| Summary: | 博士 === 國立中興大學 === 食品科學系 === 89 === Abstract
Bacillus cereus is one of the major foodborne pathogens in Taiwan. The diarrheal type of diseases is attributed to enterotoxins produced during vegetative growth of B. cereus. The B. cereus group comprises B. cereus, B. thuringiensis, B. mycoides and B. anthracis. The virulence properties of B. cereus group cells are of interest. One hundred and one strains of B. cereus group were examined for the presence of four enterotoxin genes: the hemolysin BL (hbl), the non-hemolytic enterotoxin (nhe), the Bacillus cereus enterotoxin T (bceT) and enterotoxin FM (entFM) using polymerase chain reaction (PCR). Different PCR primers were developed for the detection of these genes. Two B. anthracis strains were found to be PCR-negative for these four enterotoxin genes. At least one of the four enterotoxin genes was detected in any of the 89 B. cereus, 7 B. thuringiensis and 3 B. mycoides strains. Thirty of 89 B. cereus strains, all of the 7 B. thuringiensis and 3 B. mycoides strains carried hbl operon genes i.e., hblA, hblC and hblD. Two genes of hblC and hblD were detected in B. cereus strain BCN32, but hblA was not found. The results from the amplification of hblC correlated well with results obtained with the BCET-RPLA kit (Oxoid; Denka Seiken, Japan). Except for two strains (B. cereus strains BCN32 and BCN58), all strains that were positive in PCR amplification using primers L2F/L2b were also positive when tested with the BCET-RPLA kit. The bceT gene was found in 45 of the 101 strains of B. cereus group and entFM in 95 strains. The nhe operon was the most common enterotoxin gene detected in strains examined (97/101; 96%). These results showed that there were 8 enterotoxigenic profiles for the 101 strains of B. cereus group collected. Profile III that carrying the entFM and nhe genes was the most prevalent type (41/101; 41%). Culture supernatants from all strains of B. cereus, B. thuringiensis and B. mycoides were cytotoxic to HEp-2, CHO and Vero cells. Of the three cell lines tested, the HEp-2 cell was less susceptible to the enterotoxin of B. cereus than the CHO and Vero cells. Cell cytotoxicity was detectable only after the cell concentration of B. cereus reached ＞5 × 107 cfu/ml and the Cytotoxicity was about 1/2 ~ 1/8 at the stationary phase. PCR-RFLP analysis and PCR-directed sequencing were performed to examine the heterogeneity of the hbl and nhe genes. The PCR products of hbl genes were found to be heterogeneous by the PCR-RFLP analysis (17/40; 42%). PCR-RFLP analysis also confirmed that there was genetic diversity within the nheA and nheB genes. The sequences different genes of hbl and nhe were found to be highly conserved for different strains. However, most of the base pair substitution occurred at the third base position of the genetic codon and did not affect the amino acid sequence. The hblA, hblC and hblD sequences were used to construct a phylogenetic tree. The strains of B. thuringiensis and B. cereus were closely related species, also B. cereus strain BC13 and B. mycoides strains BMY1, BMY3 were revealed to be closely related species. B. cereus strains were typed by antibiotic susceptibility testing, plasmid profile, RAPD (Randomly Amplified Polymorphic DNA) and PFGE (Pulsed-field gel electrophoresis).Of the 83 B. cereus strains tested, there were 24 antibiogram and 46 plasmid profiles. Plasmid was not founded in 27 B. cereus strains. Digestion with NotI generated 58 PFGE profiles for these 83 B. cereus strains. RAPD with 5 different primers yielded 42 to 55 RAPD patterns. Results obtained from RAPD and PFGE were closely related among the B. cereus strains BCN29 ~ BCN58. Although the RAPD method was rapid and easier to perform, PFGE produced most discriminative and reproducible results.
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