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Indian Journal of Medical Microbiology, Vol. 26, No. 2, April-June, 2008, pp. 163-166 Brief Communication Detection of Enterobacteriaceae producing CTX-M extended spectrum β-lactamases from a tertiary care hospital in south India Padmini SBaby, Raju BAppala, Mani KR School of Biotechnology, Chemical and Biomedical Engineering, VIT University, Vellore - 632 014, Tamil Nadu Date of Submission: 11-Jun-2007 Code Number: mb08047 Abstract A total of 23 clinical isolates (15 Escherichia coli and 8 Klebsiella pneumoniae ), resistant to cefotaxime and ceftazidime recovered during 2002 and 2003, were investigated for production of CTX-M extended spectrum β-lactamase (ESBL) by phenotypic and molecular methods. The presence of ESBL was tested by NCCLS phenotypic confirmatory test using cephalosporin/clavulanate combination discs and E-test ESBL strips. Determination of MIC of cefotaxime and ceftazidime was done with and without the presence of clavulanic acid by agar dilution technique. Polymerase chain reaction revealed the presence of CTX-M type ESBLs in 19 isolates. Further sequencing resulted in identification of CTX-M-15 ESBLs. This is the first report identifying CTX-M type ESBL from clinical isolates of E. coli and K. pneumoniae from a tertiary care hospital in south India.Keywords: CTX-M type ESBLs, Enterobacteriaceae, South India Resistance to extended spectrum cephalosporins can occur in Escherichia coli and Klebsiella pneumoniae via the production of extended spectrum β-lactamases that are capable of hydrolyzing the oxyiminocephalosporins and monobactams. [1] In recent years, a new family of plasmid-mediated CTX-M extended spectrum β lactamase (ESBL) called CTX-M has arisen and reported in the literature with increasing frequency from Europe, Africa, Asia, South America, and North America [2] . CTX-M type ESBLs show only 40% identity to TEM or SHV ESBLs, but they are closely related to β-lactamase of the Kluyvera spp. These ESBLs were named CTX-M type β-lactamases, owing to their high activity against cefotaxime. [2] However, unlike most CTX-Ms, some CTX-M variants, including CTX-M-15, CTX-M-16 and CTX-M-19, also hydrolyze ceftazidime efficiently, which may complicate their phenotypic recognition. [3] In India, a multicentric study has reported 55-61% of ESBL prevalence among clinical isolates of Enterobacteriaceae from various hospitals. [4] Isolates producing ESBLs have not been characterized in most of the earlier studies except two studies from North India, which have reported CTX-M-15 β lactamases in isolates of Enterobacteriaceae . [5],[6] Although ESBL phenotypes have been reported from south India, there is no information on their molecular types. Hence, the present study was undertaken to characterize the β-lactamases in multidrug-resistant clinical isolates of Enterobacteriaceae by molecular techniques. Materials and Methods A total of 23 ESBL-producing isolates of E. coli (15) and K. pneumoniae (8) obtained from clinical specimens of inpatients and outpatients of PSG Hospitals, Coimbatore, Tamil Nadu (during August 2002 to August 2003) were included for the study. Antibiotic susceptibility testing was performed on Mueller-Hinton agar with the following antibiotic discs: cefotaxime (30 μg), ceftazidime (30 μg), piperacillin-tazobactam (100 μg/10 μg), imipenem (10 μg), meropenem (10 μg), ciprofloxacin (5 μg), amikacin (30 μg), gentamicin (10 μg), and co-trimoxazole (1.25-23.75 μg).Detection of ESBL Confirmation of ESBL was also done by E-test ESBL strips (AB Biodisc, Solna, Sweden), and the test was performed in accordance with the guidelines of the manufacturer. Double-ended strips containing gradient of cefotaxime (CT) or ceftazidime (TZ) at one end and cefotaxime or ceftazidime plus clavulanic acid (CTL and TZL) at the other end were tested in parallel. The presence of ESBL was confirmed by the appearance of phantom zone below CT or deformation of TZ inhibition ellipse or when clavulanate caused a more than or equal to three doubling concentration decrease (ratio of ≥8)in the MIC values of cefotaxime and ceftazidime. Transconjugation Isoelectric focusing PCR and DNA sequencing Purified amplicon products from few strains (6, 9 and 14) and a whole gene from a transconjugant (strain l7) were sequenced with the same primers as used for amplification using an ABI prism 3700 DNA analyzer (Applied Biosystem, CA, USA). Results Twenty-three clinical isolates included were obtained from clinical samples of urine (6), pus (10), sputum (3), endotracheal aspirate (3), and blood (1). Antibiotic susceptibility test results by disc diffusion method revealed very high susceptibility to piperacillin-tazobactam (100%), imipenem (100%), and meropenem (100%) followed by amikacin (82.6%). Resistance to cefotaxime, ceftazidime, gentamicin, ciprofloxacin, and co-trimoxazole was found to be 100, 100, 91, 82.6, and 82.6%, respectively. The results of phenotypic and molecular characterization of 23 isolates are summarized in table.In NCCLS phenotypic confirmatory test using cephalosporin/clavulanate combination discs, all the strains showed enhanced susceptibility to ceftazidime and/or cefotaxime in the presence of clavulanic acid, a typical finding for an ESBL