• Detection of presumptive Bacillus cereus in the Irish dairy farm environment

      O'Connell, Aine; Lawton, Elaine M.; Leong, Dara; Cotter, Paul D.; Gleeson, David E; Guinane, Caitriona M.; Teagasc Walsh Fellowship Programme (Teagasc (Agriculture and Food Development Authority), Ireland, 30/01/2016)
      The objective of the study was to isolate potential Bacillus cereus sensu lato (B. cereus s.l.) from a range of farm environments. Samples of tap water, milking equipment rinse water, milk sediment filter, grass, soil and bulk tank milk were collected from 63 farms. In addition, milk liners were swabbed at the start and the end of milking, and swabs were taken from cows’ teats prior to milking. The samples were plated on mannitol egg yolk polymyxin agar (MYP) and presumptive B. cereus s.l. colonies were isolated and stored in nutrient broth with 20% glycerol and frozen at -80 °C. These isolates were then plated on chromogenic medium (BACARA) and colonies identified as presumptive B. cereus s.l. on this medium were subjected to 16S ribosomal RNA (rRNA) sequencing. Of the 507 isolates presumed to be B. cereus s.l. on the basis of growth on MYP, only 177 showed growth typical of B. cereus s.l. on BACARA agar. The use of 16S rRNA sequencing to identify isolates that grew on BACARA confirmed that the majority of isolates belonged to B. cereus s.l. A total of 81 of the 98 isolates sequenced were tentatively identified as presumptive B. cereus s.l. Pulsed-field gel electrophoresis was carried out on milk and soil isolates from seven farms that were identified as having presumptive B. cereus s.l. No pulsotype was shared by isolates from soil and milk on the same farm. Presumptive B. cereus s.l. was widely distributed within the dairy farm environment.
    • Tracking the Dairy Microbiota from Farm Bulk Tank to Skimmed Milk Powder

      McHugh, Aoife; Feehily, Conor; Fenelon, Mark; Gleeson, David E; Hill, Colin; Cotter, Paul D.; Department of Agriculture, Food and the Marine; Science Foundation Ireland; European Union; 14/F/883; et al. (American Society for Microbiology, 2020-04-07)
      Microorganisms from the environment can enter the dairy supply chain at multiple stages, including production, milk collection, and processing, with potential implications for quality and safety. The ability to track these microorganisms can be greatly enhanced by the use of high-throughput DNA sequencing (HTS). Here HTS, both 16S rRNA gene amplicon and shotgun metagenomic sequencing were applied to investigate the microbiomes of fresh mid- and late-lactation milk collected from farm bulk tanks, collection tankers, milk silos, skimmed milk silos, a cream silo, and powder samples to investigate the microbial changes throughout a skim milk powder manufacturing process. 16S rRNA gene analysis established that the microbiota of raw milks from farm bulk tanks and in collection tankers were very diverse but that psychrotrophic genera associated with spoilage, Pseudomonas and Acinetobacter, were present in all samples. Upon storage within the whole-milk silo at the processing facility, the species Pseudomonas fluorescens and Acinetobacter baumannii became dominant. The skimmed milk powder generated during the mid-lactation period had a microbial composition that was very different from that of raw milk; specifically, two thermophilic genera, Thermus and Geobacillus, were enriched. In contrast, the microbiota of skimmed milk powder generated from late-lactation milk more closely resembled that of the raw milk and was dominated by spoilage-associated psychrotrophic bacteria. This study demonstrates that the dairy microbiota can differ significantly across different sampling days. More specifically, HTS can be used to trace microbial species from raw milks through processing to final powdered products.
    • Tracking the Dairy Microbiota from Farm Bulk Tank to Skimmed Milk Powder.

      McHugh, Aoife; Feehily, Conor; Fenelon, Mark; Gleeson, David E; Hill, Colin; Cotter, Paul D.; Department of Agriculture, Food and the Marine; Science Foundation Ireland; European Union; 14/F/883; et al. (American Society for Microbiology, 2020-04-07)
      Microorganisms from the environment can enter the dairy supply chain at multiple stages, including production, milk collection, and processing, with potential implications for quality and safety. The ability to track these microorganisms can be greatly enhanced by the use of high-throughput DNA sequencing (HTS). Here HTS, both 16S rRNA gene amplicon and shotgun metagenomic sequencing were applied to investigate the microbiomes of fresh mid- and late-lactation milk collected from farm bulk tanks, collection tankers, milk silos, skimmed milk silos, a cream silo, and powder samples to investigate the microbial changes throughout a skim milk powder manufacturing process. 16S rRNA gene analysis established that the microbiota of raw milks from farm bulk tanks and in collection tankers were very diverse but that psychrotrophic genera associated with spoilage, Pseudomonas and Acinetobacter, were present in all samples. Upon storage within the whole-milk silo at the processing facility, the species Pseudomonas fluorescens and Acinetobacter baumannii became dominant. The skimmed milk powder generated during the mid-lactation period had a microbial composition that was very different from that of raw milk; specifically, two thermophilic genera, Thermus and Geobacillus, were enriched. In contrast, the microbiota of skimmed milk powder generated from late-lactation milk more closely resembled that of the raw milk and was dominated by spoilage-associated psychrotrophic bacteria. This study demonstrates that the dairy microbiota can differ significantly across different sampling days. More specifically, HTS can be used to trace microbial species from raw milks through processing to final powdered products.IMPORTANCE Microorganisms can enter and persist in dairy at several stages of the processing chain. Detection of microorganisms within dairy food processing is currently a time-consuming and often inaccurate process. This study provides evidence that high-throughput sequencing can be used as an effective tool to accurately identify microorganisms along the processing chain. In addition, it demonstrates that the populations of microbes change from raw milk to the end product. Routine implementation of high-throughput sequencing would elucidate the factors that influence population dynamics. This will enable a manufacturer to adopt control measures specific to each stage of processing and respond in an effective manner, which would ultimately lead to increased food safety and quality.