Development and validation of a high throughput Neisseria gonorrhoeae genotyping method

Background Neisseria gonorrhoeae genotyping by whole-genome sequencing (WGS) is expensive for a large sample set, a less expensive and more efficient genotyping method is required. We developed a high-throughput genotyping method for N. gonorrhoeae to improve molecular epidemiological typing and antimicrobial-resistant identification in N. gonorrhoeae antimicrobial susceptibility surveillance. Methods We used multiplex-tailed PCR to amplify and sequence 15 alleles from multilocus sequence typing (MLST), N. gonorrhoeae multiantigen sequence typing (NG-MAST), and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR). After indexing-PCR, we sequenced the DNA library using the MiSeq platform (Illumina). Sequencing reads were de novo assembly or constructing consensus sequences of alleles, then assigned sequence type. We used 54 previously characterized strains of N. gonorrhoeae and WGS data to validate our method. Results The allele identification results of MLST and NG-STAR in all strains agreed with the draft WGS. However, in NG-MAST, only 35 strains agreed. Disagreement was found in the NG-MAST of porB in 15 strains and of tbpB in seven strains. QRDR analysis perfectly predicted levofloxacin resistance. But was less successful in predicting reduced susceptibility or resistance phenotype to penicillin G, cefixime, or ceftriaxone using penA, porB, ponA , or mtrR alleles. Conclusions The successful performance in MLST and NG-STAR of our method was validated in this study. This method may be useful for large-scale genotyping for N. gonorrhoeae surveillance in a cost- and labor-saving manner. Phenotypic prediction of antimicrobial susceptibility by combining multiple alleles may be necessary for other than fluoroquinolones. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This research was supported in part by AMED under Grant Numbers JP15fk0108014h0001, JP18fk0108062j0001, and JP21fk0108605j0001. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All sequencing data produced are available under the National Center for Biotechnology Information under BioProject number PRJNA901191.