Isolation of a foodborne Bacillus cereus strain and its effect on intestinal mucosal immunity-associated factors and gut microbial community in mice / 生物工程学报

Bacillus cereus is a common foodborne pathogen. Accidently eating food contaminated by B. cereus will cause vomiting or diarrhea, and even death in severe cases. In the present study, a B. cereus strain was isolated from spoiled rice by streak culture. The pathogenicity and drug resistance of the isolated strain were analyzed by drug sensitivity test and PCR amplification of virulence-associated gene respectively. Cultures of the purified strain were injected intraperitoneally into mice to examine their effects on intestinal immunity-associated factors and gut microbial communities, to provide references for the pathogenic mechanism and medication guidance of these spoilage microorganisms. The results showed that the isolated B. cereus strain was sensitive to norfloxacin, nitrofurantoin, tetracycline, minocycline, ciprofloxacin, spectinomycin, clindamycin, erythrocin, clarithromycin, chloramphenicol, levofloxacin, and vancomycin, but resistant to bactrim, oxacillin and penicillin G. The strain carries seven virulence-associated genes including hblA, hblC, hblD, nheA, nheB, nheC and entFM, which are involved in diarrhea-causing toxins production. After infecting mice, the isolated B. cereus strain was found to cause diarrhea in mice, and the expression levels of immunoglobulins and inflammatory factors in the intestinal mucosae of the challenged mice were significantly up-regulated. Gut microbiome analysis showed that the composition of gut microbial community in mice changed after infection with B. cereus. The abundance of the uncultured_bacterium_f_Muribaculaceae in Bacteroidetes, which is a marker of body health, was significantly decreased. On the other hand, the abundance of uncultured_bacterium_f_Enterobacteriaceae, which is an opportunistic pathogen in Proteobacteria and a marker of dysbacteriosis, was significantly increased and was significantly positively correlated with the concentrations of IgM and IgG. These results showed that the pathogenic B. cereus carrying diarrhea type virulence-associated gene can activate the immune system by altering the composition of gut microbiota upon infection.

Epidemiological Study and Virulence Associated Genes of Enteropathogenic Escherichia coli Isolated from Diarrheal Neonates

A total of 136 strains of Escherichia coli, isolated from rectal swabs of neonates at the neonatal intensive care unit of Pusan National University Hospital during the period from February to April of 2001 and March to April of 2002 were serotyped. The presence of eaeA, aggA, bfpA, astA, LT, and ST genes was test by PCR. Four enteroaggregative E. coli (EAggEC) strains were isolated in 2001 and eight in 2002. In 2001, three strains of enterotoxigenic E. coli (ETEC) were isolated and these strains were tested for antimicrobial susceptibility. ETEC isolates were detected by a reverse passive latex agglutination (RPLA) test for the production of heat-labile enterotoxin. The strains were analyzed by using plasmid profiling, random amplified polymorphic DNA (RAPD), and pulsed field gel electrophoresis (PFGE) methods. The O serotypes could be assigned to 29 isolates (21.3%): O166, 6; O167, 5; O86a, O6 and O127a, 4 each; O8, 2; and O28ac, O44, O158, O20, 1 each. There were 107 untypable isolates. EAggEC isolates were typed as O86a in 4, O127a in 4, and untypable in 4 strains. Three isolates of ETEC were typed as O6. The PCR detected aggA gene in 12 strains (8.8%), but bfpA, eaeA, EAST-1, and ST genes were not detected. LT gene was detected in 3 strains (2.2%) by PCR and DNA probe hybridization, LT production was confirmed in those strains by a latex bead aggregation method. Out of the 15 EAggEC and ETEC strains, eleven (80.0%) were multi-drug resistant to more than 3 antibiotics. Thirteen groups were classed by antibiogram and most strains were susceptible to gentamicin, kanamycin, and cefoperazone, but resistant to ampicillin (100%), cephalothin (66.7%), cefuroxime (46.6%), and tetracycline (46.7%). All isolates possessed a 60 kbp plasmid and 15 strains possessed smaller plasmids. Twelve EAggEC and 3 ETEC strains were differentiated into 5 and 3 groups by the plasmid profiling. RAPD and PFGE grouped the EAggEC isolates into 7 and 6 groups, respectively. The RAPD and PFGE patterns matched in 11 isolates (91.7%) among the 12 EAggEC strains, but the plasmid profile analysis and antibiogram showed no correlation. Three ETEC strains showed different plasmid profiles, and RAPD and PFGE patterns.

Detection of the Virulence-associated Genes in Vibrio Cholerae by Multiplex PCR Assay / 环境与健康杂志

Objective To study a rapid and sensitive method for determination of virulence-associated genes in O1El Tor,O139,non-O1/non-O139Vibrio cholerae strains.Methods Five pairs of primers were designed respectively ac-cording to cholera toxin sub-unit A gene(ctxA),accessory cholera enterotoxin gene(ace),zonula occludens toxin gene(zot),toxin coregulated pilus A gene(tcpA)and toxR regulatory protein gene(toxR).Multiplex PCR(MPCR)procedures for simultaneously detecting those five genes were established.The gene information of the virulence-associated genes in the Vibrio cholerae strains was obtained through agar gel electrophoresis for products of single amplification of the MPCR.Results The five virulence-associated genes in the positive control Vibrio cholerae O139(MO45strain)could be detected and the results were correct,which could meet the designed request for the method.In the other tested strains(O1EL Tor,O139,non-O1/non-O139)could be detected1to5kinds of the virulence-associated genes.Based on the results of the variety of carried virulence-associated genes,the tested Vibrio cholerae strains could be classified as5kinds of genetypes,and the Vibrio cholerae could be distinguished between toxic and non-toxic strains.The sensitivity of the MPCR approach reached to10 2 cfu/ml.Conclusion This method is rapid,specific and sensitive,which possess great value for practical application.