producer. The isolates were also demonstrated> 3 log 2, dilution reduction in MIC values of cefotaxime and ceftazidime in the presence of clavulanic acid by agar dilution and E-test ESBL tests, confirming the presence of ESBLs. Among the 23 isolates tested, transconjugants were obtained for 13 isolates and all the transconjugants were resistant to cefotaxime. β-lactamase bands were obtained for 19 of 23 isolates tested for β-lactamases by isoelectric focusing. All the isolates produced minimum of two β-lactamases with pI of 5.4 and additional β-lactamases with pI value of 8.2/8.5. Two strains ( K. pneumoniae ) displayed another band with a pI value of 7.6 [Table - 1]. Out of 23 isolates screened for bla CTX-M genes, 19 yielded positive amplicons and four isolates did not. Out of three isolates tested by PCR with SHV-specific primers, one isolate was positive (strain no. 24) for SHV gene and other two strains (strain no. 17 and 25) were negative. Sequence analysis from three transconjugants (strain no. 6, 9, and 14) and whole gene amplified from a transconjugant (strain no. 17) matched the sequence of CTX-M type enzyme in the GenBank Data base (AYO 13478) called UOE-1 or CTX-M-15. Discussion Among the "newer" ESBL families, the CTX-M type ESBLs have become widely dispersed in many parts of the world. [2] Antibiotic susceptibility test results of the above isolates illustrated an alarming trend of associated resistance to gentamicin (91%), co-trimoxazole (82.6%), and ciprofloxacin (82.6%). Such resistance has been reported in recent surveys from Canada, Italy, Spain, Greece, and UK. The bla CTX-M genes are found in association with genetic structures such as sul 1 type integrons, and this might explain the multidrug-resistant nature of organism producing these enzymes. This structure is genetically linked to class 1 integrons known to integrate antibiotic-resistant gene cassettes responsible for resistance to β-lactams, aminoglycosides, chloramphenicol, sulphonamides, and to a lesser extent rifampicin. [3] The belief that organisms producing CTX-M enzymes display higher levels of resistance to cefotaxime than ceftazidime is not universal among all CTX-M producers. Majority of the isolates in our study conferred high-level resistance to cefotaxime (>64 μg/mL) as well as to ceftazidime (>32 μg/mL) as evidenced by MIC determination [Table - 1]. These findings correlated well with other studies. [5],[11],[12] Therefore, MIC determinations to cefotaxime and ceftazidime are not a reliable approach for identifying CTX-M type ESBLs. In our study, commercially available E-test ESBL test results were 100% in agreement with standard NCCLS phenotypic confirmatory test, although varied sensitivity (87-100%) and specificity (95-100%) of E-test ESBL for confirmation of ESBLs have been reported earlier. [13] Transconjugation experiments expressed evidence of transfer of plasmid-mediated cefotaxime resistance in all the isolates. However, transconjugants have not been analyzed for plasmid content. Test based on isoelectric focusing and molecular detection of ESBL genes by PCR is more conclusive in defining ESBL productions. IEF results for β-lactamases detection using Pharmacia PhastSystem revealed multiple bands in most of the isolates. All the isolates had β-lactamases with pI value of 5.4 (TEM-1 enzyme). In addition, some isolates exhibited β-lactamases with a pI value of ~8.2 and others with pI value of 8.5 consistent with the presence of SHV type ESBLs and CTX-M ESBLs. Two isolates of K. pneumoniae displayed additional β-lactamase with a pI value of 7.6 (SHV-1 enzymes). As the PhastSystem procedure did not generate an accurate pI, the presence of β-lactamases was further confirmed by PCR screening using specific CTX-M and SHV primers. Nineteen isolates were found positive for bla CTX-M genes by PCR using specific primers. ESBLs were not characterized in the remaining four CTX-M negative strains. One isolate was positive for both CTX-M and SHV gene. As majority of isolates (19/23) belonged to CTX-M type ESBLs, sequence analysis was performed only for a few strains. Amplified DNA from three isolates (strain no. 6, 9, and 14) and a whole gene from a transconjugant (strain no. 17) revealed the presence of CTX-M-15 type ESBLs. Although extensive molecular characterization was not done in all the isolates, our preliminary findings suggest the presence of CTX-M β-lactamases as predominant ESBLs in isolates of E. coli and K. pneumoniae . Identification of CTX-M-15 in four strains of Enterobacteriaceae by sequence analysis correlated well with the earlier findings from north India. [5],[6] In conclusion, this study documents the presence of CTX-M type β lactamase among the ESBLs in this tertiary care hospital in south India. Acknowledgments We gratefully acknowledge Dr. George A. Jacoby MD, Lahey Clinic, Burlington, MA, USA, for excellent technical support for molecular characterization of ESBL positive isolates. We also thank Dr. M.K. Lalitha for the valuable guidance in MIC determination and providing Klebsiella pneumoniae ATCC 700603 (ESBL positive control) for the study.References
Copyright 2008 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb08047t1.jpg] |
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