Detection of presumptive Bacillus cereus in the Irish dairy farm environment

Abstract

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.