JAC Advance Access originally published online on October 28, 2006
Journal of Antimicrobial Chemotherapy 2006 58(6):1260-1263; doi:10.1093/jac/dkl422
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Occurrence, prevalence and genetic environment of CTX-M ß-lactamases in Enterobacteriaceae from Indian hospitals
1 Antimicrobial Agents Research Group, Institute of Biomedical Research, University of Birmingham Vincent Drive, Edgbaston, Birmingham B15 2TT, UK 2 West Midlands Health Protection Agency, Heart of England NHS Foundation Trust Bordesley Green East, Birmingham B9 5SS, UK 3 Department of Microbiology, Jawaharlal Nehru Medical College & Hospital (JNMCH), Aligarh Muslim University Aligarh-202002, Uttar Pradesh, India
*Correspondence address. Health Protection Agency, Heartlands Hospital, Birmingham B9 5SS, UK. Tel: +44-121-424-1240; Fax: +44-121-772-6229; E-mail: peter.hawkey{at}heartofengland.nhs.uk
Received 18 July 2006; returned 12 September 2006; revised 22 September 2006; accepted 25 September 2006
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Abstract |
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Objectives: To determine occurrence, prevalence and CTX-M genotypes produced by Enterobacteriaceae from clinical samples from three geographically distant Indian hospitals and to detect linkage of IS26 with blaCTX-M and map its precise insertion position.
Methods: A total of 130, non-duplicate Escherichia coli and Klebsiella pneumoniae resistant to a third-generation cephalosporin (3GC) from three Indian centres were screened for extended-spectrum ß-lactamase (ESBL) production using phenotypic detection methods. All isolates were screened for blaCTX-M using multiplex PCR. Precise CTX-M genotype was identified using reverse-line hybridization. All CTX-M-producing isolates were screened for linkage of IS26 with blaCTX-M. DNA sequencing was used to map the exact insertion position of this mobile element.
Results: Ninety-five of 130 3GC-resistant (73%) (73% of total E. coli, 72% of total K. pneumoniae) isolates were found to carry blaCTX-M-15. No other CTX-M genotype was detected. IS26 linkage with blaCTX-M-15 was detected in 31% of isolates carrying blaCTX-M-15. DNA sequencing revealed variable insertion of this mobile element within tnpA of ISEcp1. RAPD–PCR typing demonstrated great diversity in isolates carrying blaCTX-M-15; no predominant clone was identified.
Conclusions: In contrast with other studies where greater diversity exists, CTX-M-15 was the only CTX-M ESBL produced in this Indian collection of unrelated E. coli and K. pneumoniae. This is the first systematic survey report from India detecting CTX-M-type ß-lactamases This is also the first report indicating such high mobility/diversity of insertion of IS26 in close association with blaCTX-M in a single bacterial collection.
Keywords: ESBLs , IS26 , CTX-M-15 , India
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Introduction |
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In recent years CTX-M extended-spectrum ß-lactamases (ESBLs) have very rapidly disseminated and are now frequently reported from countries all over Europe and much of Asia.1 More than 50 blaCTX-M genotypes are described (http://www.lahey.org/studies/inc_webt.asp). Epidemiological reports demonstrate that some enzymes are more frequently reported than others, that predominant enzyme type varies with country and that diverse CTX-M types often exist within a single country.2
In India, the very first report of the presence of CTX-M-producing Enterobacteriaceae came from New Delhi.3 Six isolates from 2000 were investigated, all of which were found to be unrelated and all produced CTX-M-15. Since then several Indian surveys have reported the presence of ESBLs in clinical isolates based on phenotypic tests. Recent reported rates vary widely (12.6–71%),4,5 with most studies reporting a prevalence rate of around 50% and upwards. Considering a population of 
1.1 billion in India, this represents a very large reservoir of resistance genes. We were unable to find any report in our search of the literature using the Entrez PubMed database provided by the National Centre for Biotechnology Information (www.ncbi.nlm.nih.gov/entrez) that specifically looked for blaCTX-M and then determined the precise genotype present. Therefore, we planned this study to identify the occurrence, species distribution and CTX-M genotypes present in clinical isolates from a large tertiary care hospital in North India. A collection of isolates from two geographically distant centres was also included to determine any diversity in isolates and CTX-M-genotype present in the three geographically separated regions of India.
