Antibiotic Susceptibility Profile , ESBL Production and bla CTX-M 1 , bla SHV and bla TEM Types Among Escherichia coli Blood Isolates

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. 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 /trimethoprim.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


Objectives
The aim of this study was to determine the ESBL positive blood E. coli strains and the prevalence of bla CTX-M , bla SHV , and bla TEM types among ESBL positive blood isolates among 3 military hospitals in Tehran, Iran.

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 Laboratory 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 susceptibil ity 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.

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, and also in the combined disk test, 19 (80.1% of all) isolates were ESBL producers.On the other hand, there was a correlation between ceftazidime resistance and MIC results for ESBL production.The results of ceftazidime MIC confirmed the disk diffusion for guidance in the phenotypic production of ESBL by the isolates.

Genotypic Detection of ESBL Enzymes
The prevalence of bla CTX-M1 , bla SHV , and bla TEM genes among ESBL producing isolates was 26% (n = 6), 8% (n = 2) and 0%, respectively (Figure 1).The bla CTXM1 and bla SHV genes were associated with higher MIC against ceftazidime.The relationship among the MIC, characteristics of isolates, and presence of beta-lactamase genes for 10 adopted isolates is indicated in Table 4.

Discussion
In the current study, the level of ESBL production and MDR phenotype among E. coli blood isolates was high.Analysis of susceptibility of the strains to antibiotics demonstrated that among beta-lactam classes, the most effective drugs were meropenem (67%) and piperacillin (85%).Most patients were aged between 2-22 years old.In addition, among non-beta-lactam antibiotics, amikacin exhibited the highest activity against the isolates (63%).4][15] Recent publications have uncovered the predominant interference of bla CTXM1 in ESBL positive isolates all over the world.In the present study, bla CTXM1 group was accounted for 26% of blood ESBL production.This was the first study detecting the ESBL production in blood E. coli as well as molecular detection of related beta-lactamases in 3 military hospitals of Tehran.7][18][19] The bla CTXM1 was detected in the range of 4-32 ug/mL ceftazidime, although higher MIC was associated with MDR and presence of both bla CTXM1 and bla SHV genes.It has been demonstrated that bla CTXM1/14 and bla CTXM-15 genes cause high level of resistance to cefepime/ceftriaxone and ceftazidime, respectively. 12On the other, despite most of these studies, blood isolates exhibited a low frequency of ESBL encoding genes, supposed that other beta-lactamase classes interfere in the resistance to third generation cephalosporins.Although the prevalence of MDR rate and ESBL production were high, bla genes were not highly detected.The isolates with higher MIC amplified both bla CTXM-I and bla SHV type beta-lactamases.Ceftazidime MIC was in accordance with the disk resistance for detecting phenotypic ESBL production.There may be multiple mechanisms for resistance to the third generation cephalosporin drugs.

Table 2 .
The Specific Primers Used in the Present Study

Table 3 .
The Results Of Disk Diffusion Test Compared Between ESBL Negative and Positive Isolates
Our results were different from those of a study by Mohajeri et al in the West of Iran in which bla CTXM1 , bla SHV types and none of them could amplify bla TEM gene.Moreover, this study showed a correlation between higher MIC and co-resistance to multiple antibiotics, except for ciprofloxacin.The MIC, ESBL phenotype, and related genes were not significantly in relation with the age or genus of patients (P = 0.14 and 0.31, respectively).