Bacillus cereus acid stress responses

Bacillus cereus is a ubiquitous Gram-positive organism, which frequently causes foodborne illnesses. The widespread prevalence of B. cereus makes it a common contaminant in fresh foods where it also can cause spoilage. To prevent food-borne diseases and food spoilage, foods are often processed and/or preserved. In recent years, consumers’ preferences have directed to fresher and tastier foods and this has acted as a driver for food industry to use milder processing and preservation techniques. Examples of hurdles that can be applied to preserve foods are low pH and the addition of organic acids. B. cereus may overcome these adverse conditions by displaying an adaptive stress response. The response of B. cereus upon exposure to these hurdles was investigated using two model strains, ATCC 14579 and ATCC 10987. Comparative analysis revealed numerous strain-specific genes and differences in metabolic capacities, including a urease encoding gene cluster in ATCC 10987 and a nitrate respiration cluster in ATCC 14579. A survey including ATCC 10987 and 48 environmental and outbreak-associated isolates revealed urease activity, i.e., the conversion of urea in ammonia and carbon dioxide, to be present in 10 isolates. However, the activity appeared to be too low to contribute to acid resistance in the strains tested. To search for other acid resistance mechanisms, comparative phenotype and transcriptome analyses of strains ATCC 14579 and ATCC 10987 cells exposed to organic and/or inorganic acid shocks were performed. Upon exposure to low pH with or without the addition of lactic acid or acetic acid, common acid resistance mechanisms and induction of the nitrate reductase cluster in the more acid resistant strain ATCC 14579 were revealed. Furthermore, a major oxidative response was displayed, which included the induction of several oxidative stress related genes and the production of inactivation-associated reactive oxygen species (ROS), such as hydroxyl radicals, peroxynitrite, and superoxide. ROS were detected using fluorescent probes in combination with flow cytometry, including a newly developed method using a specific probe that enables superoxide detection in Grampositive and Gram-negative bacteria. The formation of ROS was also shown upon exposure to heat and was found to be oxygen dependant. Correspondingly, assessment of B. cereus stress survival capacity revealed increased heat- and acid-resistance with cells grown and exposed to stresses in the absence of oxygen. The excess ROS may originate from stressinduced dysfunction of the aerobic electron transfer chain, which was indicated by the induction of alternative electron transfer chain components upon exposure to organic and inorganic acid shocks. Upon exposure to stress, superoxide is generated through the premature leakage of electrons to oxygen at sites in the electron transfer chain at elevated rates. Subsequently, superoxide may promote the formation of other ROS, which can cause cellular damage leading to cell death. The induction of oxidative stress related genes has been reported in numerous other studies involving a wide range of bacteria exposed to different adverse conditions. However, a clear relation between the formation of ROS and the applied environmental stress was up to now not established. Secondary oxidative responses, including the formation of ROS, are possibly common bacterial responses to severe stresses under aerobic conditions. This thesis describes genomic differences between B. cereus strains and the acid stress response of these strains on transcriptome and phenotype levels, including measurements of intracellular ROS. The findings in this study can contribute to further understanding of bacterial stress responses and secondary oxidative responses. Furthermore, the results obtained may aid to optimize and select (combinations of) stresses to apply in hurdle technology, thus enabling design of safe, milder food processing and preservation techniques.