL producing isolates was 26% (n = 6), 8.6% (n = 2), and 0%, respectively. Isolates that showed higher MIC were positive for these genes. Conclusion: The prevalence of multidrug-resistant blood isolates and ESBL phenotype was high in military hospitals. A low number of blood strains amplified blaCTXM1 and blaSHV type beta–lactamases. There was a relationship between the MIC and the presence of betalactamase genes. Received May 14, 2016; Revised August 25, 2016; Accepted October 9, 2016 Background Escherichia coli Blood isolates can initiate from gastrointestinal or urinary tracts and cause fatal infections. In addition, multidrug-resistant isolates harbor plasmids such as Inc FII/IncI1 and so on, leading to the resistance to several classes of antibiotics besides cephalosporins. The genetic location of extended-spectrum beta-lactamases (ESBLs) is the mobile elements and the chromosome of Enterobacteriaceae.1 Recent data have shown that blaCTX-M1 clones are mostly widespread at an endemic status worldwide and in Iran.2 The ESBLs are increasing everywhere.3 These ESBLs are inhibited by clavulanic acid, sulbactam, and tazobactam that help their detection.4 On the other hand, resistance due to ESBLs is often accompanied with resistance to other antibiotics, including fluoroquinolones, aminoglycosides, and sulfamethoxazole /trimetho- prim.5 The pandemic E. coli clone of ST131 with a high virulent potential encoding CTX-M-15 was characterized by the multidrug resistance (MDR) through the co-production of OXA-1 or TEM-1b as well as aac(6’)-Ibeta-cr. This clone produces blaCTX-M-15 beta-lactamase.6-8 CTXM-type ESBLs are complex and heterogeneous families and may be subdivided into 5 major groups (CTX-M-1, 2, 8, 9 and CTX-M-25).9,10 These enzymes have spread worldwide and are the most ESBLs detected in Enterobacteriaceae. They are not only found in hospitals, but also have been detected in the community, thus changing the epidemiology of ESBLs.11 The blaCTX-M and blaTEM ESBLs can hydrolyze third and fourth generation cephalosporins. Several studies have demonstrated a relationship between ESBL enzymes and minimum inhibitory concentration (MIC) to third and fourth generation cephalospo- Copyright © 2017 The Author(s); Published by Alborz University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited. Nojoomi et al rins, including ceftazidime, cefepime, and cefotaxime.12 Objectives The aim of this study was to determine the ESBL positive blood E. coli strains and the prevalence of blaCTX-M, blaSHV, and blaTEM types among ESBL positive blood isolates among 3 military hospitals in Tehran, Iran. Patients and Methods Bacterial Isolates Twenty-three E. coli blood isolates were collected during 2015-2016, from 3 hospitals of Tehran, Iran. These isolates were obtained from the patients 2-23 years of age. Fourteen patients were female (mean age of 14.62) and 9 were male (mean age of 12.33). The isolates were identified by both confirmatory biochemical and molecular tests advised for E. coli. Susceptibility Tests and Detection of ESBLs Susceptibility testing was performed using the disk diffusion method as per the guidelines of Clinical and Labo ratory Standards Institute (CLSI). Seventeen antimicrobial disks were used as indicated in Table 1. The standard ATCC25922 E. coli was used as the quality control of antimicrobial susceptibility testing. The ESBL phenotype determination was done using combined disk and synergy test methods employing cefotaxime and ceftazidime with and without clavulanic acid disk. The MICs of blood isolates were determined using broth microdilution method against ceftazidime in the range of 0.25–128 ug/mL (CLSI 2014). Isolates with MIC ≥1 were further tested for ESBL production in addition to ceftazidime resistant strains. DNA Extraction Total DNA was isolated with the boiling Method. Briefly, 200 µL of bacterial suspension was prepared in distilled water. After boiling in 95°C for 10 minutes, the centrifugation of tubes was performed and the supernatant was preserved at -20°C for later use. Amplification of ESBL Genes The Polymerase chain reaction (PCR) detection of CTX-M, SHV, and TEM ESBLs was done with the specific primers shown in Table 2. Statistical Analysis The relations were compared by t test using SPSS version 20.0. The value of P < 0.05 was considered to be significant. Results The Susceptibility Testing and ESBL Production The susceptibility testing profile between ESBL positive and negative blood strains is depicted in Table 3. In the broth microdilution procedure, 21 (92.7%) isolates showed MIC ≥1, (...truncated)