Prevalence of Toxigenic Bacillus cereus in Chili and White Pepper from Bogor Area, Indonesia

Abstract

Chili and white pepper are widely used as spices and/or condiments in many Indonesian cuisines, especially Ready-to-eat (RTE) foods. These spices are often associated with pathogenic microorganisms such as Bacillus (B.) cereus, and have the potential to contaminate foods to which they are added. Bacillus cereus is a spore-forming bacterium widely found in the environment and is responsible for causing diarrheal and emetic syndromes. The organism has been implicated in various food borne outbreaks in different parts of the world. This study aimed to determine the prevalence and concentration of B. cereus in chili and white pepper from markets around Bogor, and to determine the potential toxicity of these isolates based on ces and nheA genes. The study consisted of several stages including sample collection and preparation, isolation and enumeration of Bacillus cereus on Mannitol-egg yolk- polymyxin agar (MYP), presumptive confirmation of the isolates based microscopic observations and biochemical tests, molecular confirmation by detection of 16S rRNA gene, 16S rRNA gene sequencing, phylogenetic analysis and analysis for the possession of nheA and ces toxigenic genes. Out of the forty (20 chili and 20 white pepper) samples analyzed, 33 (82.5%) contained presumptive Bacillus cereus. Ninety percent of white pepper samples (18/20) and 75% of chili samples (15/20) contained presumptive B. cereus. The highest B. cereus concentration in white pepper was 5.77 log10 CFU/g and the lowest was 1.70 log10 CFU/g while the highest in chili was 3.93 log 10 CFU/g and the lowest was 0.70 log10 CFU/g. Ninety five percent (19/20) of samples from the traditional markets and 70% (14/20) from supermarkets contained presumptive B. cereus. Samples from the traditional market had a higher mean log10 CFU/g of 3.25 compared to 2.55 log10 CFU/g from the supermarkets. However, the difference was not statistically significant (P > 0.05). Samples containing B. cereus of >5 log10 CFU/g were found in both market types. Twelve of 13 isolates tested were confirmed as B. cereus based on 16S rRNA gene and BLAST identification. The isolates shared similarity (>98%) with B. cereus strains from GenBank and were phylogenetically related to B cereus. All twelve isolates (100%) possessed the nheA gene but none of them owned the ces gene. Absence of ces gene suggests a reduced risk for emesis from chili and white pepper but the 100% prevalence of nheA gene presents a potential food safety concern and risk for B. cereus diarrheal food borne disease.