Resistance to moxifloxacin in toxigenic Clostridium difficile isolates is associated with mutations in gyrA

Abstract

Clostridium difficile is the etiological agent of antibiotic-associated colitis and the most common cause of hospital-acquired infectious diarrhea. Fluoroquinolones such as ciprofloxacin are associated with lower risks of C. difficile-associated diarrhea. In this study, we have analyzed 72 C. difficile isolates obtained from patients with different clinical courses of disease, such as toxic megacolon and relapses; the hospital environment; public places; and horses. They were investigated for their susceptibilities to moxifloxacin (MXF), metronidazole (MEO), and vancomycin (VAN). Mutants highly resistant to fluoroquinolones were selected in vitro by stepwise exposure to increasing concentrations of MXF. The resulting mutants were analyzed for the presence of mutations in the quinolone resistance-determining regions of DNA gyrase (gyrA), the production of toxins A and B, and the epidemiological relationship of these isolates. These factors were also investigated using PCR-based methods. All strains tested were susceptible to MEO and VAN. Twenty-six percent of the clinical isolates (19 of 72) were highly resistant to MXF (MIC > or = 16 microg/ml). Fourteen of these 19 strains contained nucleotide changes resulting in amino acid substitutions at position 83 in the gyrA protein. Resistant strains selected in vitro did not contain mutations at that position. These findings indicate that resistance to MXF in a majority of cases may be due to amino acid substitution in the gyrA gene.

FIG. 1

QRDR nucleotide sequence of the amplified fragment of the gyrA gene of C. difficile ATCC 17857, 1542 and 1001 and a part of the published sequence for Staphylococcus epidermidis (St. epi.). Italics indicate the wild-type sequence at amino acid position 83; boldface indicates nucleotide mutation leading to amino acid substitution; E. coli coordinates are given for amino acid sequence.

FIG. 2

Genotyping of C. difficile isolates by AP-PCR with a T-7 primer. Lane 11, 123 bp-ladder; lanes 1 to 10 and 12 to 20, AP-PCR banding patterns of MXF-resistant isolates. Lanes and isolates: 1, 2932; 2, 1542; 3, 2897; 4, 2518; 5, 1111/1; 6, 1111/2; 7, 1009; 8, 1002; 9, 1001; 10, 1000; 12, E 214; 13, E 213; 14, E 511; 15, E 510; 16, E 498; 17, E 497; 18, E 496; 19, E 287; and 20, E 239. AP-PCR types are indicated by numbers beneath the lanes.