Identification, subtyping, and tracking of dairy spoilage-associated Pseudomonas by sequencing the ileS gene

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.