David J. Kent

Evaluation of dairy powder products implicates thermophilic sporeformers as the primary organisms of interest

Dairy powder products (e.g., sweet whey, nonfat dry milk, acid whey, and whey protein concentrate-80) are of economic interest to the dairy industry. According to the US Dairy Export Council, customers have set strict tolerances (<500 to <1,000/g) for thermophilic and mesophilic spores in dairy powders; therefore, understanding proliferation and survival of sporeforming organisms within dairy powder processing plants is necessary to control and reduce sporeformer counts. Raw, work-in-process, and finished product samples were collected from 4 dairy powder processing facilities in the northeastern United States over a 1-yr period. Two separate spore treatments: (1) 80°C for 12min (to detect sporeformers) and (2) 100°C for 30min (to detect highly heat resistant sporeformers) were applied to samples before microbiological analyses. Raw material, work-in-process, and finished product samples were analyzed for thermophilic, mesophilic, and psychrotolerant sporeformers, with 77.5, 71.0, and 4.6% of samples being positive for those organisms, respectively. Work-in-process and finished product samples were also analyzed for highly heat resistant thermophilic and mesophilic sporeformers, with 63.7 and 42.6% of samples being positive, respectively. Sporeformer prevalence and counts varied considerably by product and plant; sweet whey and nonfat dry milk showed a higher prevalence of thermophilic and mesophilic sporeformers compared with acid whey and whey protein concentrate-80. Unlike previous reports, we found limited evidence for increased spore counts toward the end of processing runs. Our data provide important insight into spore contamination patterns associated with production of different types of dairy powders and support that thermophilic sporeformers are the primary organism of concern in dairy powders.

Spore populations among bulk tank raw milk and dairy powders are significantly different.

To accommodate stringent spore limits mandated for the export of dairy powders, a more thorough understanding of the spore species present will be necessary to develop prospective strategies to identify and reduce sources (i.e., raw materials or in-plant) of contamination. We characterized 1,523 spore isolates obtained from bulk tank raw milk (n=33 farms) and samples collected from 4 different dairy powder-processing plants producing acid whey, nonfat dry milk, sweet whey, or whey protein concentrate 80. The spores isolated comprised 12 genera, at least 44 species, and 216 rpoB allelic types. Bacillus and Geobacillus represented the most commonly isolated spore genera (approximately 68.9 and 12.1%, respectively, of all spore isolates). Whereas Bacillus licheniformis was isolated from samples collected from all plants and farms, Geobacillus spp. were isolated from samples from 3 out of 4 plants and just 1 out of 33 farms. We found significant differences between the spore population isolated from bulk tank raw milk and those isolated from dairy powder plant samples, except samples from the plant producing acid whey. A comparison of spore species isolated from raw materials and finished powders showed that although certain species, such as B. licheniformis, were found in both raw and finished product samples, other species, such as Geobacillus spp. and Anoxybacillus spp., were more frequently isolated from finished powders. Importantly, we found that 8 out of 12 genera were isolated from at least 2 different spore count methods, suggesting that some spore count methods may provide redundant information if used in parallel. Together, our results suggest that (1) Bacillus and Geobacillus are the predominant spore contaminants in a variety of dairy powders, implying that future research efforts targeted at elucidating approaches to reduce levels of spores in dairy powders should focus on controlling levels of spore isolates from these genera; and (2) the spore populations isolated from bulk tank raw milk and some dairy powder products are significantly different, suggesting that targeting in-plant sources of contamination may be important for achieving low spore counts in the finished product. These data provide important insight regarding the diversity of spore populations isolated from dairy powders and bulk tank raw milk, and demonstrate that several spore genera are detected by multiple spore count methods.

