Typing And Heat-resistance Of Bacillus Cereus In The Processing Environment Of Milk Powder

Bacillus cereus sensu lato is Gram-positive,spore-forming bacteria.It is an important foodborne pathogen that can cause food poisoning.The pathogenic bacteria can cause immunocompromised infants with pericarditis,bacteremia,meninges and other complications.It is one of the most important microorganisms affect the quality and safety of powder infant formula(PIF).In this study,samples collected from the processing environment of PIF were the research objects,including the raw materials,environmental air and the surfaces of equipments.MYP agar was used to isolate the suspected colonies of B.cereus in the samples.A total of 84 strains were identified as B.cereus by sequencing analysis,physiological and biochemical tests.The seven housekeeping genes glpF,gmk,ilvD,pta,pur,pycA and tpi were selected as target genes for multilocus sequence typing(MLST).The sequences were analyzed by PCR and were detected to identify the sequence type(ST)of 84 B.cereus strains.It can dissect the profile of different alleles and phylogenetic relationships of B.cereus group,revealing the diversity and population characteristics of B.cereus in the processing environment of PIF.According to the MLST results,STs from different processing areas in PIF factory were determined to trace the pathogenic bacteria,with the correlation of STs and various environmental sources of the pathogenic bacteria.Based on the results and sequencing analysis of panC gene,the heat-resistance of spores from different STs and different phylogenetic groups were investigated.The results were as follows:(1)84 suspected strains of B.cereus group were isolated from the processing environment of PIF.The isolates were identified as B.cereus by 16 S rRNA sequence analysis,panC gene sequence analysis,and biochemical test.(2)84 B.cereus strains were divided into 24 STs by MLST.ST-999(19/84,22.62%),ST-1343(13/84,15.48%),ST-1335(6/84,7.14%)and ST-1345(6/84,7.14%)were the main STs in the processing environment of PIF.The nucleic acid diversity index(?)for seven housekeeping genes ranged from 0.01439(tpi)to 0.07258(ilvD),indicating that tpi gene and ilvD gene showing the lowest and the highest diversity of nucleic acids,respectively.The phylogenetic tree of 2829 bp based on the seven housekeeping genes implicated that 24 STs were mainly divided into two phylogenetic branches.The 84 isolates were more close to the species of B.cereus,B.thuringiensis and B.anthracis.(3)According to the correlation analysis,the packaging process was related to ST-32,the production process were related to ST-1343,ST-1344,ST-1336,and ST-1347,the raw materials,pre-processing,production and packaging process were related to ST-1337,ST-1333,ST-1334 and ST-1335.(4)The spores from different STs showed different heat-resistance by the analysis of D value and Z value.The results indicated the spores from ST-62,ST-1284,ST-144 and ST-1334 were more heat resistant.On the whole,the spores from different groups showed different heat-resistance,and the spores from groups III were more heat-resistant than group II and IV.The understanding of B.cereus strains in the processing environment of PIF was improved,and the theoretical basis was provided to reduce and control the contamination of B.cereus during PIF processing.