Virulence, UHT survival, and biofilms in Bacillus cereus and other Bacillus spp.in milk

摘要

We isolated a Bacillus amyloliquefaciens strain present in ultra-high temperature (UHT) pasteurized organic whole milk in order to ascertain virulence determinants present in this species,and potential for biofilm formation compared to the mesophilic type strain Bacillus cereus ATCC 14579.The overall goal of this project was to genotypically and phenotypically characterize thermoduric Bacillus arnyloliquefaciens virulence and biofiim potential,including the presence of the global regulator effector PIcR,when cultures are sublethally-stressed by growth in subinhibitory concentrations of carvacrol,an antimicrobial extract from essential oil of oregano that has been shown to increase virulence of Bacillus spp.in other contexts.Recovery of bacteria from milk necessitated a nonselective enrichment in Brain Heart Infusion (BHI) broth,incubated aerobically (shaking) for ＞24h,after which time samples were serially diluted and spread-plated onto Tryptic Soy Agar (TSA) plates to recover Bacillus spp.Resulting colonies were streak plated onto TSA to ensure purity of culture before catalase testing,Gram and spore-staining to presumptively identify to the genus level.Pure cultures were biochemically identified to the species level using the Microgen Bacillus ID system (Hardy Diagnostics),and validated further using fatty acid profiling and 16S rDNA sequencing.In order to confirm presence of the target virulence and regulator genes in each UHT milk isolate,DNA was extracted from TSB-grown pure cultures during late log phase.Quantified DNA template was used in real-time (SYBR Green-based) PCR with primers specific for each of the target genes: plcR(encoding a pleiotropic extracellular virulence factor regulator),codY (encoding another global effector protein),nheA and hblC (encoding enterotoxins),and the16S rRNA gene is a control.PCR indicated that plcR,codY,nheA,hblC genes were all present in the Bacillus amyloliquefaciensisolate.This was determined by comparing mean melting temperatures for each PCR product.Both B.cereus ATCC14579 andB.amyloliquefaciens produce biofilms,with the former producing 10％ more relative biofilm material,with a slight (but not significantly different) decrease upon exposure of cultures to subinhibitorycarvacrol.The significance of this project will be to determine if parameters regarding shipment,storage and shelf life of UHT organic milk should be revisited,in order to ensure quality before consumption of product that may harbor thermoduric toxigenic Bacillus spp.