Detection of OXA-2 group extended-spectrum-β-lactamase-producing clinical isolates of Escherichia coli from India

Sir, Since the first report of an extended-spectrum β-lactamase (ESBL) in the early 1980s, several classes of ESBLs have been described, along with their respective genes in the chromosomal/plasmid DNA, among clinical bacterial isolates, particularly, Escherichia coli and Klebsiella species. OXA-type β-lactamases, belonging to molecular class D and functional group 2d, are characterized by their high hydrolytic activity against oxacillin and cloxacillin and are poorly inhibited by clavulanic acid. Extension of the hydrolytic spectrum of oxacillinase to oxyimino cephalosporins has been reported in OXA-2 and OXA-10 extended-spectrum derivatives.1 Unfortunately, there are very few studies on the epidemiology and geographical spread of OXA-type ESBLs. Here, we present the first report of an incidence of isolation of three E. coli isolates harbouring OXA-2 group ESBLs in a university hospital in northern India. These strains were isolated within a period of 3 months, i.e. from August 2005 to October 2005, from patients who were admitted to the surgical ward of Sir Sunderlal Hospital, Banaras Hindu University, Varanasi, India. The first one (Ec 461) was isolated from the urine of a catheterized 30-year-old female. The second isolate (Ec 614) was recovered from pus of a 70-year-old male and the third one (Ec 782) was from pus of a 20-year-old female. The strains were subjected to susceptibility testing (Kirby–Bauer method) against a total of 25 different β-lactam, non-β-lactam and β-lactam/β-lactamase inhibitor combinations. The isolates were subjected to an initial screening test (MIC) for ESBL production and further confirmed phenotypically by a combined disc diffusion and MIC reduction method according to CLSI guidelines.2 MICs of cefotaxime, ceftazidime, ceftriaxone, cefpodoxime and aztreonam were also determined. For partial gene PCR amplification, primers TEM F 5′-ATGAGTATTCAACATTTCCG-3′/TEM R 5′-CTGACAGTT ACCAATGCTTA-3′ (867 bp),1 SHV F 5′-AGGATTGACTG CCTTTTTG-3′/SHV R 5′-ATTTGCTGATTTCGCTCG-3′ (392 bp),3 CTX-M-A 5′-CGCTTTGCGATGTGCAG-3′/CTX-M-B 5′-ACCGCGATATCGTTGGT-3′ (550 bp),4 OXA I F 5′-TCAACAAATCGCCAGAGAAG-3′/OXA I R 5′-TCCCAC ACCAGAAAAACCAG-3′ (276 bp)1 and OXA II F 5′-AAGAA ACGCTACTCGCCTGC-3′/OXA II R 5′-CCACTCAACCCATC CTACCC-3′ (478 bp),1 specific for blaTEM, blaSHV, blaCTX-M-1,-2,-9, blaOXA-10 and blaOXA-2, respectively, were used for reaction with bacterial DNA as template. Each single reaction mixture contained 1 µg of DNA, 15 pmol of each primer, 10 mM dNTPs and 1 U of Taq DNA polymerase in 25 mM MgCl2Taq buffer, and the reactions were run under the following conditions: initial denaturation at 94°C for 5 min; 40 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min; and a final elongation at 72°C for 7 min. Detection of class I and class II integrons by integrase gene PCR was also performed as described previously.5 Amplicons obtained using OXA II primers were further analysed by sequencing (Genei, Bangalore, India) on both strands. The same set of primers was used for PCR analysis and for sequencing purposes. DNA sequence was compared with gene bank databases of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/entrez). All the three isolates were typed by both random amplification of polymorphic DNA (RAPD) using primer 7 (GTGGATGCGA) and enterobacterial repetitive intergenic consensus (ERIC) PCR. The first and second isolates were susceptible only to imipenem whereas the third isolate was susceptible to piperacillin/tazobactam in addition to imipenem. All of them had similar MIC profiles and, when characterized genotypically, all of them possessed multiple β-lactamase genes. The first (Ec 461) and second (Ec 614) isolates harboured CTX-M, TEM and OXA-2 group β-lactamase genes whereas TEM was absent in the third isolate (Ec 782) (Figure 1). All of the three strains also possessed a class I integron. Sequencing of the PCR product of the OXA II primers revealed that all of the three isolates harboured an OXA-2 variant gene. Both RAPD and ERIC PCR were equally discriminative; strains Ec 614 and Ec 782 had an identical pattern (pattern A), whereas Ec 461 was different (pattern B) by both of the typing methods. Detection of ESBLs using multiplex PCR. Lanes 1 and 7, 100 bp DNA ladder (Genei); lane 2, SHV (392 bp) positive control; lane 3, negative control; lanes 4 and 5, OXA (478 bp), CTX-M (550 bp) and TEM (867 bp) amplicons of Ec 461 and Ec 614, respectively; lane 6, OXA and CTX-M amplicons of Ec 782. The presence of the OXA-2 gene has been frequently detected in Pseudomonas1,4 and it was first reported in E. coli from Israel in 2005.6 To the best of our knowledge, the presence of this gene in E. coli is the first report from India and the second in the world. The implication/s of the recent detection of the OXA-2 group gene in E. coli needs further investigation and action. We would like to acknowledge the Head, Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi and the University Grants Commission, India, for providing financial support to carry out the study. None to declare.