Identification of a novel mutation at the primary dimer interface of GyrA conferring fluoroquinolone resistance in Clostridium difficile

Micheál Mac Aogáin; Shauna Kilkenny; Claire Walsh; Sinéad Lindsay; Geraldine Moloney; Trefor Morris; Sophie Jones; Thomas R Rogers

doi:10.1016/j.jgar.2015.09.007

Identification of a novel mutation at the primary dimer interface of GyrA conferring fluoroquinolone resistance in Clostridium difficile

J Glob Antimicrob Resist. 2015 Dec;3(4):295-299. doi: 10.1016/j.jgar.2015.09.007. Epub 2015 Nov 3.

Authors

Micheál Mac Aogáin¹, Shauna Kilkenny², Claire Walsh², Sinéad Lindsay², Geraldine Moloney², Trefor Morris³, Sophie Jones³, Thomas R Rogers²

Affiliations

¹ Department of Clinical Microbiology, Sir Patrick Dun Translational Research Laboratory, School of Medicine, Trinity College Dublin, Ireland. Electronic address: m.macaogain@tcd.ie.
² Department of Clinical Microbiology, Sir Patrick Dun Translational Research Laboratory, School of Medicine, Trinity College Dublin, Ireland.
³ Anaerobe Reference Laboratory, Public Health Wales, University Hospital of Wales, Cardiff, UK.

PMID: 27842877
DOI: 10.1016/j.jgar.2015.09.007

Abstract

The aim of this study was to determine whether alternative resistance mechanisms, other than mutation in the quinolone resistance-determining region (QRDR) of DNA gyrase, could confer fluoroquinolone resistance in Clostridium difficile. An in vitro-generated C. difficile mutant exhibiting increased fluoroquinolone resistance was isolated through antibiotic selection on ciprofloxacin. The QRDR of this mutant was investigated by chain-termination sequencing and was found to be devoid of mutation. To determine the nature of the non-QRDR resistance mechanism in this strain, the genomes of the mutant and wild-type strains were sequenced. The gyrBA region from a collection of clinical isolates exhibiting variable fluoroquinolone resistance levels was also sequenced and was compared with that present in 918 publicly available C. difficile genomic data sets. Whole-genome sequence analysis of the fluoroquinolone-resistant mutant revealed a single non-synonymous substitution (Ala384Asp) at the predicted primary dimer interface of GyrA, far beyond the classically defined QRDR. This novel mutation caused increased resistance to ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin while conferring hypersusceptibility to novobiocin. Several novel extra-QRDR polymorphisms in C. difficile DNA gyrase were identified among clinical isolates, whilst observed fluoroquinolone resistance in strains devoid of gyrBA mutations confirmed the existence of DNA gyrase-independent resistance mechanisms in this species. In conclusion, we report the first non-QRDR mutation to confer fluoroquinolone resistance in C. difficile. Although the Ala384Asp substitution was not detected in clinical isolates, this study revealed a diversity of alternative extra-QRDR polymorphisms in DNA gyrase whose association with fluoroquinolone resistance warrants further investigation.

Keywords: Clostridium difficile; DNA gyrase; Fluoroquinolone resistance; Genomics.