The development of a multiplex polymerase chain reaction assay for direct detaection of enterotoxigenic bacillus cereus in food

Bacillus cereus food poisoning has been implicated in outbreak episodes in the world. Bacillus cereus is a potential food safety hazard causesing two different types of food poisoning, the diarrhea type, caused by hemolysin BL and non-hemolytic enterotoxin, and the emetic type, caused by the emetic toxin cereulide. In Kenya the organism has been isolated from pasteurized milk at 41.2%, 26% of which were enterotoxigenic. The aim of this study was to, develop a multiplex polymerase chain reation (PCR) technique that can be used to detect enterotoxigenic B. cereus in food, determine the level of contamination in various foods that will enable gene detection, and to develop suitable food processing procedures that can reduce PCR inhibitory substances to enable use of direct multiplex PCR for detection of toxins genes in various foods. In this study, seven pairs of primers for multiplex PCR were used for simulteneous detection of 6 genes and a specific sequence encording hemolysin BL, non-hemolytic enterotoxin, and an emetic-specific sequence respectively. Bacillus cereus standard strains 11145, a diarrhea type strain and BC 68 an emetic type strain were used as reference strains. The suitability of the multiplex PCR was evaluated for direct use in B. cereus free cheese, pastuerized milk and cooked rice, spiked with varing concentrations of standard B. cereus strains and 108 non-spiked food samples obtained from Nairobi central business district (NCBD) food outlets, comprising of 36 cooked rice, 36 cheese and 36 pastuerized milk. The samples were tested for the presence of the six genes and the emetic-specific sequence using the direct multiplex PCR. The results were confirmed using the conventional colony multiplex PCR. It was therefore possible to detect presence of toxigenic B. cereus in spiked foods by direct multiplex PCR with a sensitivity level of 4x I0 colony forming units (CFU) per gram for cooked rice homogenate, and 4x I03 CFU per ml or gram for both milk and cheese respectively. Of the 108 food samples tested, fifty three (49.1 %) samples were contaminated with B. cereus. Fifty one (96.2 %) of the contaminating B. cereus were toxigenic. A total of 14 (27.5 %) of the 51 samples contaminated with toxigenic B. cereus had B. cereus counts within the direct multiplex PCR detection levels (4x I0 CFU per gram), and tested positive. The direct multiplex PCR assay was complete within 8 hours and was therefore more rapid compared to the convectional colony multiplex PCR that takes 24 to 48 hours. In conclusion, testing for genes directly in foods will eliminate the need to carry out time consuming and costly culture, isolation and identification procedures which will drastically reduce the cost and time required to identify foods contaminated with toxigenic B. cereus for both diagnosis and food quality control. In addition this technique will be necessary in confirming the pathogenicity of B. cereus by detecting presence or absence of toxins gene profiles in contaminating bacterial cells. vr'UVA"I"'T lIa""rnt"'ITV I I ADV CHAPTER ONE: INTRODUCTION