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2013年美国与一起与奶酪相关的疫情有关的单核细胞增生李斯特菌分离株的全基因组和核心基因组多位点序列分型及单核苷酸多态性分析

Whole Genome and Core Genome Multilocus Sequence Typing and Single Nucleotide Polymorphism Analyses of Listeria monocytogenes Isolates Associated with an Outbreak Linked to Cheese, United States, 2013.

作者信息

Chen Yi, Luo Yan, Carleton Heather, Timme Ruth, Melka David, Muruvanda Tim, Wang Charles, Kastanis George, Katz Lee S, Turner Lauren, Fritzinger Angela, Moore Terence, Stones Robert, Blankenship Joseph, Salter Monique, Parish Mickey, Hammack Thomas S, Evans Peter S, Tarr Cheryl L, Allard Marc W, Strain Errol A, Brown Eric W

机构信息

Food and Drug Administration, College Park, Maryland, USA

Food and Drug Administration, College Park, Maryland, USA.

出版信息

Appl Environ Microbiol. 2017 Jul 17;83(15). doi: 10.1128/AEM.00633-17. Print 2017 Aug 1.

Abstract

Epidemiological findings of a listeriosis outbreak in 2013 implicated Hispanic-style cheese produced by company A, and pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) were performed on clinical isolates and representative isolates collected from company A cheese and environmental samples during the investigation. The results strengthened the evidence for cheese as the vehicle. Surveillance sampling and WGS 3 months later revealed that the equipment purchased by company B from company A yielded an environmental isolate highly similar to all outbreak isolates. The whole genome and core genome multilocus sequence typing and single nucleotide polymorphism (SNP) analyses results were compared to demonstrate the maximum discriminatory power obtained by using multiple analyses, which were needed to differentiate outbreak-associated isolates from a PFGE-indistinguishable isolate collected in a nonimplicated food source in 2012. This unrelated isolate differed from the outbreak isolates by only 7 to 14 SNPs, and as a result, the minimum spanning tree from the whole genome analyses and certain variant calling approach and phylogenetic algorithm for core genome-based analyses could not provide differentiation between unrelated isolates. Our data also suggest that SNP/allele counts should always be combined with WGS clustering analysis generated by phylogenetically meaningful algorithms on a sufficient number of isolates, and the SNP/allele threshold alone does not provide sufficient evidence to delineate an outbreak. The putative prophages were conserved across all the outbreak isolates. All outbreak isolates belonged to clonal complex 5 and serotype 1/2b and had an identical sequence which did not have premature stop codons. In this outbreak, multiple analytical approaches were used for maximum discriminatory power. A PFGE-matched, epidemiologically unrelated isolate had high genetic similarity to the outbreak-associated isolates, with as few as 7 SNP differences. Therefore, the SNP/allele threshold should not be used as the only evidence to define the scope of an outbreak. It is critical that the SNP/allele counts be complemented by WGS clustering analysis generated by phylogenetically meaningful algorithms to distinguish outbreak-associated isolates from epidemiologically unrelated isolates. Careful selection of a variant calling approach and phylogenetic algorithm is critical for core-genome-based analyses. The whole-genome-based analyses were able to construct the highly resolved phylogeny needed to support the findings of the outbreak investigation. Ultimately, epidemiologic evidence and multiple WGS analyses should be combined to increase confidence levels during outbreak investigations.

摘要

2013年一次李斯特菌病暴发的流行病学调查结果表明,涉事产品为A公司生产的西班牙风味奶酪。在调查过程中,对临床分离株以及从A公司奶酪和环境样本中收集的代表性分离株进行了脉冲场凝胶电泳(PFGE)和全基因组测序(WGS)。结果进一步证明奶酪是传播媒介。3个月后的监测采样和WGS结果显示,B公司从A公司购买的设备产生了一个与所有暴发分离株高度相似的环境分离株。对全基因组和核心基因组多位点序列分型以及单核苷酸多态性(SNP)分析结果进行比较,以证明使用多种分析方法所获得的最大鉴别力,这对于区分暴发相关分离株与2012年在非涉事食品来源中收集的PFGE无法区分的分离株是必要的。这个不相关的分离株与暴发分离株仅相差7至14个SNP,因此,全基因组分析的最小生成树以及基于核心基因组分析的特定变异检测方法和系统发育算法无法区分不相关的分离株。我们的数据还表明,SNP/等位基因计数应始终与基于系统发育有意义算法对足够数量分离株生成的WGS聚类分析相结合,仅靠SNP/等位基因阈值并不能提供足够的证据来划定一次暴发。假定的前噬菌体在所有暴发分离株中都是保守的。所有暴发分离株都属于克隆复合体5和血清型1/2b,并且具有相同的序列,没有提前终止密码子。在这次暴发中,使用了多种分析方法以获得最大鉴别力。一个PFGE匹配但流行病学上不相关的分离株与暴发相关分离株具有高度的遗传相似性,SNP差异少至7个。因此,SNP/等位基因阈值不应作为定义暴发范围的唯一证据。至关重要的是,SNP/等位基因计数应辅以基于系统发育有意义算法生成的WGS聚类分析,以区分暴发相关分离株与流行病学上不相关的分离株。仔细选择变异检测方法和系统发育算法对于基于核心基因组的分析至关重要。基于全基因组的分析能够构建高度解析的系统发育树,以支持暴发调查的结果。最终,在暴发调查期间,应将流行病学证据和多种WGS分析结合起来,以提高可信度。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/44c8/5514676/9d54b2142d63/zam9991179660001.jpg

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