Correspondence this research from the CIBER en Epidemiologı´a y Salud Pu´blica (CIBERESP) in Spain.
Transparency declarations None to declare.
References 1. Ding YF, Zhang JH, Mi ZH et al. Study on the molecular epidemiology of b-lactamase TEM gene in isolated Streptococcus pneumoniae. Zhonghua Liu Xing Bing Xue Za Zhi 2004; 25: 970– 2. 2. del Campo R, Cafini F, Morosini MI et al. Combinations of PBPs and MurM protein variants in early and contemporary high-level penicillin-resistant Streptococcus pneumoniae isolates in Spain. J Antimicrob Chemother 2006; 57: 983– 6. 3. Mabilat C, Courvalin P. Development of ‘oligotyping’ for characterization and molecular epidemiology of TEM b-lactamases in members of the family Enterobacteriaceae. Antimicrob Agents Chemother 1990; 34: 2210–6. 4. Lin TL, Tang SI, Fang CT et al. Extended-spectrum b-lactamase genes of Klebsiella pneumoniae strains in Taiwan: recharacterization of shv-27, shv-41, and tem-116. Microb Drug Resist 2006; 12: 12 – 5. 5. Song JS, Lee JH, Lee JH et al. Removal of contaminating TEM-1a b-lactamase gene from commercial Taq DNA polymerase. J Microbiol 2006; 44: 126– 8. 6. Chiang CS, Liu CP, Weng LC et al. Presence of b-lactamase gene TEM-1 DNA sequence in commercial Taq DNA polymerase. J Clin Microbiol 2005; 43: 530–1.
Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dkm267 Advance Access publication 10 July 2007
Detection of OXA-2 group extended-spectrumb-lactamase-producing clinical isolates of Escherichia coli from India Amitabha Bhattacharjee, Malay Ranjan Sen*, Shampa Anupurba, Pradyot Prakash and Gopal Nath Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India Keywords: E. coli, ESBLs, OXA-type *Corresponding author. Tel: þ91-9415820675; E-mail:
[email protected] Sir, Since the first report of an extended-spectrum b-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 b-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 b-lactam, non-b-lactam and b-lactam/b-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 50 -ATGAGTATTCAACATTTCCG-30 /TEM R 50 -CTGACAGTT ACCAATGCTTA-30 (867 bp),1 SHV F 50 -AGGATTGACTG CCTTTTTG-30 /SHV R 50 -ATTTGCTGATTTCGCTCG-30 3 0 (392 bp), CTX-M-A 5 -CGCTTTGCGATGTGCAG-30 /CTXM-B 50 -ACCGCGATATCGTTGGT-30 (550 bp),4 OXA I F 50 -TCAACAAATCGCCAGAGAAG-30 /OXA I R 50 -TCCCAC ACCAGAAAAACCAG-30 (276 bp)1 and OXA II F 50 -AAGAA ACGCTACTCGCCTGC-30 /OXA II R 50 -CCACTCAACCCATC CTACCC-30 (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 mg of DNA, 15 pmol of each primer, 10 mM dNTPs and 1 U of Taq DNA polymerase in 25 mM MgCl2 Taq buffer, and the reactions were run under the following conditions: initial denaturation at 948C for 5 min; 40 cycles of 948C for 1 min, 558C for 1 min and 728C for 1 min; and a final elongation at 728C 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 b-lactamase genes. The first (Ec 461) and second (Ec 614) isolates harboured CTX-M, TEM and OXA-2 group b-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
703
Correspondence Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dkm261 Advance Access publication 16 July 2007
Plasmid-mediated quinolone resistance determinant qnrS1 detected in Salmonella enterica serovar Corvallis strains isolated in Denmark and Thailand L. M. Cavaco1,2*, R. S. Hendriksen1 and F. M. Aarestrup1 1
Figure 1. 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.