Different management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk

Bacterial endospores (also referred to as spores) present in raw milk are capable of surviving pasteurization and other adverse conditions encountered during dairy powder production. Therefore, requiring low spore levels in raw ingredients (e.g., raw milk) may be necessary for producing dairy powders with low spore counts. To identify potential associations between management practices and spore levels in raw milk, we sampled bulk tank raw milk from 33 farms throughout New York State every other month for 1yr. Following spore pasteurization (80°C for 12min), samples were incubated at 3 different temperatures to enumerate psychrotolerant (6°C for 10 d), mesophilic (32°C for 48h), and thermophilic (55°C for 48h) spores. An additional enrichment procedure was used to detect spores present at low levels (<10 spores/mL). Overall, psychrotolerant, mesophilic, and thermophilic spores were detected (at levels ≥10 spores/mL) in 1, 74, and 58% of bulk tank raw milk samples, respectively. Although thermophilic spore levels could not be quantified (due to bacterial swarming), mesophilic spore levels ranged from below detection (<10 spores/mL) to 680 spores/mL. Data collected through surveys were used to identify management practices associated with either mesophilic or thermophilic spore levels. We found that different management practices are associated with mesophilic and thermophilic spore levels. Low mesophilic spore levels in bulk tank raw milk samples were associated with (1) large herd size, (2) use of sawdust or sand bedding, and (3) not fore stripping during the premilking routine. Management practices that were associated with lower odds of having a thermophilic spore level ≥10 spores/mL are (1) large herd size, (2) spray-based application of the postmilking disinfectant, (3) dry massaging the udder during the premilking routine, and (4) the use of straw bedding. Collectively, these results suggest that different management practices may influence mesophilic and thermophilic spore levels in raw milk.

Bacillus wiedmannii sp. nov., a psychrotolerant and cytotoxic Bacillus cereus group species isolated from dairy foods and dairy environments.

A facultatively anaerobic, spore-forming Bacillus strain, FSL W8-0169T, collected from raw milk stored in a silo at a dairy powder processing plant in the north-eastern USA was initially identified as a Bacillus cereus group species based on a partial sequence of the rpoB gene and 16S rRNA gene sequence. Analysis of core genome single nucleotide polymorphisms clustered this strain separately from known B. cereus group species. Pairwise average nucleotide identity blast values obtained for FSL W8-0169T compared to the type strains of existing B. cereus group species were <95 % and predicted DNA-DNA hybridization values were <70 %, suggesting that this strain represents a novel B. cereus group species. We characterized 10 additional strains with the same or closely related rpoB allelic type, by whole genome sequencing and phenotypic analyses. Phenotypic characterization identified a higher content of iso-C16 : 0 fatty acid and the combined inability to ferment sucrose or to hydrolyse arginine as the key characteristics differentiating FSL W8-0169T from other B. cereus group species. FSL W8-0169T is psychrotolerant, produces haemolysin BL and non-haemolytic enterotoxin, and is cytotoxic in a HeLa cell model. The name Bacillus wiedmannii sp. nov. is proposed for the novel species represented by the type strain FSL W8-0169T (=DSM 102050T=LMG 29269T).

Coliform detection in cheese is associated with specific cheese characteristics, but no association was found with pathogen detection

