title: High-resolution melting analysis of the single nucleotide polymorphism hot-spot region in the rpoB gene as an indicator of reduced susceptibility to rifaximin in Clostridium difficile.
abstract: Clostridium difficile, 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 C. difficile infections (CDI). However, exposure of C. difficile to this antibiotic has led to the development of rifaximin-resistance due to point mutations in the β-subunit of the RNA polymerase (rpoB) gene. In the present study, 348 C. difficile strains with known PCR-ribotypes were investigated for respective single nucleotide polymorphisms (SNPs) within the proposed rpoB 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 rpoB section. In the present study, we were able to identify 16 different rpoB 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 rpoB gene of C. difficile is a fast and cost-effective method for the identification of C. difficile samples with reduced susceptibility to rifaximin and even allows simultaneous SNP subtyping of the respective C. difficile isolates.
authors.affiliation: Austrian Agency for Health and Food Safety (AGES), Institute for Medical Microbiology and Hygiene, Waehringerstrasse 25a, A-1090 Vienna, Austria.
authors.forename: Verena
authors.initials: V
authors.lastname: Pecavar
authors.affiliation: Austrian Agency for Health and Food Safety (AGES), Institute for Medical Microbiology and Hygiene, Waehringerstrasse 25a, A-1090 Vienna, Austria.
authors.forename: Marion
authors.initials: M
authors.lastname: Blaschitz
authors.affiliation: Austrian Agency for Health and Food Safety (AGES), Institute for Medical Microbiology and Hygiene, Waehringerstrasse 25a, A-1090 Vienna, Austria.
authors.forename: Peter
authors.initials: P
authors.lastname: Hufnagl
authors.affiliation: Austrian Agency for Health and Food Safety, Institute for Medical Microbiology and Hygiene, Spargelfeldstrasse 191, A-1220 Vienna, Austria.
authors.forename: Josef
authors.initials: J
authors.lastname: Zeinzinger
authors.affiliation: Austrian Agency for Health and Food Safety (AGES), Institute for Medical Microbiology and Hygiene, Waehringerstrasse 25a, A-1090 Vienna, Austria.
authors.forename: Anita
authors.initials: A
authors.lastname: Fiedler
authors.affiliation: Paracelsus Medical University, Institute for Medical Microbiology, Hygiene and Infectious Diseases, Muellner Hauptstrasse 48, A-5020 Salzburg, Austria.
authors.forename: Franz
authors.initials: F
authors.lastname: Allerberger
authors.affiliation: Institut für Laboratoriumsmedizin, Mikrobiologie und Umwelthygiene, Klinikum Augsburg, Stenglinstrasse 2, D-86156 Augsburg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Maass
authors.affiliation: Paracelsus Medical University, Institute for Medical Microbiology, Hygiene and Infectious Diseases, Muellner Hauptstrasse 48, A-5020 Salzburg, Austria.
authors.forename: Alexander
authors.initials: A
authors.lastname: Indra
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D016360:Clostridioides difficile / Q000187:drug effects / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000191:economics / Q000379:methods; D058889:Molecular Typing / Q000191:economics / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D012294:Rifamycins / Q000494:pharmacology*; D000078262:Rifaximin; D017422:Sequence Analysis, DNA; D013997:Time Factors; D044366:Transition Temperature
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D012294:Rifamycins; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D000078262:Rifaximin