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Novel reverse hybridisation assay to identify CTX-M genotype in cephalosporin-resistant isolates from UK and India

Abstract number: p1750

Ensor  V.M., Shahid  M., Hope  R., Warner  M., Woodford  N., Livermore  D.M., Hawkey  P.M.

Objectives: 

a) To develop a novel reverse hybridisation assay for identification of CTX-M genotypes among large collections of cephalosporin-resistant Enterobacteriaceae isolated during surveillance studies in South-East England and North India.

b) To validate the assay results by DNA sequencing.

Methods: 

Isolate collection 1: 110 Enterobacteriaceae resistant to extended-spectrum cephalosporins, isolated in London and South-East England. These isolates were known to carry phylogenetic group 1 blaCTX-M, but precise genotypes had not been determined. Isolate collection 2: 130 Enterobacteriaceae resistant to extended-spectrum cephalosporins, isolated in Aligarh, North India. Resistance determinants had not been investigated previously. A novel multiplex PCR was used to amplify blaCTX-M. Reverse hybridisation was carried out using biotinylated PCR amplicon and sequence-specific oligonucleotides designed to identify members of CTX-M phylogenetic group 1. Hybridisation results were validated by DNA sequencing for 20 representative isolates from each collection.

Results: 

109/110 London and SE England isolates known to carry group 1 blaCTX-M gave a consistent profile, corresponding to that for CTX-M-15 and CTX-M-28; 1/110 gave a profile corresponding to CTX-M-3 and CTX-M-22. 82/130 Indian isolates had blaCTX-M genes, all of which belonged to group 1, and all these gave a hybridisation profile corresponding to CTX-M-15 or CTX-M-28. CTX-M-28 and CTX-M-22 are rare variants, suggesting that the enzymes present were more likely to be CTX-M-15 and CTX-M-3, and this was confirmed by DNA sequencing.

Conclusions: 

This is the first reported application of this novel reverse hybridisation assay to the analysis of large numbers of cephalosporin-resistant Enterobacteriaceae. Results were validated by DNA sequencing. The assay is cheap and convenient, enables reasonable throughput, provides results within one day and can be used in place of DNA sequencing. We believe it will be valuable for monitoring the prevalence and genotypes of blaCTX-M genes in Enterobacteriaceae.

Session Details

Date: 01/08/2007
Time: 00:00-00:00
Session name: XXIst ISTH Congress
Subject:
Location: Oxford, UK
Presentation type:
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