Phenotypic and Molecular Characterization of Plasmid- Encoded Extended Spectrum Beta-Lactamases Produced by Escherichia coli and Klebsiella spp from Lahore, Pakistan

S Riaz,Mf Bashir

doi:10.4314/tjpr.v14i9.8

Abstract

Purpose: To investigate the distribution of plasmid-encoded extended spectrum beta-lacatamases (ESBLs) in Lahore, Pakistan using different phenotypic and molecular methods.Methods: Escherichia coli and Klebsiella spp were obtained over a period of nineteen months (June 2007 to December 2008). Both were tested by the double disk synergy test, combined disk test and Epsiometer-test (E-test) to evaluate their ability to detect ESBLs. The genotypes of ESBLs were analyzed by monoplex polymerase chain reaction (PCR), multiplex PCR, DNA sequencing and isoelectric focusing.Results: 662 E. coli and 153 Klebsiella spp were analyzed. Among these isolates, 39.3 % E. coli and 26.1 % Klebsiella spp were positive for extended spectrum beta-lactamases (ESBLs).71.9 % E. coli and 79.6 % Klebsiella spp showed minimum inhibitory concentration (MIC) in the range > 32/0.064 = 500 μl/mL for cetatzidime/cetatzidime + clavulanic acid, while 66.5 % E. coli and 69.1 % Klebsiella spp revealed MIC in the range of > 16/0.016 = 1000 μl/mL for cefotaxime/cefotaxime + clavulanic acid. Antibiotic susceptibility testing revealed that imipemem, meropenem and tazocine were the most effective in the management of such infections. The most frequent genotype of ESBL was OXA (19.2 %) for E. coli and SHV (92.5 %) for Klebsiella spp. The highest genotypic combination found was the combination of TEM/OXA (44.2 %) for E. coli.Conclusion: The resistance of E. coli and Klebsiella spp-producing ESBLs in Pakistan is a serious issue, and TEM, OXA and SHV type ESBL were the most common genotypes. Some isolates produced two or three genotypes at a time. Multiplex PCR of ESBL may help in early detection as well as phenotypic antibiotic therapy of these infections.Keywords: Beta-lactamases, Escherichia coli, Klebsiella spp, Antibiotic susceptibility, Plasmidencoded, Structural genes, Imipemem, Meropenem, Tazocine

Highlights

Antimicrobial resistance is a very serious issue all over the world
Such bacteria are frequently reported as multidrug resistant bacteria (MDR) and some of the MDRs produce extended spectrum beta-lacatamases (ESBLs)
Total 300 bacterial strains were positive for beta-lactamases, 39.2 % E. coli and 26.10 % Klebsiella species were found to express ESBLs

Summary

INTRODUCTION

Antimicrobial resistance is a very serious issue all over the world. Pathogenic and opportunistic bacteria, which become highly resistant to drugs are causing untreatable infections [1]. Plasmid-mediated extended-spectrum betalactamase (ESBL) confers resistance to cephalosporin group, such as ceftazidime, ceftriaxone and others These enzymes occur in genera of the family Enterobacteriaceae for example Citrobacter, Enterobacter but predominantly present in E. coli and Klebsiella species [4]. The panel used in this study consists of 21 antibiotics that belong to three major groups of antibiotics according to mechanism of action Such as antibiotic disks containing amikacin 30 μg, ampicillin 10 μg , amoxicillin clavulanic acid (20 μg/10 μg) 30 μg, carbencilin 100 μg, cephredine 30 μg, cefaperazone 75 μg, ciprofloxacin 5 μg, gentamicin 10 μg, nitrofurontoin 300 μg, imepenem 10 μg, meropenem 10 μg, norfloxacin 10 μg, cotrimoxazole 25 μg, ceftizoxime 30 μg, cefotaxime 30 μg, cefoxitin 30 μg, cetazidime 30 μg, ceftriaxone 30 μg, cefixime 5 μg, aztreonam 10 μg, and pipracilin-tazobactam 100/10 μg were tested. E. coli was more resistant to cefotaxime (99.5 %) and less resistant to cefotaxime+ clavulanic acid (disc) (3.84 %)

RESULTS

DISCUSSION

CONCLUSION