National Food Institute, Technical University of Denmark, Bu¨lowsvej 27, DK-1790 Copenhagen V, Denmark; 2 Department for Veterinary Pathobiology, LIFE—Faculty of Life Sciences, University of Copenhagen, Stigbojlen 4, DK-1870 Frederiksberg C, Denmark Keywords: imported meat, qnr, QRDR
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. The presence of the OXA-2 gene has been frequently detected in Pseudomonas 1,4 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.
Acknowledgements 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.
Transparency declarations None to declare.
References 1. Bert F, Branger C, Zechovsky NL. Identification of PSE and OXA b-lactamase genes in Pseudomonas aeruginosa using PCR-restriction fragment length polymorphism. J Antimicrob Chemother 2002; 50: 11 –8. 2. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disc Susceptibility Tests: M100-S15. CLSI, Wayne, PA, USA, 2005. 3. Colom K, Perez J, Alonso R et al. Simple and reliable multiplex PCR assay for detection of blaTEM, blaSHV and blaOXA-1 genes in Enterobacteriaceae. FEMS Microbiol Lett 2003; 223: 147–51. 4. Lee S, Park YJ, Kim M et al. Prevalence of Ambler class A and D b-lactamases among clinical isolates of Pseudomonas aeruginosa in Korea. J Antimicrob Chemother 2005; 56: 122– 7. 5. Johannes G, Koeleman M, Stoof J et al. Identification of epidemic strains of Acinetobacter baumannii by integrase gene PCR. J Clin Microbiol 2001; 39: 8– 13. 6. Chmelnitsky I, Carmeli Y, Leavitt A et al. CTX-M-2 and a new CTX-M-39 enzyme are the major extended spectrum b-lactamases in multiple Escherichia coli clones isolated in Tel Aviv, Israel. Antimicrob Agents Chemother 2005; 49: 4745– 50.
*Corresponding author. Tel: þ45-72-34-62-69; Fax: þ45-72-3463-41; E-mail:
[email protected] Sir, Until recently, chromosomal mutations in the topoisomerase genes involved in DNA transcription and replication were considered the main mechanisms of quinolone resistance in Enterobacteriaceae. A new and transferable mechanism was described in 1998 in a Klebsiella pneumoniae isolate obtained from a patient in 1994 in Alabama, USA. Other qnr genes (A, B and S) and several variants encoding plasmid-mediated quinolone resistance have recently been described.1 These encode Qnr proteins that are members of the pentapeptide family and are able to protect topoisomerases, and thus reduce their susceptibility to fluoroquinolones.1 Plasmid-mediated quinolone resistance, which was originally found very rarely, seems to have spread more rapidly than expected and is now found in the US, Africa, Asia and also in Europe.1 The genes are often located on transferable plasmids and co-transmitted with other important resistance genes, especially genes encoding resistance to cephalosporins.1 We have recently described the characterization of a collection of 59 S. Corvallis isolates from Denmark, Bulgaria and Thailand.2 A total of 23 isolates in this collection showed reduced susceptibility to ciprofloxacin (MIC .0.06 mg/L) but were found to be susceptible or intermediate to nalidixic acid (MIC 8 –16 mg/L). All isolates showing this type of resistance profile were examined for the presence of qnrA, qnrB, qnrS and aac(60 )Ib by PCR amplification and sequencing of the gyrA and parC genes. Briefly, qnrA and qnrS were amplified using primers based on published sequences and qnrB using primers described before as FQ1 and FQ2.3 As positive control strains, we used E. coli J53 pMG252 positive for qnrA, E. coli J53 pMG298 positive for qnrB (both strains kindly provided by Dr George Jacoby) and for qnrS E. coli MT102 pBC-H2.6, obtained by electroporating the plasmid pBC-H2.6 (obtained from Dr M. Hata, through the Rikken DNA Bank) into E. coli MT102. For aac(60 )Ib, PCR was performed as described by Park et al.4 As positive control, we used the strain Salmonella enterica S74 (GenBank accession number: DQ278189).5
704
