Molecular epidemiology of bacillus cereus food poisoning

Abstract

In this study, a survey was carried out to determine the prevalence of food poisoning outbreaks in Kenya by use of medical records and annual reports of the Ministry of Health. Thirty seven outbreaks were reported to the Ministry of Health from various parts of the country between 1970 and 1994, and only 13 of these outbreaks, involving a total of 926 people, were confirmed to be due to particular aetiological agents. The confirmed outbreaks were due to Staphylococcus aureus (5); Clostridium perfringens (1); Clostridiun botulinum (2); Plant poisoning (3); and chemical poisoning (2). Vehicle foods that were involved in these outbreaks included milk and milk products (6), meat and meat products (2), cassava (2), white ants(1) and maize flour contaminated with chemicals(2). This study indicated that the occurrence of food poisoning outbreaks in the country is more than was recorded by the Ministry of Health considering the high number (309,191) of food poisoning cases at were treated as outpatients in the period 1988to 1991(Anon, 1993). The study further explored the potential of antibiogram analysis and genetic based techniques in identifying and establishing the toxigenicity of B. cereus strains. A total of 153 milk samples consisting of 80 pasteurized and 73 raw were studied. Bacillus cereus was isolated from 33(41.2 %) pasteurized and from 27(37 %) raw milk samples. The 60 isolates were tested for production of diarrhoeal enterotoxin and ability to hydrolyse starch. Fifty percent of them produced diarrhoeal enterotoxin and 65 % hydrolysed starch. Although there was a strong correlation between production of diarrhoeal enterotoxin and hydrolysis of starch, the latter cannot be used in diagnosis of enterotoxigenic B. cereus. Antimicrobial susceptibility tests found B. cereus isolates to be resistant to one or more drugs with all strains being resistant to ampicillin and susceptible to streptomycin. The test for carriage of plasmids found 71.7 % of the isolates to harbour between 1 and 5 plasmids with sizes ranging from 0.1 to 60 megadaltons (mDa). No relationship was apparent between carriage of plasmids, antimicrobial susceptibility patterns and enterotoxin production. Bacterial restriction endonuclease DNA analysis (BRENDA) with EcoRl, Hind III, BamHl, Mb01 and Dral restriction enzymes revealed no common BRENDA type for either enterotoxin positive or enterotoxin negative strains. A polymerase chain reaction (Pf'R) test detected the bceT gene in 41.1 % of the strains, while a 741 base pair (bp) probe detected bee T gene in 44.6 % of the strains. All the bceT gene positive strains with the peR were also positive with the 741 bp DNA probe. In conclusion, this study has demonstrated that plasmid profile analysis and BRENDA are not useful in differentiating enterotoxin producing strains of B. cereus, but can be useful techniques in differentiating individual strains of B. cereus. The study further demonstrated that two or more diarrhoeal enterotoxins are involved in B. cereus food poisoning, and some strains can produce more than one diarrhoeal enterotoxins. Therefore, during investigation of a suspected B. cereus food poisoning, it would be necessary to screen for production of diarrhoeal enterotoxin complex by use of Bacillus cereus enterotoxin-reverse passive latex agglutination (BeET -RPLA) test kit, or western blot, and also detect the presence of bceT gene by peR or a probe derived from the bee T gene. It is suggested that peR be used to amplify whole hblA (Beecher and Macmillan, 1990), hblC and hblD genes (Ryan et al. 1997) that code for the B, ~ and L, proteins of hemolysin BL respectively, to delineate the gene that is specific enough to characterize enterotoxigenic strains of B. cereus. The tests should involve direct detection of hblA, hblC and hblD by (1) peR, and (2) probe prepared from the peR products on a number of B. cereus strains representing both enterotoxin positive and negative strains.