Prevalence of blaCTX-M , blaSHV and blaTEM β-Lactamase Genes Among Escherichia coli Isolates in Foodborne Outbreak in Iran

© 2018 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. Background Increasing challenge of antimicrobial resistance is focused on the worldwide increasing Escherichia coli that contain extended-spectrum beta-lactamases (ESBLs). These enzymes lead to resistance against the antibiotics such as cephalosporins and monobactams by hydrolyzing the β-lactam ring.1 In E. coli, ESBL genes like blaCTX-M, blaSHV and blaTEM are typically transported by movable genetic elements that have high facility in transition of resistance genes.2 Pathogenic E. coli is one of the causes of intestinal and extraintestinal infections in humans.3 E. coli is a major producer of β-lactamase enzymes4 and one of the most important health problems worldwide, especially in developed countries where it is considered to be responsible for the death of 2.5 million children per year.5,6 Different methods such as double disk synergy test (DDST) have been suggested for the detection of ESBL in clinical isolates.7,8 In the past 15 years, many epidemics caused by ESBLpositive organisms have been reported worldwide. This prevalence is a serious threat to the treatment of infections. Experience has shown that there is no satisfactory outcome for treatment of infections caused by ESBLpositive bacteria.9 The mortality rate of this group was significantly higher than the infections caused by drugsensitive bacteria, varying from 42% to 100%. Nowadays, increasing use of antibiotics like cephalosporin has led to the emergence of a different group of β-lactamase genes that are compared with primary β-lactams (TEM-1, TEM2, SHV-1). They have a higher activity spectrum, which can be described as general ESBLs which are wider than the first β-lactamases.10 Therefore, the use of molecular Received August 14, 2017; Revised January 10, 2018; Accepted January 15, 2018


Background
Increasing challenge of antimicrobial resistance is focused on the worldwide increasing Escherichia coli that contain extended-spectrum beta-lactamases (ESBLs).These enzymes lead to resistance against the antibiotics such as cephalosporins and monobactams by hydrolyzing the β-lactam ring. 1 In E. coli, ESBL genes like bla CTX-M , bla SHV and bla TEM are typically transported by movable genetic elements that have high facility in transition of resistance genes. 2 Pathogenic E. coli is one of the causes of intestinal and extraintestinal infections in humans. 3. coli is a major producer of β-lactamase enzymes 4 and one of the most important health problems worldwide, especially in developed countries where it is considered to be responsible for the death of 2.5 million children per year. 5,6Different methods such as double disk synergy test (DDST) have been suggested for the detection of ESBL in clinical isolates. 7,8n the past 15 years, many epidemics caused by ESBLpositive organisms have been reported worldwide.This prevalence is a serious threat to the treatment of infections.Experience has shown that there is no satisfactory outcome for treatment of infections caused by ESBLpositive bacteria. 9The mortality rate of this group was significantly higher than the infections caused by drugsensitive bacteria, varying from 42% to 100%.Nowadays, increasing use of antibiotics like cephalosporin has led to the emergence of a different group of β-lactamase genes that are compared with primary β-lactams (TEM-1, TEM-2, SHV-1).They have a higher activity spectrum, which can be described as general ESBLs which are wider than the first β-lactamases. 10Therefore, the use of molecular methods for the complete identification of these enzymes is important.The aim of this study was to identify ESBLproducing enzymes of E. coli using phenotypic methods and to evaluate TEM, SHV and CTX-M1 β-lactamase genes by polymerase chain reaction (PCRmethod in foodborne disease outbreaks. 11,12terials and Methods Within 9 months, 400 stool samples were collected from 100 foodborne outbreaks that had occurred across the country.Diarrheal samples of the patients with foodborne poisoning with symptoms of diarrhea, vomiting, nausea, abdominal cramps, fever and headache were taken. Fifty E. coli isolates were identified via biochemical tests on colonies based on the following criteria: positive glucose fermentation, positive lactose fermentation, negative citrate, negative SH2, positive nitrate, methyl red positive (MR), Voges-Proskauer negative (VP), negative ureases, positive indole, and negative phenylalanine deaminase detected by using standard biochemical tests including the indole, methyl red, Voges-Proskauer, and citrate (IMViC) tests.All strains of E. coli were isolated from the samples that created a metallic mucilage colony on the Eosin Methylene Blue (EMB) agar (Merck, Germany) and stored in skim milk at -70°C until required for further tests.

Screening and Phenotypic Identification of ESBLs
According to Clinical and Laboratory Standards Institute (CLSI), disk diffusion method was carried out using Muller-Hinton agar (pH 7.4 = 7.2) for the initial screening of E. coli isolates expressing ESBLs.Microbial suspensions with concentrations of 0.5 McFarland were consistently spread on the plates with Muller-Hinton agar medium.Antibiotic disks prepared by the company (Rosco Neo-Sensitabs, Denmark) included ampicilin (10 µg), azitromysin (15 µg), cotrimoxazole (25 µg), nalidixic acid (30 µg), ciprofloxacin (5 µg), meropenem (10 µg), gentamicin (10 µg), ceftriaxone (30 μg) and ceftazidime (30 μg) which were placed with distance of at least 2.5 cm from each other and incubated for 24 hours at 37°C.The inhibition zone was measured around each disc and compared to the CLSI standard.Resistant isolates of cephalosporin representatives were selected for confirmatory testing.Combined disk was used for this purpose.Muller Hinton Agar medium was prepared.Microbial suspension with concentrations of 0.5 McFarland extended completely were diffused in the medium.Then, discs of ceftazidime (30 μg), ceftazidimeclavonic acid (10-30 μg), cefotaxime (30 μg), and cefotaxime-clavonic acid (10-30 μg) prepared from the company (Rosco Neo-Sensitabs Denmark) were placed with at least 2.5 cm from each other on the medium.After incubation for 24 hours at 37°C, an inhibition zone around the disk containing clavonic acid as an inhibitor of ESBL enzymes was measured relative to non-clavicic acid.If there was an increase in the zone of inhibition (ZOI) by ≥5 mm, strain was considered as an ESBL generator according to the CLSI standards.

