Objectives:

The increasing use of fluoroquinolones for the treatment of bacterial respiratory infections has led to the emergence of S. pyogenes isolates with resistance to this class of antibiotics. In this study, we determined the mechanisms of ciprofloxacin resistance and the emm-types associated to ciprofloxacin resistance in Spain.

Methods:

S. pyogenes pharyngeal isolates belonged to the SAUCE (standing for Susceptibility to the Antimicrobials Used Commonly in ‘España’ and is the Spanish word for the willow tree) national surveillance collection (1998–99). Sequence analysis of specific PCR products was used to deduce emm-types, and to detect mutational alterations in the QRDRs of gyrA and parC. Sequencing was done with the Big Dye terminator mix and autosequenator (Applied Biosystems). DNA sequences were subjected to homology searches against the bacterial DNA database. MICs to ciprofloxacin were determined with and without reserpine following standard procedures. Genetic diversity of the S. pyogenes ciprofloxacin resistant isolates was studied by pulse field gel electrophoresis of genomic DNA restricted with SfiI.

Results:

All the ciprofloxacin resistant S. pyogenes belonging to the SAUCE collection were analysed (n = 27). We found point mutations at Ser79 and/or at Asp91 of parC that were replaced in all the strains by Ala and Asn, respectively. By contrast, we did not identify mutations in the gyrA gene in any isolate. In addition, MIC of ciprofloxacin for these isolates (4–8 mg/mL) was not influenced by the presence of reserpine. 66.6 % of the resistant isolates were M type 6 and the rest expressed different M types. PFGE analysis revealed that 50% of the M type 6 had an identical restriction pattern. By contrast we identified up to 12 different PFGE patterns among the rest of the ciprofloxacin resistant strains.

Conclusions:

Reduced susceptibility of S. pyogenes to ciprofloxacin in Spain is essentially caused by point mutations of parC present mainly in a single M type 6 clone.