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

OBJECTIVES: To develop a reverse-line hybridization assay to identify CTX-M genotypes, potentially useful for large-scale investigation of surveillance collections. METHODS: Isolates carrying previously characterized bla(CTX-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 bla(CTX-M) open-reading frame. Oligonucleotides were designed to hybridize to regions within each amplicon, covering mutations that distinguish among bla(CTX-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 bla(CTX-M) genes among survey collections of Enterobacteriaceae.