Identification of Bacillus cereus Group Species Associated with Food Poisoning Outbreaks in British Columbia, Canada

Abstract

Food poisoning laboratories identify Bacillus cereus using routine methods that may not differentiate all Bacillus cereus group species. We recharacterized Bacillus food-poisoning strains from 39 outbreaks and identified B. cereus in 23 outbreaks, B. thuringiensis in 4, B. mycoides in 1, and mixed strains of Bacillus in 11 outbreaks. (16,19,22). They are difficult to discern using standard biochemical schemes, chemotaxonomic methods, or phylogenetically relevant target genes (1, 2), and many distinguishing pathogenicity markers in this group can be attributed to mobile plasmids (18,21,22,23). B. cereus sensu stricto carries the plasmid-borne emetic toxin cereulide (ces) (7,13,14), and B. thuringiensis carries insecticidal crystal protein (ICP) (cry) genes on one or more plasmids (3, 5, 6). The differentiation of B. cereus group members using molecular techniques is not routine in food-poisoning diagnostic methods and may cause underreporting of species such as B. thuringiensis (1,8 DNA was extracted by lysing pure culture in a heating block at 102°C for 10 min. Microcentrifuged supernatant was frozen at Ϫ80°C until required. Pathogenicity genes for emetic cereulide toxin (nonribosomal peptide synthetase [NRPS]) and ICP (cry1 or cry2) were detected in multiplex PCR assays (7, 10) shown in Fig. 1. Each master mix contained 0.8 M of each primer, hot start master mix, diethyl pyrocarbonate water (20 l), and 5 l of DNA. The PCR products were loaded onto 2% agarose gels made with 0.5ϫ Tris-borate-EDTA buffer and ethidium bromide (1 g ml Ϫ1 ). The gels were electrophoresed at 120 V for 30 min and then visualized on a Bio-Rad Gel Doc 2000.Strains positive for NRPS were designated as B. cereus, those positive for ICP (by microscopy or PCR) as B. thuringiensis, those with rhizoidal growth on nutrient agar as B. mycoides, and all other strains with the typical B. cereus phenotype as B. cereus NRPS Ϫ ICP Ϫ . PCR-negative isolates were further examined for ICP crystals using transmission electron microscopy (TEM) since B. thuringiensis strains may carry one to six cry genes and there is no universal method available to detect all cry genes (there are currently more than 150 cry1 toxins) (17; Bacillus thuringiensis toxin nomenclature [http: //www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/]). Samples of B. cereus group bacteria were prepared for electron microscopy by fixation with 2% glutaraldehyde and 1% para-

A quadruple-enterotoxin-deficient mutant of Bacillus thuringiensis remains insecticidal

“…Frederiksen et al (2006) postulated that it is therefore conceivable that a human could consume an infective dose from such produce. A recent report analysed B. cereus-group strains isolated from 39 food poisoning outbreaks and determined that in four of the outbreaks only B. thuringiensis isolates were recovered from food samples (McIntyre et al, 2008), suggesting that B. thuringiensis can be responsible for food poisoning when B. cereus is implicated, because isolates are not routinely examined for the presence of crystal toxin genes or proteins (Granum, 2002;European Food Safety Authority, 2005 For many years, B. thuringiensis bioinsecticides have been considered safe and effective despite the presence of enterotoxin genes. This work has improved upon this strain of B. thuringiensis subsp.…”

“…Moreover, heat treatment of food can result in plasmid loss, and therefore a B. thuringiensis strain that caused disease would no longer carry B. thuringiensis-specific genes at the time of diagnosis (Granum, 2002;European Food Safety Authority, 2005). A recent report re-examined 155 B. cereusgroup-like species collected from 39 foodborne outbreaks in British Columbia, Canada, between 1991(McIntyre et al, 2008. Of these B. thuringiensis isolates, 23 were identified as the only Bacillus sp.…”

Eksplorasi Bakteri Endofitik Dan Potensinya Dalam Penghambatan Jamur Akar Putih (Rigidoporus Microporus)

“…cereus dapat memiliki siklus hidup sebagai saprofit seumur hidupnya, tetapi bakteri ini juga berpotensi sebagai patogen yang menyebabkan sakit pada manusia, terutama sebagai penyebab keracunan makanan yang ditandai dengan diare, gangguan perut, mual atau muntah, yang sebagian besar terjadi pada orang yang kurang sehat, memiliki penyakit kritis atau memiliki kekebalan tubuh yang lemah (McIntyre et al, 2008;Senesi & Ghelardi, 2010;Tallent et al, 2012;). Hasil pengujian Bacillus cereus isolat A10 pada medium skim milk menunjukkan adanya aktivitas enzim protease yang sangat lemah ditandai dengan adanya zona bening yang tipis di sekitar koloni, dibandingkan dengan kontrol positif (Gambar 4).…”

About the <i>Bacillus cereus</i> Group

“…Instead, colony observation and Gram staining alone may lead to misidentification, with knowledge of species-specific clinical pictures and virulence determinants remaining fragmentary and confused (3). In fact, B. cereus (sensu strictu) is not the only microorganism responsible for human disease within this microbial group (6), and phenotypical investigations may provide only a preliminary classification of strains as presumptively belonging to the B. cereus group.…”