Clostridium difficile Isolates Resistant to Fluoroquinolones in Italy: Emergence of PCR Ribotype 018

Recent evidence strongly suggests an association between the use of fluoroquinolones and Clostridium difficile infection (CDI). Resistance to fluoroquinolones has been described not only in the hypervirulent strain 027, but also in other important PCR ribotypes circulating in hospital settings. In a European prospective study conducted in 2005, strains resistant to moxifloxacin represented 37.5% of C. difficile clinical isolates. In this study, we investigated a sample of 147 toxigenic C. difficile isolates, collected in Italy from 1985 to 2008, for the presence of mutations in gyr genes that conferred resistance to fluoroquinolones based on a LightCycler assay. Results were confirmed by the determination of MICs for moxifloxacin. Strains resistant to moxifloxacin were also investigated for resistance to three other fluoroquinolones and for a possible association between fluoroquinolone and macrolide-lincosamide-streptogramin B resistance. C. difficile isolates were typed by PCR ribotyping. In total, 50 clinical isolates showed substitutions in gyr genes and were resistant to fluoroquinolones. Ninety-six percent of the C. difficile resistant isolates showed the substitution Thr82-to-Ile in GyrA, as already observed in the majority of resistant strains worldwide. A significant increase of resistance (P < 0.001) was observed in the period 2002 to 2008 (56% resistant) compared to the period 1985 to 2001 (10% resistant). Coresistance with erythromycin and/or clindamycin was found in 96% (48/50) of the isolates analyzed and, interestingly, 84% of resistant strains were erm(B) negative. The majority of the fluoroquinolone-resistant isolates belonged to PCR ribotype 126 or 018. PCR ribotype 126 was the most frequently found from 2002 to 2005, whereas PCR ribotype 018 was predominant in 2007 and 2008 and still represents the majority of strains typed in our laboratory. Overall, the results demonstrate an increasing number of C. difficile strains resistant to fluoroquinolones in Italy and changes in the prevalence and type of C. difficile isolates resistant to fluoroquinolones circulating over time.Clostridium difficile is an anaerobic, Gram-positive, spore-forming bacillus that may cause a spectrum of diseases, ranging from uncomplicated mild diarrhea to pseudomembranous colitis, collectively known as C. difficile infections (CDIs) (3, 39). The risk of CDI appears to be greater with certain antimicrobial agents and increases if strains are resistant to administered antimicrobials (30).Recently, the new hypervirulent C. difficile strain, typed as PCR ribotype 027, toxinotype III, pulsed-field gel electrophoresis pattern NAP1, has been associated with more severe and fatal cases in the United States, Canada, Japan, and Europe (4, 16, 18, 20, 21, 22). Strain 027 isolates are characterized by a hyperproduction of toxins A and B, production of binary toxin, and resistance to erythromycin and fluoroquinolones (12, 18, 34). Resistance to these antibiotics characterizes not only strain 027 but the majority of C. difficile strains circulating in hospital settings and responsible for disease (2, 34).The use of macrolide-lincosamide-streptogramin B (MLSB) antibiotics has long been known to be one of the major risk factors for CDI (15, 25). With C. difficile, resistance to these antibiotics, in particular to erythromycin and clindamycin, is due to an erm(B) gene carried by Tn5398, a mobile element that shows heterogeneous genetic organization. Nevertheless, an increased number of C. difficile erm(B)-negative isolates resistant to MLSB has been described, including strains characterized as PCR ribotype 027 (1, 19).Historically, fluoroquinolones were considered a low risk in C. difficile diseases, but recent evidence strongly suggests an association between their use and CDI (6, 24, 26). Resistance to fluoroquinolones has been described not only in the epidemic strain 027 but also in other important PCR ribotypes, and it is increasing, since recent data have shown that resistant strains represent 37.5% of C. difficile clinical isolates in Europe (2). Two main mechanisms of fluoroquinolone resistance have been described: alterations in the targets of antibiotics, DNA gyrase and topoisomerase IV, and decreased accumulation inside bacteria (13, 29). In C. difficile, as in many other bacterial species, resistance is determined by amino acid substitutions in the quinolone resistance-determining region (QRDR) of the target enzymes (27). Since this bacterium does not have genes for topoisomerase IV, these alterations are located in the QRDR of either GyrA or GyrB, the DNA gyrase subunits (11, 34). Recent studies indicated that the replacement of Thr82 with Ile in GyrA characterized 93% of European toxigenic C. difficile isolates resistant to fluoroquinolones, including the hypervirulent epidemic clone 027/NAP1/III (12, 34). The remaining 7% showed a substitution in position 426 (Asp to Asn or Val) of GyrB (34).Few data on fluoroquinolone resistance of C. difficile clinical isolates are available for Italy (34) and, for this reason, we analyzed a convenient sample of toxigenic isolates, collected by the Istituto Superiore di Sanità (ISS) from 1985 to 2008, for substitutions in GyrA and GyrB and for their MIC values to moxifloxacin. All C. difficile resistant strains were also tested for their resistance to ciprofloxacin, gatifloxacin, and levofloxacin and investigated for a possible association between fluoroquinolone and MLSB resistance. Typing of resistant isolates was carried out using the already-described PCR ribotyping method (5).