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.

Interventions designed to control postpasteurization contamination in high-temperature, short-time-pasteurized fluid milk processing facilities: A case study on the effect of employee training, clean-in-place chemical modification, and preventive maintenance programs.

Postpasteurization contamination (PPC) with gram-negative bacteria adversely affects the quality and shelf-life of milk through the development of flavor, odor, texture, and visual defects. Through evaluation of milk quality at 4 large fluid milk processing facilities in the northeast United States, we examined the efficacy of 3 strategies designed to reduce the occurrence of PPC in fluid milk: (1) employee training (focusing on good manufacturing practices) alone and (2) with concurrent implementation of modified clean-in-place chemistry and (3) preventive maintenance (PM) focused on replacement of wearable rubber components. Despite increases in employee knowledge and self-reported behavior change, microbiological evaluation of fluid milk before and after interventions indicated that neither training alone nor training combined with modified clean-in-place interventions significantly decreased PPC. Furthermore, characterization of gram-negative bacterial isolates from milk suggested that specific bacterial taxonomic groups (notably, Pseudomonas sequence types) continued to contribute to PPC even after interventions and that no major changes in the composition of the spoilage-associated microbial populations occurred as a consequence of the interventions. More specifically, in 3 of 4 facilities, gram-negative bacteria with identical 16S rDNA sequence types were isolated on multiple occasions. Evaluation of a PM intervention showed that used rubber goods harbored PPC-associated bacteria and that PPC may have been less frequent following a PM intervention in which wearable rubber goods were replaced (reduction from 3/3 samples with PPC before to 1/3 samples after). Overall, our findings suggest that commonly used "broad stroke interventions" may have a limited effect on reducing PPC. Our case study also demonstrates the inherent complexities of identifying and successfully addressing sanitation problems in large and complex fluid milk processing facilities. For example, broad changes to sanitation practices without improvements in PM and sanitary equipment design may not always lead to reduced PPC. Our data also indicate that although short-term evaluations, such as pre- and post-tests for employee training, may suggest improvements after corrective and preventive actions, extensive microbial testing, ideally in combination with isolate characterization, may be necessary to evaluate return on investment of different interventions.

Pseudomonas fluorescens group bacterial strains are responsible for repeat and sporadic postpasteurization contamination and reduced fluid milk shelf life.

Postpasteurization contamination (PPC) of high temperature, short time-pasteurized fluid milk by gram-negative (GN) bacteria continues to be an issue for processors. To improve PPC control, a better understanding of PPC patterns in dairy processing facilities over time and across equipment is needed. We thus collected samples from 10 fluid milk processing facilities to (1) detect and characterize PPC patterns over time, (2) determine the efficacy of different media to detect PPC, and (3) characterize sensory defects associated with PPC. Specifically, we collected 280 samples of high temperature, short time-pasteurized milk representing different products (2%, skim, and chocolate) and different fillers over 4 samplings performed over 11 mo at each of the 10 facilities. Standard plate count (SPC) as well as total GN, coliform, and Enterobacteriaceae (EB) counts were performed upon receipt and after 7, 10, 14, 17, and 21 d of storage at 6°C. We used 16S rDNA sequencing to characterize representative bacterial isolates from (1) test days with SPC >20,000 cfu/mL and (2) all samples with presumptive GN, coliforms, or EB. Day-21 samples were also evaluated by a trained defect judging panel. By d 21, 226 samples had SPC >20,000 cfu/mL on at least 1 d of shelf life; GN bacteria were found in 132 of these 226 samples, indicating PPC. Crystal violet tetrazolium agar detected PPC with the greatest sensitivity. Spoilage due to PPC was predominantly associated with Pseudomonas (isolated from 101 of the 132 samples with PPC); coliforms and EB were found in 27 and 37 samples with spoilage due to PPC, respectively. Detection of Pseudomonas and Acinetobacter was associated with lower flavor scores; coagulated, fruity fermented, and unclean defects were more prevalent in d-21 samples with PPC. Repeat isolation of Pseudomonas fluorescens group strains with identical partial 16S rDNA sequence types was observed in 8 facilities. In several facilities, specific lines, products, or processing days were linked to repeat product contamination with Pseudomonas with identical sequence types. Our data show that PPC due to Pseudomonas remains a major challenge for fluid milk processors; the inability of coliform and EB tests to detect Pseudomonas may contribute to this. Our data also provide important initial insights into PPC patterns (e.g., line-specific contamination), supporting the importance of molecular subtyping methods for identification of PPC sources.