gyrA Mutations in Fluoroquinolone-resistant Clostridium difficile PCR-027

gyrA Mutations in Fluoroquinolone-resistant Clostridium difficile PCR-027 To the Editor: Clostridium diffi-cile is the most common cause of bacterial diarrhea in hospitalized patients (1). Antimicrobial drug therapy is the most important risk factor associated with the acquistion of C. difficile, and several antimicrobial agents including clindamycin, amoxicillin, and cepha-lo-sporins have been particularly associated with C. difficile infection (2). Acquisition of resistance to clin-damycin is considered 1 mechanism whereby clonal strains emerge and predominate in healthcare environments (3). Historically, fluoro-quinolone antimicrobial agents were considered low risk for C. difficile– associated-disease; however, recent studies indicate a shift in the risk associated with their use (4). Furthermore, recent outbreaks in Canada and the United States have been associated with fluoroquinolone exposure (4). Recently, several C. difficile outbreaks due to PCR ribotype 027 (PCR-027) and associated with increased disease severity and death have been reported worldwide (4). This strain type contains the genes for binary toxin and has an 18-bp deletion and a frameshift mutation in tcdC hypothesized to result in deregulated expression of toxins A and B. These strains produce 16× more toxin A and 23× more toxin B in vitro than toxino-type 0 strains (5). These isolates demonstrate universal high-level resistance to fluoroquinolones in contrast to that of PCR 027 isolates collected before 2001 (4). We report the mechanism of fluo-roquinolone resistance in a cluster (n = 5) of Irish PCR-027 C. difficile isolates that were characterized by using toxinotyping and 16–23S ribotyping. Amplification with PCR and sequenc-ing was used to identify the binary toxin gene (cdtB) and an 18-bp deletion and a frameshift mutation at position 117 in the tcdC gene. Anti-microbial susceptibility to 5 fluoro-quinolone antimicrobial drugs was determined with E-tests (AB-Biodisk, Solna, Sweden). The quinolone-resist-ance–determining region (QRDR) of gyrA and gyrB was amplified by PCR and characterized. The nucleotide sequence data for partial sequences of the gyrA gene were submitted to GenBank and assigned accession nos. DQ821481, DQ821482, DQ821483, and DQ821484. PCR ribotyping profiles identified 1 cluster of C. difficile PCR-027 with clinical isolates that showed indistinguishable profiles to the control 027 strain. PCR identified the cdtB, an 18-bp deletion, and a frameshift mutation at position 117 in the tcdC gene in all 5 isolates. These strains were universally resistant to the fluoroquinolones tested (ofloxacin, ciprofloxacin, levo-floxacin, moxifloxacin, and gatiflo-xacin, respectively, MIC >32 µg/mL [Table]). Control isolates were susceptible to moxifloxacin and gatifloxacin (MICs 0.3, 0.2 µg/mL, respectively); however, …