Clostridium difficile isolates resistant to fluoroquinolones in Italy: emergence of PCR ribotype 018

Abstract

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.

FIG. 1.

gyr gene melting curve analysis in a representative number of C. difficile clinical isolates analyzed in this study. (A) For the gyrA gene, isolates showing the substitution Thr82 to Ile had a T_m of 52.2°C, whereas isolates showing the wild-type codon in position 82 had a T_m of 59.3°C. (B) For the gyrB gene, isolates showing the substitution Asp426 to Asn had a T_m of 52.8°C, whereas those with the wild-type gene had a T_m of 58.3°C.

FIG. 2.

Dendrogram obtained from the analysis of the representative C. difficile PCR ribotype patterns identified in this study. Similarity analysis was performed with Pearson's correlation and clustering by the unweighted pair group mean association (UPGMA) method. Two international reference strains belonging to PCR ribotypes 027 and 017 were used as control strains in the analysis.