Kozyreva Varvara K, Crandall John, Sabol Ashley, Poe Alyssa, Zhang Peng, Concepción-Acevedo Jeniffer, Schroeder Morgan N, Wagner Darlene, Higa Jeffrey, Trees Eija, Chaturvedi Vishnu
California Department of Public Health, Richmond, CA, USA.
Centers for Disease Control and Prevention, Atlanta, GA, USA.
PLoS Curr. 2016 Nov 22;8:ecurrents.outbreaks.1bb3e36e74bd5779bc43ac3a8dae52e6. doi: 10.1371/currents.outbreaks.1bb3e36e74bd5779bc43ac3a8dae52e6.
Recently, serovar Poona caused a multistate outbreak, with 245 out of 907 cases occurring in California. We report a comparison of pulsed-field gel electrophoresis (PFGE) results with whole genome sequencing (WGS) for genotyping of Poona isolates.
CA Poona isolates, collected from July to August 2015, were genotyped by PFGE using XbaI restriction enzyme. WGS was done using Nextera XT library kit with 2x300 bp or 2x250 bp sequencing chemistry on the Illumina MiSeq Sequencer. Reads were mapped to the de novo assembled serovar Poona draft genome (48 contigs, N50= 223,917) from the outbreak using CLCbio GW 8.0.2. The phylogenetic tree was generated based on hqSNPs calling. Genomes were annotated with CGE and PHAST online tools. In silico MLST was performed using the CGE online tool.
Human (14) and cucumber (2) Poona isolates exhibited 3 possibly related PFGE patterns (JL6X01.0018 [predominant], JL6X01.0375, JL6X01.0778). All isolates that were related by PFGE also clustered together according to the WGS. One isolate with a divergent PFGE pattern (JL6X01.0776) served as an outlier in the phylogenetic analysis and substantially differed from the outbreak clade by WGS. All outbreak isolates were assigned to MLST sequence type 447. The majority of the outbreak-related isolates possessed the same set of Pathogenicity Islands with few variations. One outbreak isolate was sequenced and analyzed independently by CDC and CDPH laboratories; there was 0 SNP difference in results. Additional two isolates were sequenced by CDC and the raw data was processed through CDPH and CDC analysis pipelines. Both data analysis pipelines also generated concordant results. Discussion: PFGE and WGS results for the recent CA serovar Poona outbreak provided concordant assignment of the isolates to the outbreak cluster. WGS allowed more robust determination of genetic relatedness, provided information regarding MLST-type, pathogenicity genes, and bacteriophage content. WGS data obtained independently at two laboratories showed complete agreement.
最近，波纳血清型引发了一场多州疫情，907例病例中有245例发生在加利福尼亚州。我们报告了用于波纳分离株基因分型的脉冲场凝胶电泳（PFGE）结果与全基因组测序（WGS）的比较。
2015年7月至8月收集的加利福尼亚州波纳分离株，使用XbaI限制性内切酶通过PFGE进行基因分型。使用Nextera XT文库试剂盒，在Illumina MiSeq测序仪上采用2x300 bp或2x250 bp测序化学方法进行全基因组测序。使用CLCbio GW 8.0.2将读数映射到疫情中从头组装的波纳血清型基因组草图（48个重叠群，N50 = 223,917）。基于hqSNP调用生成系统发育树。使用CGE和PHAST在线工具对基因组进行注释。使用CGE在线工具进行计算机辅助多位点序列分型（MLST）。
人类（14株）和黄瓜（2株）波纳分离株呈现出3种可能相关的PFGE模式（JL6X01.0018 [主要模式]、JL6X01.0375、JL6X01.0778）。所有通过PFGE相关的分离株在WGS分析中也聚集在一起。一株具有不同PFGE模式（JL6X01.0776）的分离株在系统发育分析中作为外群，并且通过WGS与疫情分支有显著差异。所有疫情分离株均被指定为MLST序列型447。大多数与疫情相关的分离株具有相同的一套致病岛，仅有少数差异。一株疫情分离株由疾病控制与预防中心（CDC）和加利福尼亚州公共卫生部（CDPH）实验室独立测序和分析；结果中SNP差异为0。另外两株分离株由CDC测序，原始数据通过CDPH和CDC分析流程进行处理。两个数据分析流程也产生了一致的结果。
最近加利福尼亚州波纳血清型疫情的PFGE和WGS结果为将分离株归入疫情集群提供了一致的分类。WGS允许更有力地确定遗传相关性，提供有关MLST类型、致病基因和噬菌体含量的信息。在两个实验室独立获得的WGS数据显示完全一致。
