1887

Abstract

, a Gram-positive, spore-forming, anaerobic bacterium, is the main causative agent of hospital-acquired diarrhoea worldwide. In addition to metronidazole and vancomycin, rifaximin, a rifamycin derivative, is a promising antibiotic for the treatment of recurring infections (CDI). However, exposure of to this antibiotic has led to the development of rifaximin-resistance due to point mutations in the β-subunit of the RNA polymerase () gene. In the present study, 348 strains with known PCR-ribotypes were investigated for respective single nucleotide polymorphisms (SNPs) within the proposed hot-spot region by using high-resolution melting (HRM) analysis. This method allows the detection of SNPs by comparing the altered melting behaviour of dsDNA with that of wild-type DNA. Discrimination between wild-type and mutant strains was enhanced by creating heteroduplexes by mixing sample DNA with wild-type DNA, leading to characteristic melting curve shapes from samples containing SNPs in the respective section. In the present study, we were able to identify 16 different sequence-types (ST) by sequencing analysis of a 325 bp fragment. The 16 PCR STs displayed a total of 24 different SNPs. Fifteen of these 24 SNPs were located within the proposed 151 bp SNP hot-spot region, resulting in 11 different HRM curve profiles (CP). Eleven SNPs (seven of which were within the proposed hot-spot region) led to amino acid substitutions associated with reduced susceptibility to rifaximin and 13 SNPs (eight of which were within the hot-spot region) were synonymous. This investigation clearly demonstrates that HRM analysis of the proposed SNP hot-spot region in the gene of is a fast and cost-effective method for the identification of samples with reduced susceptibility to rifaximin and even allows simultaneous SNP subtyping of the respective isolates.

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2012-06-01
2020-07-04
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