Primer Design
Submitted sequences related to the E. coli TEM, CTX-M1, and SHV are present in GenBank, NCBI.All sequences were achieved and aligned using a MEGA 4 multiplealignment program to identify analogs.The conserved sequences that had a high degree of homology were selected and used to design universal primers via the gene runner software (Table 1).Designed primers were evaluated using PCR as a final point.

PCR and Sequencing of the β-Lactamase Genes
For identification of the β-lactamase genes of SHV, CTX-M1, and TEM, genomic DNA of the strains were extracted according to the manufacturer's instructions (Bioneer Company).PCR process was followed on β-lactamase SHV (747 bp), CTX-M1 (593 bp) and TEM (445 bp) genes, using 3 pairs of universal primers under the following conditions: each PCR reaction contained 2.5 µL 10X buffer, 1µL 50 mM MgCl 2 , 1 µL 10 mM dNTP, 1.5 µL 50 pmol primer each for the forward and reverse primers, 1 µL 5 U Taq DNA polymerase, 2 µL 50 pmol template DNA, and 14.5 µL H2O in a final volume of 25 µL.Markedly a mixture of template material was used as a negative control for SHV, CTX-M1 and TEM genes.In addition, Klebsiella pneumoniae ATCC 7881 , E. coli strain DF39TA and E. coli ATCC 25923 were used as positive controls for all genes.PCR was carried out under the following conditions: initial denaturation at 94°C for 3 minutes, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 62°C for 1 minute, and  elongation at 72°C for 1 minute.The final elongation step (72°C) occurred for 10 minutes.A multiplex PCR for the SHV and CTX-M1 genes was also performed, similar to the one abovementioned by adding both pairs of primers.Afterward, electrophoresis of PCR products was performed using a 0.8% agarose gel with 100 bp DNA ladder (Fermentas).

Results
Fifty E. coli were recognized from 400 stool samples that were collected from 100 foodborne outbreaks in the country.Achieved resistance pattern of isolates against 10 antibiotics is shown in Table 2. Their resistance pattern is shown in Figure 1.

Discussion
Contaminated foods that possess E. coli with antimicrobial resistance genes can transiently inhabit the gastrointestinal tract for consequent infection. 13E. coli is considered as an important foodborne pathogen that causes diarrhea both in children and adults. 14,15he aim of this study was to investigate the prevalence of resistance genes which express ESBL enzymes.These genes are repeatedly associated with the plasmid-encoded mechanisms. 16We focused on bla CTX-M , bla SHV and bla TEM genes which are recognized to exhibit resistance to β-lactam antibiotics and are a major public health issue worldwide. 17The use of broad-spectrum beta-lactam antibiotics has increased rapidly along with resistance to these antibiotics. 18Currently, there are several phenotypic tests recommended by the CLSI to detect ESBLs. 19owever, they are not sufficient for ultimate diagnosis of antibiotic resistant pathogens.In this study, recognizing ESBL-producing strains was performed according to the recommended tests by the CLSI.PCR was used to evaluate the prevalence of 13 lactamase enzymes and was successfully applied for the identification of ESBLs. 20This method is useful for detection of foodborne outbreaks which require rapid detection to prevent broadening and spread of the disease and to control outbreak well timed.In this study we gathered all samples from foodborne outbreaks that had happened in the country and recognized the genes which played a significant role in creating the resistance to β-lactam antibiotics.
Tested samples showed high resistance to ampicillin.In the study conducted by Soltan Dallal et al, the frequency of broad-spectrum beta-lactamase genes of TEM, DHA, and MOX of 200 isolates of E. coli were tested and 74 (57%) of them were reported as having the TEM gene.3][24] Based on these results, having accurate data about the resistance genes could be helpful in achieving a correct pattern for taking helpful antibiotics.Previous results on prevalence of gram-negative bacteria, generators of broad-spectrum beta-lactamases, have shown that ESBLs are rising rapidly. 25,26In view of our survey, it was indicated that spread of ESBLs and their expression was associated with failures in treatment and increased mortality.

Conclusion
In order to control the proliferation of resistance genes in E. coli strains causing infection and to choose a rapid and appropriate treatment in emergency conditions like foodborne illnesses, it is suggested to gain more knowledge about the prevalence of different virulence genes in ESBL-producing E. coli.Therefore, molecular methods like PCR that has high sensitivity and specificity can be effective and helpful.

Table 1 .
Primers used for amplification

Table 2 .
Pattern of Resistance Among 50 Escherichia coli Isolates Against 9 Antimicrobial Agents Figure 1.Pattern of Resistance Among Escherichia coli Isolates Against Antimicrobial Agents.