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

Abstract

Objectives: 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.Methods: 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.Results: 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 diversity¼ 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.Conclusions: 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.

A new Multi Locus Variable Number of Tandem Repeat Analysis Scheme for epidemiological surveillance of Xanthomonas vasicola pv. musacearum, the plant pathogen causing bacterial wilt on banana and enset

“…Due to their different evolutionary dynamics, MLVAs and SNPs offer complementary information [65]. MLVA is considered suitable for short-term epidemiological analysis, while SNPs are suited to long-term or global epidemiological analyses [66].…”

Genomic epidemiology and population structure of Neisseria gonorrhoeae from remote highly endemic Western Australian populations

“…Among WHO GASP countries, isolates with decreased susceptibility or resistance to the extended-spectrum cephalosporins (ESCs) and isolates with resistance to azithromycin have been reported in 51% and 75% of these countries respectively [6]. To understand the epidemiology underlying the appearance and spread of antimicrobial resistance (AMR) in gonococci, several molecular approaches such as whole genome sequencing (WGS), multilocus sequence typing (MLST), N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and most recently N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) have been developed [7–9]. Resistance to ESCs has spread primarily through clonal expansion and is highly correlated with the presence of the penA mosaic allele in NG-MAST sequence type (ST)1407, MLST STs 1901 and 7363 and NG-STAR ST90 clusters [8, 10, 11].…”

Mini-Multilocus Sequence Typing Scheme for the Global Population of Neisseria gonorrhoeae

“…This extension could facilitate the genotyping of gonococci in the future. Whiley et al and Trembizki et al previously analyzed local samples of N. gonorrhoeae isolates using 14 informative polymorphisms [26,27]. The samples were examined through RT-PCR and the MassARRAY iPLEX platform [26,27].…”

“…Whiley et al demonstrated the feasibility of predicting the MLST genotype of N. gonorrhoeae using a small number of informative single-nucleotide polymorphisms (SNPs) in the corresponding genes [ 26 ]. Using real-time PCR and high-resolution melting curve analysis, 14 informative polymorphisms in abcZ , adk , aroE , fumC , gdh , pdhC , and pgm genes were identified [ 26 ]. After completing the amplification process, a profile of informative SNPs was obtained for the isolates.…”