Milk Quality Improvement Program, Department of Food Science, Cornell University, Ithaca, NY 14853.
Milk Quality Improvement Program, Department of Food Science, Cornell University, Ithaca, NY 14853.
J Dairy Sci. 2021 Mar;104(3):2668-2683. doi: 10.3168/jds.2020-19283. Epub 2021 Jan 15.
Pseudomonas spp. are important spoilage bacteria that negatively affect the quality of refrigerated fluid milk and uncultured cheese by generating unwanted odors, flavors, and pigments. They are frequently found in dairy plant environments and enter dairy products predominantly as postpasteurization contaminants. Current subtyping and characterization methods for dairy-associated Pseudomonas are often labor-intensive and expensive or provide limited and possibly unreliable classification information (e.g., to the species level). Our goal was to identify a single-copy gene that could be analyzed in dairy spoilage-associated Pseudomonas for preliminary species-level identification, subtyping, and phenotype prediction. We tested 7 genes previously targeted in a Pseudomonas fluorescens multilocus sequence typing scheme for their individual suitability in this application using a set of 113 Pseudomonas spp. isolates representing the diversity of typical pasteurized milk contamination. For each of the 7 candidate genes, we determined the success rate of PCR and sequencing for these 113 isolates as well as the level of discrimination for species identification and subtyping that the sequence data provided. Using these metrics, we selected a single gene, isoleucyl tRNA synthetase (ileS), which had the most suitable traits for simple and affordable single-gene Pseudomonas characterization. This was based on the number of isolates successfully sequenced for ileS (113/113), the number of unique allelic types assigned (83, compared with 50 for 16S rDNA), nucleotide and sequence diversity measures (e.g., number of unique SNP and Simpson index), and tests for genetic recombination. The discriminatory ability of ileS sequencing was confirmed by separation of 99 additional dairy Pseudomonas spp. isolates, which were indistinguishable by 16S rDNA sequencing, into 28 different ileS allelic types. Further, we used whole-genome sequencing data to demonstrate the similarities in ileS-based phylogenetic clustering to whole-genome-based clustering for 27 closely related dairy-associated Pseudomonas spp. isolates and for 178 Pseudomonas type strains. We also found that dairy-associated Pseudomonas within an ileS cluster typically shared the same proteolytic and lipolytic activities. Use of ileS sequencing provides a promising strategy for affordable initial characterization of Pseudomonas isolates, which will help the dairy industry identify, characterize, and track Pseudomonas in their facilities and products.
铜绿假单胞菌是一种重要的腐败菌，会产生不良气味、味道和色素，从而降低冷藏液态奶和未培养奶酪的质量。它们经常在乳制品厂环境中被发现，并主要作为巴氏杀菌后的污染物进入乳制品。目前用于乳制品相关铜绿假单胞菌的亚分型和特征描述方法通常劳动强度大、成本高，或者提供的分类信息有限且可能不可靠（例如，仅到种的水平）。我们的目标是鉴定一个单拷贝基因，以便在乳制品腐败相关的铜绿假单胞菌中进行初步的种水平鉴定、亚分型和表型预测。我们使用了一组 113 株代表典型巴氏杀菌奶污染的铜绿假单胞菌分离株，测试了先前在荧光假单胞菌多位点序列分型方案中靶向的 7 个基因，以确定它们在该应用中的单独适用性。对于这 7 个候选基因中的每一个，我们确定了 113 个分离株中 PCR 和测序的成功率，以及序列数据提供的种鉴定和亚分型的区分水平。使用这些指标，我们选择了一个单基因，即异亮氨酸 tRNA 合成酶（ileS），它具有最适合简单且经济实惠的单基因铜绿假单胞菌特征描述的特性。这是基于成功测序 ileS 的分离株数量（113/113）、分配的独特等位基因类型数量（83，而 16S rDNA 为 50）、核苷酸和序列多样性衡量标准（例如，独特 SNP 的数量和 Simpson 指数）以及遗传重组测试。通过将 99 株无法通过 16S rDNA 测序区分的额外乳制品铜绿假单胞菌分离株分离成 28 种不同的 ileS 等位基因类型，证实了 ileS 测序的区分能力。此外，我们使用全基因组测序数据证明了基于 ileS 的系统发育聚类与基于全基因组的聚类对于 27 株密切相关的乳制品相关铜绿假单胞菌分离株和 178 株铜绿假单胞菌模式株的相似性。我们还发现，ileS 聚类中的乳制品相关铜绿假单胞菌通常具有相同的蛋白水解和脂肪水解活性。使用 ileS 测序为经济实惠的铜绿假单胞菌分离株初始特征描述提供了一种有前景的策略，这将有助于乳制品行业识别、特征描述和跟踪其设施和产品中的铜绿假单胞菌。