Coliform detection in finished products, including cheese, has traditionally been used to indicate whether a given product has been manufactured under unsanitary conditions. As our understanding of the diversity of coliforms has improved, it is necessary to assess whether coliforms are a good indicator organism and whether coliform detection in cheese is associated with the presence of pathogens. The objective of this study was (1) to evaluate cheese available on the market for presence of coliforms and key pathogens, and (2) to characterize the coliforms present to assess their likely sources and public health relevance. A total of 273 cheese samples were tested for presence of coliforms and for Salmonella, Staphylococcus aureus, Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and other Listeria species. Among all tested cheese samples, 27% (75/273) tested positive for coliforms in concentrations >10cfu/g. Pasteurization, pH, water activity, milk type, and rind type were factors significantly associated with detection of coliforms in cheese; for example, a higher coliform prevalence was detected in raw milk cheeses (42% with >10cfu/g) compared with pasteurized milk cheese (21%). For cheese samples contaminated with coliforms, only water activity was significantly associated with coliform concentration. Coliforms isolated from cheese samples were classified into 13 different genera, including the environmental coliform genera Hafnia, Raoultella, and Serratia, which represent the 3 genera most frequently isolated across all cheeses. Escherichia, Hafnia, and Enterobacter were significantly more common among raw milk cheeses. Based on sequencing of the housekeeping gene clpX, most Escherichia isolates were confirmed as members of fecal commensal clades of E. coli. All cheese samples tested negative for Salmonella, Staph. aureus, and Shiga toxin-producing E. coli. Listeria spp. were found in 12 cheese samples, including 5 samples positive for L. monocytogenes. Although no association was found between coliform and Listeria spp. detection, Listeria spp. were significantly more likely to be detected in cheese with the washed type of rind. Our data provide information on specific risk factors for pathogen detection in cheese, which will facilitate development of risk-based strategies to control microbial food safety hazards in cheese, and suggest that generic coliform testing cannot be used to assess the safety of natural cheese.

Peroxide test strips detect added hydrogen peroxide in raw milk at levels affecting bacterial load.

Hydrogen peroxide (H2O2) has a long-established history of use as a preservative in milk worldwide. The use of H2O2 to activate the inherent lactoperoxidase enzyme system has dramatically improved the quality of raw dairy products in areas in which cooling is not widely available. In the United States, however, where refrigeration is widely available, the addition of H2O2 to milk is not permitted, with the exception of certain applications prior to cheesemaking and during the preparation of modified whey. Due to the relatively quick deterioration of H2O2 in fluid milk, the detection of raw milk adulterated with the compound can be challenging. In this study we evaluated (i) total aerobic bacterial counts and (ii) ability of peroxide test strips to detect H2O2 in raw milk with various concentrations (0, 100, 300, 500, 700, and 900 ppm) of added H2O2, incubated at both 6 and 21°C for 0, 24, and 48 h. Results showed that at both 6 and 21°C the H2O2 concentration and time had a significant effect on bacterial loads in raw milk. Additionally, commercially available test strips were able to detect H2O2 in raw milk, with predicted probability of >90%, immediately after addition and after 24 and 48 h for the higher concentrations used, offering a viable method for detecting raw milk adulteration with H2O2.

Spore test parameters matter: Mesophilic and thermophilic spore counts detected in raw milk and dairy powders differ significantly by test method

United States dairy industry exports have steadily risen in importance over the last 10yr, with dairy powders playing a particularly critical role. Currently, approximately half of US-produced nonfat dry milk and skim milk powder is exported. Reaching new and expanding existing export markets relies in part on the control of endospore-forming bacteria in dairy powders. This study reports baseline mesophilic and thermophilic spore counts and spore populations from 55 raw material samples (primarily raw milk) and 33 dairy powder samples from dairy powder processors across the United States. Samples were evaluated using various spore testing methodologies and included initial heat treatments of (1) 80°C for 12 min; (2) 100°C for 30 min; and (3) 106°C for 30 min. Results indicate that significant differences in both the level and population of spores were found for both raw milk and dairy powders with the various testing methods. Additionally, on average, spore counts were not found to increase significantly from the beginning to the end of dairy powder processing, most likely related to the absence of biofilm formation by processing plant-associated sporeformers (e.g., Anoxybacillus sp.) in the facilities sampled. Finally, in agreement with other studies, Bacillus licheniformis was found to be the most prevalent sporeformer in both raw materials and dairy powders, highlighting the importance of this organism in developing strategies for control and reduction of spore counts in dairy powders. Overall, this study emphasizes the need for standardization of spore enumeration methodologies in the dairy powder industry.

