title: Epidemiological typing of Neisseria gonorrhoeae and detection of markers associated with antimicrobial resistance directly from urine samples using next generation sequencing.
abstract: OBJECTIVE
  To investigate the potential for next generation sequencing (NGS) to be used directly on clinical specimens that have tested positive for Neisseria gonorrhoeae by nucleic acid amplification testing (NAAT), to generate information on epidemiological genotyping and antimicrobial resistance (AMR) markers.
  
  
  METHODS
  DNA was extracted from 13 N. gonorrhoeae NAAT-positive urine specimens, enriched for microbial DNA and sequenced using the Ion Torrent PGM workflow. Sequences that aligned to the human genome were filtered out and the remaining sequences were de novo assembled. The resulting contigs were searched for regions of interest using Ridom SeqSphere. MLST and NG-MAST alleles were assigned according to the schemes at PubMLST.org and NG-MAST.net, respectively.
  
  
  RESULTS
  In total, 11 of the 13 samples tested generated a sufficient number of N. gonorrhoeae sequence reads to provide full coverage of the genome at a depth of 6-130×. Complete MLST and NG-MAST sequence types could be generated for each of these samples. The presence of 10 different AMR markers was investigated, and both previously reported and novel mutations were identified in genes associated with reduced susceptibility to several antimicrobials.
  
  
  CONCLUSIONS
  We found that sequencing the entire genome of N. gonorrhoeae directly from clinical samples is possible using NGS, and that multiple levels of N. gonorrhoeae typing information can be generated. As NAAT only testing becomes more common, this method could be used to detect both known and novel mutations associated with AMR and to generate genotyping information, supporting AMR and epidemiological surveillance in the absence of culturing.
authors.affiliation: Public Health Microbiology, Public and Environmental Health, Forensic and Scientific Services, Queensland Department of Health, Brisbane, Queensland, Australia.
authors.forename: R M A
authors.initials: RM
authors.lastname: Graham
authors.affiliation: Public Health Microbiology, Public and Environmental Health, Forensic and Scientific Services, Queensland Department of Health, Brisbane, Queensland, Australia.
authors.forename: C J
authors.initials: CJ
authors.lastname: Doyle
authors.affiliation: Public Health Microbiology, Public and Environmental Health, Forensic and Scientific Services, Queensland Department of Health, Brisbane, Queensland, Australia.
authors.forename: A V
authors.initials: AV
authors.lastname: Jennison
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial / Q000187:drug effects* / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D006069:Gonorrhea / Q000453:epidemiology* / Q000382:microbiology / Q000652:urine*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008826:Microbial Sensitivity Tests; D017720:Molecular Epidemiology; D009344:Neisseria gonorrhoeae / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques
chemical_list: D000900:Anti-Bacterial Agents; D005819:Genetic Markers
keywords: ANTIMICROBIAL RESISTANCE; GONORRHOEA; MOLECULAR EPIDEMIOLOGY; MOLECULAR TECHNIQUES; NEISSERIA GONORRHOEA