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

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.