Table_4_Genes Associated With Psychrotolerant Bacillus cereus Group Isolates.DOCX

The Bacillus cereus 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 log10 increase in cfu/mL after 21 days incubation at 6°C) among dairy-associated B. cereus 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 log10 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 B. mycoides/weihenstephanensis). 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 B. cereus 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 B. cereus group isolates.

蜡样芽孢杆菌群（Bacillus cereus group）包含18个不同物种，其中既涵盖人类致病菌，也包含耐冷菌株——这类菌株是引发液态牛乳变质的重要致病因子。为深化我们对乳制品源蜡样芽孢杆菌群分离株中，与耐冷性（本文定义为6℃下培养21天后，菌落形成单位/mL增量>1 log₁₀）相关的遗传标记的认知，我们采用遗传学手段（全基因组测序）与表型分析方法[即在脱脂乳肉汤（Skim Milk Broth, SMB）和脑心浸液肉汤（Brain Heart Infusion, BHI）中进行生长实验]，对503株乳制品源分离株中的23株遗传背景各异的代表性分离株开展了表征分析。经质量阈值聚类分析，菌株被划分为三类耐冷表型：(i) 14株在BHI与SMB中均未表现出耐冷性；(ii) 6株仅在BHI中呈现耐冷性，但在SMB中无耐冷表型；(iii) 2株在BHI与SMB中均表现出耐冷性。另有1株虽在BHI肉汤中表现出耐冷性，但在SMB中的菌落形成单位/mL增量恰好低于>1 log₁₀的临界阈值，未被归入任何聚类类别。基于核心基因组单核苷酸多态性构建的最大似然系统发育树，将所有耐冷菌株（即BHI中表现出耐冷性的菌株）归类至进化枝VI（对应B. mycoides/weihenstephanensis）。对基因同源物存在/缺失模式与耐冷性的相关性分析显示，共有206个同源基因簇在耐冷菌株中显著富集，其中包含2个冷休克蛋白簇，分别在8/9和7/9的耐冷菌株中被检出。基因本体（Gene Ontology, GO）富集分析发现，耐冷菌株中共富集得到36个GO条目，涵盖腐胺分解代谢过程、腐胺跨膜转运活性等功能类别。最后，隐马尔可夫模型（Hidden Markov Model, HMM）检索鉴定出3个蛋白质家族基序，其中冷休克结构域蛋白与脂肪酸羟化酶与BHI肉汤中的耐冷性显著相关。对CspA序列的分析显示，耐冷菌株与此前已报道的“耐冷型”CspA序列存在显著正相关关联。综上，本研究明确了乳制品源蜡样芽孢杆菌群分离株间耐冷性的遗传与表型差异，并证实耐冷性依赖于生长培养基类型。本研究还鉴定出多个可用于特异性检测或防控耐冷蜡样芽孢杆菌群分离株的基因靶点。