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.

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 ß-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.

Real-time PCR for single-nucleotide polymorphism detection in the 16S rRNA gene as an indicator for extensive drug resistance in Mycobacterium tuberculosis.

OBJECTIVES: A real-time PCR screening system was established for rapid detection of single-nucleotide polymorphisms (SNPs) at positions 1401, 1402 and 1484 of the 16S rRNA gene of Mycobacterium tuberculosis leading to resistance to amikacin, kanamycin and capreomycin. Resistances to the respective drugs may indicate the presence of an extensively drug-resistant (XDR) strain of M. tuberculosis. METHODS: Fifty-seven M. tuberculosis isolates that tested phenotypically susceptible or resistant to amikacin, capreomycin or both were subjected to 1401-2/1484 real-time PCR to screen for SNPs in the respective rrs region. RESULTS: 1401-2 and 1484 wild-type and mutant M. tuberculosis strains displayed distinct melting peaks. Of the cross-resistant strains, 86.7% displayed A1401G SNPs, 76.9% of amikacin-resistant strains did not display rrs SNPs and one capreomycin-resistant strain showed a C1402T SNP. CONCLUSIONS: Phenotypic drug susceptibility testing takes several weeks, but with the 1401-2/1484 real-time PCR a preliminary diagnosis can be made within a few hours. SNPs in the rrs region are not exclusively involved in the development of resistances to amikacin and capreomycin. However, 80.0% of XDR-tuberculosis samples tested were detected with the real-time PCR screening assay of the present study.

Rifaximin disc diffusion test for in vitro susceptibility testing of Clostridium difficile.

Rifaximin is a rifampicin derivative, poorly absorbed by the gastro-intestinal tract. We studied the in vitro susceptibility to rifamixin of 1082 Clostridium difficile isolates; among these, 184 isolates from a strain collection were tested by an in-house rifaximin disc (40 µg) diffusion test, by an in-house rifaximin broth microdilution test, by rifampicin Etest and by rpoB gene sequencing. In the absence of respective CLSI or EUCAST MIC breakpoints for rifaximin and rifampicin against C. difficile we chose MIC ≥32 µg ml(-1) as criterion for reduced in vitro susceptibility. To further validate the disc diffusion test 898 consecutive clinical isolates were analysed using the disc diffusion test, the Etest and rpoB gene sequence analysis for all resistant strains. Rifaximin broth microdilution tests of the 184 reference strains yielded rifaximin MICs ranging from 0.001 (n = 1) to ≥1024 µg ml(-1) (n = 61); 62 isolates showed a reduced susceptibility (MIC ≥32 µg ml(-1)). All of these 62 strains showed rpoB gene mutations producing amino acid substitutions; the rifampicin- and rifaximin-susceptible strains showed either a wild-type sequence or silent amino acid substitutions (19 strains). For 11 arbitrarily chosen isolates with rifaximin MICs of >1024 µg ml(-1), rifaximin end-point MICs were determined by broth dilution: 4096 µg ml(-1) (n = 2), 8192 µg ml(-1) (n = 6), 16,384 µg ml(-1) (n = 2) and 32,678 µg ml(-1) (n = 1). Rifampicin Etests on the 184 C. difficile reference strains yielded MICs ranging from ≤0.002 (n = 117) to ≥32 µg ml(-1) (n = 59). Using a 38 mm inhibition zone as breakpoint for reduced susceptibility the use of rifaximin disc diffusion yielded 59 results correlating with those obtained by use of rifaximin broth microdilution in 98.4 % of the 184 strains tested. Rifampicin Etests performed on the 898 clinical isolates revealed that 67 isolates had MICs of ≥32 µg ml(-1). There were no discordant results observed among these isolates with reduced susceptibility using an MIC of ≥32 µg ml(-1) as breakpoint for reduced rifampicin susceptibility and a <38 mm inhibition zone as breakpoint for reduced rifaximin susceptibility. The prevalence of reduced susceptibility was 7.5 % for all isolates tested. However, for PCR ribotype 027 the prevalence of reduced susceptibility was 26 %. Susceptibility testing in the microbiology laboratory therefore could have an impact on the care and outcome of patients with infection. Our results show that rifaximin--despite its water-insolubility--may be a suitable candidate for disc diffusion testing.