Research On The Virulence Genes And Antibiotic Resistance Phenotypes Of Bacillus Cereus Detected From Fermented Bean Curd In Beijing

Fermented bean curd is a traditional fermented soybean product in China.It has unique flavor,rich nutrition and delicate texture,so many consumers like fermented bean curd very much.However,the opportunistic pathogen of Bacillus cereus has been reported in some kinds of these products in recent years,which may result in the damage of the consumers' health.Therefore,it is vital to detect Bacillus cereus in fermented bean curd,master virulence genes and antibiotic resistance phenotypes of Bacillus cereus.In this paper,an identification method for Bacillus cereus in food by matrix assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS)was established.Based on the method developed,Bacillus cereus in fermented bean curd was detected and identified.At the same time,Bacillus cereus isolates were typed by MALDI-TOF-MS.Finally,the virulence genes and antibiotic resistance phenotypes of Bacillus cereus were detected.Contents and results of the research are as follows:(1)An identification method for Bacillus cereus by MALDI-TOF-MS was established.This study optimized sample handling methods,focusing on Bacillus cereus type strain,and 17 standard strains of Bacillus cereus were used to supplement a MALDI-TOF-MS database,the sensitivity,stability,accuracy of the method were evaluated.The results showed that the optimal sample handling method of Bacillus cereus was formic acid treatment method,the credibility of identification results of Bacillus cereus was improved by supplementing the database.Based on these conditions,the MALDI-TOF-MS method showed excellent accuracy,stability and appropriate sensitivity(104 cfu).The identification method for Bacillus cereus by MALDI-TOF-MS is accurate,rapid,and it can provide technical support for identification and typing of Bacillus cereus in food.(2)Bacillus cereus in fermented bean curd was isolated,identified and typed.112 samples of fermented bean curd from different brands,producing areas,kinds,batches were selected;the national standard method was used for isolation,identification and counting of Bacillus cereus in fermented bean curd,and MALDI-TOF-MS was used for typing of Bacillus cereus.The results showed that 204 strains of Bacillus cereus were isolated from fermented bean curd,and 100%fermented bean curd was contaminated by Bacillus cereus with considerable level over 103 CFU/g;when the distance was 0.9,204 strains of Bacillus cereus were divided into four types byMALDI-TOF-MS,and the Bacillus cereus isolated from fermented bean curd of same brand or producing area had certain aggregation,which could provide a basis for traceability of Bacillus cereus.(3)Virulence genes in Bacillus cereus were detected.The PCR technique and electrophoresis technique were used for detection of hbl A,hbl C,hbl D,nhe A,nhe B,nhe C,ent FM,cyt K,bce T,ces,EM1 of Bacillus cereus.The results showed that the detection rates of virulence genes among all isolates were 34.31%,25.98%,7.84%,100.00%,99.51%,99.02%,99.51%,35.29%,40.69%,4.90%,4.90%,respectively,indicating non-hemolytic enterotoxin and enterotoxin FM may be the main virulence factors of Bacillus cereus in fermented bean.Based on the detection results of virulence genes,204 strains of Bacillus cereus were divided into 30 genetypes,indicating the Bacillus cereus isolated from fermented bean curd appeared diversified.Almost all the strains of Bacillus cereus carried four virulence genes and above,indicating the Bacillus cereus isolated from fermented bean curd may produce composite toxins.(4)Antibiotic resistance phenotypes of Bacillus cereus were detected.204 strains of Bacillus cereus were tested for susceptibility to penicillin,gentamicin,trimethoprim-sulfamethoxazole,tetracycline,erythromycin,chloramphenicol,ciprofloxacin,oxacillin,clindamycin,rifampin,amikacin based on the paper disk diffusion method.The results showed that the rates of antibiotic resistance among all isolates were 100.00%,0.00%,11.27%,8.82%,0.00%,0.00%,0.00%,100.00%,0.00%,7.35%,0.00%,respectively,indicating penicillin and oxacillin couldn't be used for foodborne illness caused by Bacillus cereus.