Lambert Dominic, Carrillo Catherine D, Koziol Adam G, Manninger Paul, Blais Burton W
Research and Development, Ottawa Laboratory Carling, Science Branch, Canadian Food Inspection Agency, Ottawa, Ontario, Canada.
PLoS One. 2015 Apr 10;10(4):e0122928. doi: 10.1371/journal.pone.0122928. eCollection 2015.
The timely identification and characterization of foodborne bacteria for risk assessment purposes is a key operation in outbreak investigations. Current methods require several days and/or provide low-resolution characterization. Here we describe a whole-genome-sequencing (WGS) approach (GeneSippr) enabling same-day identification of colony isolates recovered from investigative food samples. The identification of colonies of priority Shiga-toxigenic Escherichia coli (STEC) (i.e., serogroups O26, O45, O103, O111, O121, O145 and O157) served as a proof of concept. Genomic DNA was isolated from single colonies and sequencing was conducted on the Illumina MiSeq instrument with raw data sampling from the instrument following 4.5 hrs of sequencing. Modeling experiments indicated that datasets comprised of 21-nt reads representing approximately 4-fold coverage of the genome were sufficient to avoid significant gaps in sequence data. A novel bioinformatic pipeline was used to identify the presence of specific marker genes based on mapping of the short reads to reference sequence libraries, along with the detection of dispersed conserved genomic markers as a quality control metric to assure the validity of the analysis. STEC virulence markers were correctly identified in all isolates tested, and single colonies were identified within 9 hrs. This method has the potential to produce high-resolution characterization of STEC isolates, and whole-genome sequence data generated following the GeneSippr analysis could be used for isolate identification in place of lengthy biochemical characterization and typing methodologies. Significant advantages of this procedure include ease of adaptation to the detection of any gene marker of interest, as well as to the identification of other foodborne pathogens for which genomic markers have been defined.
为进行风险评估而及时鉴定和表征食源细菌是疫情调查中的关键操作。目前的方法需要数天时间且/或提供低分辨率的表征。在此，我们描述了一种全基因组测序（WGS）方法（GeneSippr），可实现从调查性食品样本中回收的菌落分离株的同日鉴定。对优先鉴定的产志贺毒素大肠杆菌（STEC）（即血清型O26、O45、O103、O111、O121、O145和O157）菌落的鉴定作为概念验证。从单个菌落中分离基因组DNA，并在Illumina MiSeq仪器上进行测序，测序4.5小时后从仪器获取原始数据样本。建模实验表明，由代表基因组约4倍覆盖率的21个核苷酸读段组成的数据集足以避免序列数据出现重大缺口。使用一种新颖的生物信息学流程，基于短读段与参考序列文库的比对来鉴定特定标记基因的存在，并检测分散的保守基因组标记作为质量控制指标，以确保分析的有效性。在所有测试的分离株中均正确鉴定出STEC毒力标记，且在9小时内鉴定出单个菌落。该方法有潜力对STEC分离株进行高分辨率表征，并且GeneSippr分析后生成的全基因组序列数据可用于分离株鉴定，以取代冗长的生化表征和分型方法。此程序的显著优点包括易于适用于检测任何感兴趣的基因标记，以及鉴定已定义基因组标记的其他食源性病原体。
