Beno Sarah M, Orsi Renato H, Cheng Rachel A, Kent David J, Kovac Jasna, Duncan Diana R, Martin Nicole H, Wiedmann Martin
Department of Food Science, Cornell University, Ithaca, NY, United States.
Department of Food Science, Penn State University, University Park, PA, United States.
Front Microbiol. 2019 Mar 29;10:662. doi: 10.3389/fmicb.2019.00662. eCollection 2019.
The group comprises 18 different species, including human pathogens as well as psychrotolerant strains that are an important cause of fluid milk spoilage. To enhance our understanding of the genetic markers associated with psychrotolerance (defined here as > 1 log increase in cfu/mL after 21 days incubation at 6°C) among dairy-associated group isolates, we used genetic (whole genome sequencing) and phenotypic methods [growth in Skim Milk Broth (SMB) and Brain Heart Infusion (BHI) broth] to characterize 23 genetically-distinct representative isolates from a collection of 503 dairy-associated isolates. Quality threshold clustering identified three categories of psychrotolerance: (i) 14 isolates that were not psychrotolerant in BHI or SMB, (ii) 6 isolates that were psychrotolerant in BHI but not in SMB, and (iii) 2 isolates that were psychrotolerant in BHI and SMB. One isolate, which was psychrotolerant in BHI broth but was just below the cut-off of >1 log cfu/mL increase in SMB was not assigned to a cluster. A maximum likelihood phylogeny constructed with core genome single nucleotide polymorphisms classified all psychrotolerant isolates (i.e., psychrotolerant in BHI) into clade VI (representing ). Analysis of correlations between gene ortholog presence or absence patterns and psychrotolerance identified 206 orthologous gene clusters that were significantly overrepresented among psychrotolerant strains, including two clusters of cold shock proteins, which were identified in 8/9 and 7/9 psychrotolerant isolates. Gene ontology analyses revealed 36 gene ontology terms that were overrepresented in psychrotolerant isolates, including putrescine catabolic processes and putrescine transmembrane transporter activity. Lastly, Hidden Markov Model searches identified three protein family motifs, including cold shock domain proteins and fatty acid hydroxylases that were significantly associated with psychrotolerance in BHI broth. Analyses of CspA sequences revealed a positive association between psychrotolerant strains and a previously identified "psychrotolerant" CspA sequence. Overall, our data highlight genetic and phenotypic differences in psychrotolerance among group dairy-associated isolates and show that psychrotolerance is dependent on the growth medium. We also identified a number of gene targets that could be used for specific detection or control of psychrotolerant group isolates.
该菌群由18个不同的物种组成，包括人类病原体以及嗜冷菌株，嗜冷菌株是液态奶变质的一个重要原因。为了增进我们对与乳制品相关菌群分离株中嗜冷性（此处定义为在6°C下培养21天后cfu/mL增加>1 log）相关的遗传标记的理解，我们使用了遗传学方法（全基因组测序）和表型方法[在脱脂乳肉汤（SMB）和脑心浸液（BHI）肉汤中生长]来对来自503株乳制品相关分离株集合中的23株遗传上不同的代表性分离株进行表征。质量阈值聚类确定了三类嗜冷性：（i）14株在BHI或SMB中不嗜冷的分离株，（ii）6株在BHI中嗜冷但在SMB中不嗜冷的分离株，以及（iii）2株在BHI和SMB中嗜冷的分离株。有一株在BHI肉汤中嗜冷但在SMB中仅略低于>1 log cfu/mL增加的临界值的分离株未被归入某一聚类。用核心基因组单核苷酸多态性构建的最大似然系统发育树将所有嗜冷分离株（即在BHI中嗜冷的分离株）归入进化枝VI（代表 ）。对基因直系同源物存在或缺失模式与嗜冷性之间的相关性分析确定了206个直系同源基因簇在嗜冷菌株中显著富集，包括两簇冷休克蛋白，在8/9和7/9的嗜冷分离株中被鉴定出来。基因本体分析揭示了36个在嗜冷分离株中富集的基因本体术语，包括腐胺分解代谢过程和腐胺跨膜转运蛋白活性。最后，隐马尔可夫模型搜索确定了三个蛋白质家族基序，包括冷休克结构域蛋白和脂肪酸羟化酶，它们与BHI肉汤中的嗜冷性显著相关。对CspA序列的分析揭示了嗜冷菌株与先前鉴定的“嗜冷”CspA序列之间存在正相关。总体而言，我们的数据突出了乳制品相关菌群分离株在嗜冷性方面的遗传和表型差异，并表明嗜冷性取决于生长培养基。我们还鉴定了一些可用于特异性检测或控制嗜冷性菌群分离株的基因靶点。