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Materials and methods |
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A total of 130 non-duplicate third-generation cephalosporin (3GC)-resistant Escherichia coli or Klebsiella pneumoniae were investigated in this study. Of these, 111 isolates were cultured from clinical samples taken from patients at JNMCH, Aligarh, Uttah Pradesh Province, North India, 74 of which were collected in a 4 week period in 2005 and 37 of which were collected between 2003 and 2004. Of the remaining 19 isolates, 9 were collected from a centre 560 km away in Varanasi, Uttar Pradesh, North India, and 10 were collected from a centre in Hubli, Karnataka, South India, and 1430 km from Aligarh. These isolates were included to determine any genetic diversity or difference in CTX-M types from the geographically distant centres compared with the main study centre. Most isolates (98/130, 75%) were cultured from urine samples. The remaining isolates were cultured from respiratory or pus samples.
Susceptibility testing to 12 antimicrobial agents (Table 1) was performed using the BSAC standardized disc diffusion method (http://www.bsac.org.uk/susceptibility_testing/bsac_standardized_disc_susceptibility_method.cfm). ESBL production was determined using the combination disc test method.6 Discs containing cefotaxime, ceftazidime and cefepime were used alone and in combination with clavulanate, and tests were set up and results interpreted according to the manufacturer's instructions (Oxoid, Basingstoke, UK).
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All isolates were screened for the presence of blaCTX-M using a new multiplex PCR designed to detect and identify phylogenetic group in one PCR.7 Precise CTX-M genotype was identified using reverse line hybridization (RLH).7 To determine whether Indian isolates carried IS26 in association with blaCTX-M, the primers and PCR protocol previously described by Woodford et al.8 were used. In this protocol, the forward primer is designed within IS26 and the reverse primer within blaCTX-M, therefore a product confirms linkage. Products obtained were then sequenced to determine the precise insertion point of IS26 upstream of blaCTX-M.
RAPD–PCR9 was used to determine any genetic relatedness amongst isolates. Banding patterns were analysed using BioNumerics software version 4.5 (Applied Maths, Sint-Martens-Latem, Belgium). Position tolerance was set at 1%. Strains with at least 85% similarity (UPGMA, Dice Coefficient) were regarded as being highly related and were grouped into clusters.
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Results and discussion |
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ESBL production was confirmed in 115 (89%) of the 130 3GC-resistant isolates investigated using the combination disc test method. Of these, 72 of 98 (73%) E. coli and 23 of 32 (72%) K. pneumoniae were found to carry blaCTX-M by multiplex PCR, all of which identified as blaCTX-M-15 by RLH. This is interesting and is in contrast to other surveys that have reported variation of CTX-M genotypes present within a single country.2,10,11
Denominator data were only available for the isolates collected in Aligarh over the 4 week study period in 2005. During this time, 52% of all E. coli and K. pneumoniae isolated at the Aligarh centre were found to be resistant to a 3GC. This is similar to previous reports from India but much higher than the ESBL prevalence rates in most European countries.12 Results of antimicrobial susceptibility testing are shown in Table 1. Concomitant resistance to ciprofloxacin, trimethoprim and gentamicin was frequent, and a higher proportion of both E. coli and K. pneumoniae with blaCTX-M were resistant to the antibiotics tested than those isolates found not to carry blaCTX-M. All isolates were susceptible to both ertapenem and meropenem. Isolates found not to carry blaCTX-M most likely carry another ESBL gene (blaTEM or blaSHV) although this has not yet been investigated further. Resistance to cefoxitin was noted in 35% of E. coli and 41% of K. pneumoniae. Using a previously published protocol,13 this resistance in strains tested so far is attributed to the production of an Amp-C ß-lactamase. Further work is required to characterize genotypes present more fully.
