Molecular basis of high-level ciprofloxacin resistance in Neisseria gonorrhoeae strains isolated in Denmark from 1995 to 1998

In Denmark surveillance of the in vitro susceptibility to ciprofloxacin of Neisseria gonorrhoeae was established in 1990. The proportion of N. gonorrh oeae strains with decreased susceptibility or resistance to ciprofloxacin ( MIC greater than or equal to 0.06 mug/ml) was low (0.3 to 2.3%) up to 1995. Between 1995 and 1998 the rate of less-susceptible and resistant strains r ose from 6.9 to 13.2%. Among ciprofloxacin-resistant strains (MIC greater t han or equal to 1 mug/ml), 81% were highly resistant (MIC greater than or e qual to 4 mug/ml). Thirty-five N. gonorrhoeae strains (40 isolates) for whi ch ciprofloxacin MICs were 4 to 32 mug/ml were investigated for the frequen cy and patterns of mutations within the gyrA and parC genes. The quinolone resistance-determining regions of the gyrA and parC genes were amplified by PCR, and the amplicons were directly sequenced. Alterations at Ser-91 and Asp-95 in GyrA and a single or double alteration in ParC were identified in 32 strains (91%). Ser-91-to-Phe and Asp-95-to-Gly alterations in GyrA were detected in 28 strains (80%). The most common ParC alteration, Asp-86 to A sn, was found in 19 strains (54%). The strains were analyzed for genetic re lationship by pulsed-field gel electrophoresis (PFGE). The analysis showed that nine strains with the same mutation pattern in the gyrA and parC genes , originating from different geographical areas over 3 years, had the same PFGE patterns after SpeI as well as NheI digestion (only one strain with on e band difference in the NheI pattern), suggesting that a resistant clone h ad spread worldwide. The results from this study strongly suggest that doub le gyrA mutations plus a parC mutation(s) play an important role in the dev elopment of high-level fluoroquinolone resistance in N. gonorrhoeae.