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ABSTRACT: Antimicrobial use is recognized as a risk factor for Clostridium difficile infection (CDI) and outbreaks. We studied the relationship between PCR ribotype, antimicrobial susceptibility and the genetic basis of resistance in response to exposure to antimicrobial agents. C. difficile isolates were cultured from 133 CDI patients for whom recent antimicrobial drug exposure had been recorded. Isolates were ribotyped by PCR and assessed for their susceptibility to the macrolide-lincosamide-streptogramin B (MLS(B)) group of compounds (erythromycin and clindamycin) and fluoroquinolone antimicrobials (ciprofloxacin, levofloxacin and moxifloxacin). Where relevant, the genetic basis of resistance was determined. Prevalent ribotypes (including 027, 001 and 106) exhibited significantly greater antimicrobial resistance compared with ribotypes 078 and 014, among others. Clindamycin-resistant ribotype 078 was detected for the first time. Ribotypes 027 and 001 were more likely to exhibit MLS(B) resistance, a feature that was associated with the erm(B) gene. Exposure to MLS(B) or fluoroquinolone antimicrobial compounds in the 8 weeks prior to the onset of infection was not associated with specific genetic markers of resistance. Single amino acid substitutions in the A and B subunits of DNA gyrase were noted and were ribotype specific and linked to resistance to moxifloxacin. Resistance to MLS(B) and fluoroquinolone antimicrobial compounds is common among prevalent ribotypes of C. difficile. The genetic basis for antimicrobial resistance appears to be ribotype specific and conserved in the absence of recent antimicrobial selection pressure.