A novel reverse-line hybridization assay for identifying genotypes of CTX-M-type extended-spectrum -lactamases

useful for large-scale investigation of surveillance collections. Methods: Isolates carrying previously characterized blaCTX-M genes were used to develop the method. In addition, 334 isolates from five separate surveys were used to validate the method. CTX-M group was known from an independent multiplex PCR for 122 isolates and genotype was confirmed for 80 isolates by DNA sequencing. A multiplex PCR was designed to amplify a genotype-specific region within the blaCTX-M open-reading frame. Oligonucleotides were designed to hybridize to regions within each amplicon, covering mutations that distinguish among blaCTX-M genotypes. Results: CTX-M phylogenetic groups were identified by the multiplex PCR with 100 % concordance. The reverse-line hybridization assay specifically identified commonly-reported variants within these groups (98.7 % concordance). Conclusions: The hybridization method enabled precise identification of CTX-M genes, rather than just to group level, without the need for DNA sequencing. In its present format, the method enables 43 isolates to be processed per membrane, giving results within one working day. It is a useful tool for the epidemiological investigation of blaCTX-M genes among survey collections of Enterobacteriaceae

Genetic Environment of Plasmid Mediated CTX-M-15 Extended Spectrum Beta-Lactamases from Clinical and Food Borne Bacteria in North-Eastern India

The study investigated the presence of CTX-M-15 type extended spectrum beta-lactamases (ESBL), compared their genetic arrangements and plasmid types in gram negative isolates of hospital and food origin in north-east India. From September 2013 to April 2014, a total of 252 consecutive, non-duplicate clinical isolates and 88 gram negative food isolates were selected. Phenotypic and molecular characterization of ESBL genes was performed. Presence of integrons and gene cassettes were analyzed by integrase and 59 base-element PCR respectively. The molecular environments surrounding blaCTX-M and plasmid types were investigated by PCR and PCR-based replicon typing respectively. Transformation was carried out to assess plasmid transfer. Southern blotting was conducted to localize the blaCTX-M-15 genes. DNA fingerprinting was performed by ERIC-PCR.Prevalence of ESBL was found to be 40.8% (103/252) in clinical and 31.8% (28/88) in food-borne isolates. Molecular characterization revealed the presence of 56.3% (58/103) and 53.5% (15/28) blaCTX-M-15 in clinical and food isolates respectively. Strains of clinical and food origin were non-clonal. Replicon typing revealed that IncI1 and IncFII plasmid were carrying blaCTX-M-15 in clinical and food isolates and were horizontally transferable. The ISEcp1 element was associated with blaCTX-M-15 in both clinical and food isolates.The simultaneous presence of resistance determinants in non-clonal isolates of two different groups thus suggests that the microbiota of common food products consumed may serve as a reservoir for some of the drug resistance genes prevalent in human pathogens