Mechanisms of resistance to ciprofloxacin in Neisseria gonorrhoeae

Abstract: A few years ago, most strains of Neisseria gonorrhoeae were susceptible to ciprofloxacin, but after introduction as a first line therapy, resistant strains emerged. Ciprofloxacin inhibit two enzymes necessary for DNA replication. Mutations in the quinolone resistance determining regions (QRDR) of genes encoding the subunit GyrA of the target enzyme DNA gyrase and ParC of topoisomerase IV are mechanisms of resistance in N. gonorrhoeae. These alterations do not explain why the minimum inhibitory concentrations (MICs) of ciprofloxacin in resistant strains vary so widely. The target enzymes also contain the subunits GyrB and ParE. Their role and other mechanisms of resistance in N. gonorrhoeae, such as increased efflux out of the cell, decreased uptake or competition of quinolone binding sites by protection, have not been fully investigated in N. gonorrhoeae. Several commercial kits for molecular diagnosis of N. gonorrhoeae are available and can be analyzed in a duplex PCR with Chlamydia trachomatis, but these methods are known to produce false positive results. Results obtained with AMPLICOR N. gonorrhoeae polymerase chain reaction (PCR) (Roche Diagnostics) were compared to cultivation results in 956 samples. In positive samples species verification of the 16S rRNA gene was compared to pyrosequencing of QRDR the gyrA gene, which was also evaluated as an indicator of ciprofloxacin susceptibility. Culture and the molecular method verified in gyrA produced two and one false negative result respectively and the molecular method verified in 16S rRNA produced four false positive results. QRDR of all eleven urine samples positive in AMPLICOR N. gonorrhoeae PCR, with corresponding isolates as well as 46 N. gonorrhoeae strains, were correctly diagnosed according to susceptibility to ciprofloxacin compared to MICs. Pyrosequencing of QRDR of gyrA of 40 isolates of nine other Neisseria spp. showed that QRDR in gyrA is not unique for N. gonorrhoeae. Sequencing of QRDR of gyrA, gyrB, parC, and parE in 25 highly ciprofloxacin resistant and five susceptible strains of N. gonorrhoeae, showed that all the resistant strains had two mutations in gyrA. Fourteen strains also had an additional mutation in parC, and 17 strains had an additional mutation in parE. No alterations were found in gyrB in any strain. In transformation experiments an alteration in GyrA was introduced in a ciprofloxacin susceptible N. gonorrhoeae strain (MIC 0.008 mg/L) and MIC increased to 0.064 mg/L. Two alterations, together, increased MIC to 0. 125 - 0.25 mg/L. Introduction of alterations in major outer membrane porin, PorB1b, and probably other alterations, in a moderately ciprofloxacin resistant strain (MIC 0.25 mg/L) gave transformants with MICs of ciprofloxacin 0.5-16 mg/L. In one transformant an alteration in ParE was also introduced. We conclude that verification of a molecular method by pyrosequencing in gyrA gene is superior to verification by PCR in 16S rRNA. The gene gyrA is not unique for N. gonorrhoeae. However, whether this region is possible to use also for verification also depends on the specificity of the primary method. QRDR of gyrA is a strong indicator of ciprofloxacin resistance in N. gonorrhoeae. Two alterations in gyrA only increases MIC of ciprofloxacin to 0. 125 - 0.25 mg/L. Additional alterations in QRDR of parC and parE as well as alterations in porB1b also contribute to ciprofloxacin resistance.