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

摘要

Pseudomonas spp. are important spoilage bacteriathat negatively affect the quality of refrigerated fluidmilk and uncultured cheese by generating unwantedodors, flavors, and pigments. They are frequently foundin dairy plant environments and enter dairy productspredominantly as postpasteurization contaminants.Current subtyping and characterization methods fordairy-associated Pseudomonas are often labor-intensiveand expensive or provide limited and possibly unreliableclassification information (e.g., to the species level).Our goal was to identify a single-copy gene that couldbe analyzed in dairy spoilage-associated Pseudomonasfor preliminary species-level identification, subtyping,and phenotype prediction. We tested 7 genes previouslytargeted in a Pseudomonas fluorescens multilocussequence typing scheme for their individual suitabilityin this application using a set of 113 Pseudomonas spp.isolates representing the diversity of typical pasteurizedmilk contamination. For each of the 7 candidategenes, we determined the success rate of PCR andsequencing for these 113 isolates as well as the level ofdiscrimination for species identification and subtypingthat the sequence data provided. Using these metrics,we selected a single gene, isoleucyl tRNA synthetase(ileS), which had the most suitable traits for simpleand affordable single-gene Pseudomonas characterization.This was based on the number of isolates successfullysequenced for ileS (113/113), the number ofunique allelic types assigned (83, compared with 50 for16S rDNA), nucleotide and sequence diversity measures(e.g., number of unique SNP and Simpson index), andtests for genetic recombination. The discriminatoryability of ileS sequencing was confirmed by separationof 99 additional dairy Pseudomonas spp. isolates, whichwere indistinguishable by 16S rDNA sequencing, into28 different ileS allelic types. Further, we used wholegenomesequencing data to demonstrate the similaritiesin ileS-based phylogenetic clustering to whole-genomebasedclustering for 27 closely related dairy-associatedPseudomonas spp. isolates and for 178 Pseudomonastype strains. We also found that dairy-associated Pseudomonaswithin an ileS cluster typically shared thesame proteolytic and lipolytic activities. Use of ileSsequencing provides a promising strategy for affordableinitial characterization of Pseudomonas isolates, whichwill help the dairy industry identify, characterize, andtrack Pseudomonas in their facilities and products.