Real-time PCR genotyping of Neisseria gonorrhoeae isolates using 14 informative single nucleotide polymorphisms on gonococcal housekeeping genes

Abstract

Neisseria gonorrhoeae multilocus sequence typing (MLST) is a key tool used to investigate the macroepidemiology of gonococci exhibiting antimicrobial resistance (AMR). However, the utility of MLST is undermined by the high workload and cost associated with DNA sequencing of seven housekeeping genes. In this study, we investigated single nucleotide polymorphism (SNP)-based profiling as a means of circumventing these problems. A total of 14 SNPs were selected following in silico analysis of available N. gonorrhoeae MLST sequence data. Real-time PCR methods were developed for characterization of each SNP and applied to 86 N. gonorrhoeae isolates exhibiting a range of ceftriaxone MICs. Twenty-one isolates had previously been characterized by MLST. The ability of the real-time PCR methods to generate SNP profiles and of the 14 SNP profiles to predict MLST types were assessed. In silico analysis of the 217 different MLST types available on the Neisseria web site showed 181 different 14 SNP profiles (Simpson's index of diversity = 0.998). When the real-time PCR methods were applied to the isolates, 29 different 14 SNP profiles were obtained for 83 isolates. Predicted MLST types were consistent with those for the 21 isolates previously characterized by MLST. For 46 isolates with raised ceftriaxone MICs (≥ 0.03 mg/L), there were 14 different 14 SNP profiles observed, with two profiles accounting for more than half of these isolates. The 14 SNP real-time PCR profiling approach is a simple and cost-effective alternative to N. gonorrhoeae MLST and could be used to complement current typing schemes in N. gonorrhoeae AMR investigations.