Whole-Genome Sequencing of Drug-Resistant Salmonella enterica Isolates from Dairy Cattle and Humans in New York and Washington States Reveals Source and Geographic Associations

ABSTRACT Multidrug-resistant (MDR) Salmonella enterica can be spread from cattle to humans through direct contact with animals shedding Salmonella as well as through the food chain, making MDR Salmonella a serious threat to human health. The objective of this study was to use whole-genome sequencing to compare antimicrobial-resistant (AMR) Salmonella enterica serovars Typhimurium, Newport, and Dublin isolated from dairy cattle and humans in Washington State and New York State at the genotypic and phenotypic levels. A total of 90 isolates were selected for the study (37 S. Typhimurium, 32 S. Newport, and 21 S. Dublin isolates). All isolates were tested for phenotypic antibiotic resistance to 12 drugs using Kirby-Bauer disk diffusion. AMR genes were detected in the assembled genome of each isolate using nucleotide BLAST and ARG-ANNOT. Genotypic prediction of phenotypic resistance resulted in a mean sensitivity of 97.2 and specificity of 85.2. Sulfamethoxazole-trimethoprim resistance was observed only in human isolates (P < 0.05), while resistance to quinolones and fluoroquinolones was observed only in 6 S. Typhimurium isolates from humans in Washington State. S. Newport isolates showed a high degree of AMR profile similarity, regardless of source. S. Dublin isolates from New York State differed from those from Washington State based on the presence/absence of plasmid replicons, as well as phenotypic AMR susceptibility/nonsusceptibility (P < 0.05). The results of this study suggest that distinct factors may contribute to the emergence and dispersal of AMR S. enterica in humans and farm animals in different regions. IMPORTANCE The use of antibiotics in food-producing animals has been hypothesized to select for AMR Salmonella enterica and associated AMR determinants, which can be transferred to humans through different routes. Previous studies have sought to assess the degree to which AMR livestock- and human-associated Salmonella strains overlap, as well as the spatial distribution of Salmonellas associated AMR determinants, but have often been limited by the degree of resolution at which isolates can be compared. Here, a comparative genomics study of livestock- and human-associated Salmonella strains from different regions of the United States shows that while many AMR genes and phenotypes were confined to human isolates, overlaps between the resistomes of bovine and human-associated Salmonella isolates were observed on numerous occasions, particularly for S. Newport. We have also shown that whole-genome sequencing can be used to reliably predict phenotypic resistance across Salmonella isolated from bovine sources.

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.

Temperature Significantly Affects the Plaquing and Adsorption Efficiencies of Listeria Phages.

Listeria-infecting phages are currently being used to control and detect the important foodborne pathogen Listeria monocytogenes; however, the influence of environmental conditions on the interactions between L. monocytogenes and its phages has not been explored in depth. Here, we examined the infective potential of four Listeria phages (two each from the P70-like and P100-like phages of Listeria) against five strains of L. monocytogenes (representing serotypes 1/2a, 1/2b, 4a, and 4b) grown under a range of temperatures (7–37°C). We show that the plaquing efficiencies for all four phages were significantly affected by temperature. Interestingly, no plaques were observed for any of the four phages at 37°C. Adsorption assays performed with the P100-like phages, LP-048 and LP-125, showed that LP-048 had a severely reduced adsorption efficiency against susceptible strains at 37°C as compared to 30°C, suggesting that there is considerably less accessible rhamnose (LP-048’s putative phage receptor) on the host at 37°C than at 30°C. LP-125 adsorbed to host cells at 37°C, indicating that the inability for LP-125 to plaque at 37°C is not due to adsorption inhibition. LP-048 showed significantly higher adsorption efficiency against a mutant strain lacking N-acetylglucosamine in its wall teichoic acids (WTA) than the parental strain at both 30 and 37°C, suggesting that N-acetylglucosamine competes with rhamnose for glycosylation sites on the WTA. The data presented here clearly shows that L. monocytogenes can gain physiological refuge from phage infection, which should be carefully considered for both the design and implementation of phage-based control and detection applications.