Unlike the situation in the UK where an epidemic E. coli clone carrying blaCTX-M-15 has been described,8 RAPD–PCR typing of these Indian blaCTX-M-15-carrying E. coli or K. pneumoniae demonstrated great diversity and no predominant type, indicating either horizontal transfer of blaCTX-M-15 and/or mobilization of blaCTX-M-15 by genetic mobile elements as well as widespread geographical dissemination in both North and South India. The association of insertion sequences with antibiotic resistance genes has been reported previously. ISEcp1 is always found upstream of blaCTX-M-15 and is strongly implicated in the mobilization of this antibiotic resistance gene.14 IS26 has also been reported in association with blaCTX-M including blaCTX-M-15 and more specifically, has been found to insert within ISEcp1, although interestingly, the insertion of IS26 differs from strain to strain.8,15 In our study of highly diverse E. coli and K. pneumoniae producing CTX-M-15, IS26 was found in association with blaCTX-M-15 in 31% of isolates, indicating high frequency of association of this element with blaCTX-M-15. DNA sequencing of IS26-blaCTX-M PCR products enabled precise size determination of the amplicons obtained. It is interesting to note that PCR products of variable sizes were detected from all three Indian study centres, and an amplicon of 863 bp was most frequently observed (Table 2). DNA sequencing of representative IS26-blaCTX-M amplicons also enabled precise determination of the insertion point of this element, which was found to be at three variable positions within tnpA, the transposase-coding gene of ISEcp1. To our knowledge this is the first report of such high mobility and diversity of IS26 insertion into tnpA of ISEcp1. It is entirely plausible to speculate that since first being reported in India in 2000,3 blaCTX-M may have been mobilized on many occasions, as seems to be the case for blaSHV,16 perhaps explaining its presence in such a highly diverse collection of isolates from the three study centres. The fact that IS26 was found to be linked to blaCTX-M-15 in as many as one-third of CTX-M-15-producing isolates suggests this mobile element may have a more central role in the catalysis of the mobilization of blaCTX-M than has thus far been recognized. We have not looked for a second copy of IS26 downstream of blaCTX-M although two copies of this insertion sequence have been found to bracket blaSHV forming a composite transposon, providing the mechanism for mobilization of this resistance gene from the chromosome of K. pneumoniae.16 Further work is planned to look for the presence of IS26 downstream of blaCTX-M-15 in our isolates. The evolutionary advantage (if any) that may be conferred by the presence of two different mobile elements in close association of a resistance gene remains to be determined. In the UK epidemic CTX-M-15-producing E. coli clone, it has been suggested that IS26 insertion separates blaCTX-M-15 from its usual promoter (provided by ISEcp1) resulting in reduced resistance to cephalosporin antibiotics.8 Perhaps the function of IS26 insertion into tnpA of ISEcp1 is to inhibit further ISEcp1-mediated blaCTX-M-15 mobilization, effectively trapping blaCTX-M onto the plasmid and ensuring that it is maintained in the gene pool.
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The bowel is a rich environment for genetic exchange between commensal Enterobacteriaceae. Faecal carriage of CTX-M-producing bacteria has been described.10,11 It is plausible to suggest that conditions of overcrowding and poor sanitation, and the selective pressure created by overuse of antibiotics in India has enabled such widespread dispersal of blaCTX-M-15. The Indian population, therefore, must represent a significant reservoir and source of blaCTX-M-15.
This is the first systematic molecular survey report from India characterizing the occurrence and prevalence of CTX-M enzymes. Our report supports the urgent need for regular screening and national surveillance characterizing the CTX-M types and to implement strict antibiotics policy to limit the irrational use of antibiotics, including cephalosporins, so as to minimize antibiotic selective pressure.
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Transparency declarations |
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None to declare.
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Acknowledgements |
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We appreciate helpful comments made by Dr M. Avison. M. Shahd is grateful to the University Grants Commission, India, and the Association of Commonwealth Universities and British Council, UK, for awarding a Commonwealth Academic Fellowship (INCF-108-05) and to A. Rattan for providing some of the bacterial isolates. Secretarial support provided by J. Moore is much appreciated, and we acknowledge receipt of BBSRC grant 6/JIF13209 awarded to the Functional Genomics Laboratory, University of Birmingham, UK, which supports DNA sequencing work.
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References |
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1 Bonnet R. (2004) Growing group of extended-spectrum ß-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother 48:1–14.
2 Livermore DM and Woodford N. (2006) The ß-lactamase threat in Enterobacteriaceae, Pseudomonas and Acinetobacter. Trends Microbiol 14:413–20.[CrossRef][Web of Science][Medline]
3 Karim A, Poirel L, Nagarajan S, et al. (2001) Plasmid-mediated extended-spectrum ß-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1. FEMS Microbiol Lett 201:237–41.[Web of Science][Medline]
4 Datta P, Thakur A, Mishra B, et al. (2004) Prevalence of clinical strains resistant to various ß-lactams in a tertiary care hospital in India. Jpn J Infect Dis 57:146–9.[Medline]
5 Grover SS, Sharma M, Chattopadhya D, et al. (2006) Phenotypic and genotypic detection of ESBL mediated cephalosporin resistance in Klebsiella pneumoniae: emergence of high resistance against cefepime, the fourth generation cephalosporin. J Infect 53:279–88.[CrossRef][Web of Science][Medline]
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Carter MW, Oakton KJ, Warner M, et al. (2000) Detection of extended-spectrum ß-lactamases in klebsiellae with the Oxoid combination disk method. J Clin Microbiol 38:4228–32.
7 Ensor V, Hope R, Warner M, et al. (2006) Novel reverse line hybridisation assay to identify CTX-M genotype in cephalosporin-resistant isolates from UK and India. Abstracts of the Sixteenth European Congress on Clinical Microbiology and Infectious Diseases, Nice, FranceClin Microbiol Infect 2006 12:Suppl 4, Abstract P1750.
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Woodford N, Ward ME, Kaufmann ME, et al. (2004) Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum ß-lactamases in the UK. J Antimicrob Chemother 54:735–43.
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