Detection, characterization and survival of Bacillus cereus group members in spices and herbs

The Gram positive spore forming bacterium B. cereus can cause foodborne diseases with diarrhoeal and emetic symptoms. Spices and herbs have frequently been found to contain spores of B. cereus group species which has been linked to several foodborne disease outbreaks. The spice market is growing and a considerable part of condiments is used in the industrial food production and in the catering sector. Thus, spices and herbs can act as a vehicle to transfer B. cereus spores to various foodstuffs that might enable germination and growth under temperature abuse conditions. The informative value of standard microbiological methods applied to spices and herbs is limited as they can be biased by a high microbial background flora and possible antimicrobial effects. Moreover, they neither provide information on the exact B. cereus group species nor on the toxinogenic potential. Hence, data on these parameters are scarce for presumptive B. cereus isolated from spices and herbs. To facilitate data gathering culture independent detection and characterization methods should be developed. In this work a multiplex real- time PCR for the quantification of spores of B. cereus group species with simultaneous discrimination of B. pseudomycoides, cry1-positive B. thuringiensis as well as B. weihenstephanensis/B. mycoides was applied to artificially contaminated spices and herbs. For this, an internal amplification control was included in the PCR-method and different DNA- extraction procedures were evaluated. The best results with pure spore suspensions were achieved with the Qiagen DNeasy Blood &amp; Tissue Kit coupled to quantitative real-time PCR (LOD ~10E2 cfu/ml). Yet, applied to spices and herbs kit based DNA-extraction yielded poor LODs of ≥10E5 cfu/g, whereas the open-formula CTAB-DNA-extraction achieved LODs of 10E2 to 10E3 cfu/g. In order to further investigate the pathogenic potential of B. cereus group species naturally present in spices and herbs, we analysed presumptive B. cereus strains isolated from six different condiments with presumptive B. cereus concentrations of 10E1 to 10E3 cfu/g. In a total of 59 isolates, 44 were classified as B. cereus, five as B. thuringiensis, four as B. toyonensis-like, two as B. pseudomycoides/B. mycoides one as B. weihenstephanensis, and three as undefined B. cereus group species. Most isolates carried the enterotoxin genes nheA and hblD and were also able to produce the respective toxins (Nhe and Hbl). Half of the isolates additionally harboured the cytK-2 gene. One isolate possessed the ces gene and was able to produce cereulide. For some isolates MLST analyses revealed disagreements between phylogenetic relationship and the classification as B. weihenstephanensis and B. mycoides based on previously described real-time PCR species markers (motB and bpm sequences). Hence, the suitability of these markers for species differentiation should be further investigated. For the purpose to investigate the long-term survival of B. cereus and B. thuringiensis in spices and herbs, tenacity studies were conducted using artificially contaminated condiments with spores of each species. After 50 weeks of storage in the dark no significant spore reduction could be observed for either strain. Beforehand, an appropriate spiking technique for spices and herbs was investigated and the suitability of the applied cultural detection method was evaluated. Using sand as a carrier proved to be a suitable method for dry-spiking. The examination of the cultural detection method indicated that the enumeration of vegetative cells in suspensions of spices and herbs can be biased due to substances with inhibitory effect on the bacteria. In contrast, spores remained unaffected unless stored for prolonged times in condiment suspension. This finding might be relevant for cultural enrichment.

Topic: Probiotics and Fermented Foods
Subfield: Food Science
Field: Agricultural and Biological Sciences
Domain: Life Sciences