Short communication: Postpasteurization hold temperatures of 4 or 6°C, but not raw milk holding of 24 or 72 hours, affect bacterial outgrowth in pasteurized fluid milk.

As fluid milk processors continue to reduce microbial spoilage in fluid milk through improved control of postpasteurization contamination and psychrotolerant sporeformer outgrowth, it is necessary to identify strategies to further improve the quality and extend the shelf life of fluid milk products that are high-temperature, short-time pasteurized. Solutions that optimize product quality, and are economically feasible, are of particular interest to the dairy industry. To this end, this study examined the effects of raw milk holding time and temperature of pasteurized milk storage over shelf life on bacterial growth. In 3 independent replicates, raw milk was stored for 24 and 72 h before pasteurization at 76°C for 25s and then incubated at 3 different storage conditions: (1) 4°C for 21d; (2) 4°C for the first 48 h, then 6°C for the duration of the 21-d shelf life; or (3) 6°C for 21d. Total bacteria counts were assessed initially and on d 7, 14, and 21. No substantial difference in bacterial growth over shelf life was observed between samples processed from raw milk held for 24 versus 72 h. A significantly lower bacterial load was seen at d 21 after pasteurization in samples held at 4°C, versus 4°C for the first 48 h followed by 6°C for the duration of the 21-d shelf life and samples held at 6°C for 21d. This work demonstrates the importance of maintaining control of the fluid milk cold chain throughout postpasteurization, transportation, and retail storage on fluid milk microbial quality.

Genomic comparison of sporeforming bacilli isolated from milk.

BACKGROUND: Sporeformers in the order Bacillales are important contributors to spoilage of pasteurized milk. While only a few Bacillus and Viridibacillus strains can grow in milk at 6°C, the majority of Paenibacillus isolated from pasteurized fluid milk can grow under these conditions. To gain a better understanding of genomic features of these important spoilage organisms and to identify candidate genomic features that may facilitate cold growth in milk, we performed a comparative genomic analysis of selected dairy associated sporeformers representing isolates that can and cannot grow in milk at 6°C. RESULTS: The genomes for seven Paenibacillus spp., two Bacillus spp., and one Viridibacillus sp. isolates were sequenced. Across the genomes sequenced, we identified numerous genes encoding antimicrobial resistance mechanisms, bacteriocins, and pathways for synthesis of non-ribosomal peptide antibiotics. Phylogenetic analysis placed genomes representing Bacillus, Paenibacillus and Viridibacillus into three distinct well supported clades and further classified the Paenibacillus strains characterized here into three distinct clades, including (i) clade I, which contains one strain able to grow at 6°C in skim milk broth and one strain not able to grow under these conditions, (ii) clade II, which contains three strains able to grow at 6°C in skim milk broth, and (iii) clade III, which contains two strains unable to grow under these conditions. While all Paenibacillus genomes were found to include multiple copies of genes encoding ß-galactosidases, clade II strains showed significantly higher numbers of genes encoding these enzymes as compared to clade III strains. Genome comparison of strains able to grow at 6°C and strains unable to grow at this temperature identified numerous genes encoding features that might facilitate the growth of Paenibacillus in milk at 6°C, including peptidases with cold-adapted features (flexibility and disorder regions in the protein structure) and cold-adaptation related proteins (DEAD-box helicases, chaperone DnaJ). CONCLUSIONS: Through a comparative genomics approach we identified a number of genomic features that may relate to the ability of selected Paenibacillus strains to cause spoilage of refrigerated fluid milk. With additional experimental evidence, these data will facilitate identification of targets to detect and control Gram positive spore formers in fluid milk.

Development and Validation of Pathogen Environmental Monitoring Programs for Small Cheese Processing Facilities.

Pathogen environmental monitoring programs (EMPs) are essential for food processing facilities of all sizes that produce ready-to-eat food products exposed to the processing environment. We developed, implemented, and evaluated EMPs targeting Listeria spp. and Salmonella in nine small cheese processing facilities, including seven farmstead facilities. Individual EMPs with monthly sample collection protocols were designed specifically for each facility. Salmonella was detected in only one facility, with likely introduction from the adjacent farm indicated by pulsed-field gel electrophoresis data. Listeria spp. were isolated from all nine facilities during routine sampling. The overall Listeria spp. (other than Listeria monocytogenes ) and L. monocytogenes prevalences in the 4,430 environmental samples collected were 6.03 and 1.35%, respectively. Molecular characterization and subtyping data suggested persistence of a given Listeria spp. strain in seven facilities and persistence of L. monocytogenes in four facilities. To assess routine sampling plans, validation sampling for Listeria spp. was performed in seven facilities after at least 6 months of routine sampling. This validation sampling was performed by independent individuals and included collection of 50 to 150 samples per facility, based on statistical sample size calculations. Two of the facilities had a significantly higher frequency of detection of Listeria spp. during the validation sampling than during routine sampling, whereas two other facilities had significantly lower frequencies of detection. This study provides a model for a science- and statistics-based approach to developing and validating pathogen EMPs.

American origin of Cupriavidus bacteria associated with invasive Mimosa legumes in the Philippines.

To identify the origins of Cupriavidus nodule symbionts associated with two invasive Mimosa species in the Philippines, 22 isolates were sequenced for portions of three chromosomal genes and two symbiotic plasmid loci. Eleven isolates were identical at all gene loci (2713 bp) to a lineage found in Central America. Four other Philippine isolates were identical to a second Cupriavidus lineage distributed both in Central America and in the Caribbean. None of the remaining Philippine strains had more than 0.6% sequence divergence from American Cupriavidus lineages. These results imply that the Philippine population was founded by multiple introductions from the native range of their Mimosa hosts.