Graham R M A, Doyle C J, Jennison A V
Public Health Microbiology, Public and Environmental Health, Forensic and Scientific Services, Queensland Department of Health, Brisbane, Queensland, Australia.
Sex Transm Infect. 2017 Feb;93(1):65-67. doi: 10.1136/sextrans-2015-052422. Epub 2016 Mar 11.
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.
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.
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.
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.
研究通过核酸扩增检测（NAAT）淋病奈瑟菌检测呈阳性的临床标本直接进行下一代测序（NGS）以生成有关流行病学基因分型和抗菌药物耐药性（AMR）标志物信息的潜力。
从13份淋病奈瑟菌NAAT阳性尿液标本中提取DNA，富集微生物DNA并使用Ion Torrent PGM工作流程进行测序。过滤掉与人类基因组比对的序列，其余序列进行从头组装。使用Ridom SeqSphere搜索所得重叠群中的感兴趣区域。分别根据PubMLST.org和NG-MAST.net上的方案指定MLST和NG-MAST等位基因。
总共，13个测试样本中有11个产生了足够数量的淋病奈瑟菌序列读数，以6 - 130倍的深度提供基因组的全覆盖。可以为每个样本生成完整的MLST和NG-MAST序列类型。研究了10种不同AMR标志物的存在情况，并在与几种抗菌药物敏感性降低相关的基因中鉴定出先前报道的和新的突变。
我们发现使用NGS直接从临床样本中对淋病奈瑟菌的全基因组进行测序是可行的，并且可以生成多个水平的淋病奈瑟菌分型信息。随着仅NAAT检测变得更加普遍，这种方法可用于检测与AMR相关的已知和新突变，并生成基因分型信息，在不进行培养的情况下支持AMR和流行病学监测。
