Bacillus cereus responses to acid stress

Coping with acid environments is one of the prerequisites for the soil saprophytic and human pathogenic lifestyle of Bacillus cereus. This minireview highlights novel insights in the responses displayed by vegetative cells and germinating spores of B. cereus upon exposure to low pH as well as organic acids, including acetic acid, lactic acid and sorbic acid. Insights regarding the possible acid-inflicted damage, physiological responses and protective mechanisms have been compiled based on single cell fluorescence microscopy, flow cytometry and transcriptome analyses

Primary and secondary oxidative stress in Bacillus

Coping with oxidative stress originating from oxidizing compounds or reactive oxygen species (ROS), associated with the exposure to agents that cause environmental stresses, is one of the prerequisites for an aerobic lifestyle of Bacillus spp. such as B. subtilis, B. cereus and B. anthracis. This minireview highlights novel insights in the primary oxidative stress response caused by oxidizing compounds including hydrogen peroxide and the secondary oxidative stress responses apparent upon exposure to a range of agents and conditions leading to environmental stresses such as antibiotics, heat and acid. Insights in the pathways and damaging radicals involved have been compiled based among others on transcriptome studies, network analyses and fluorescence techniques for detection of ROS at single cell level. Exploitation of the current knowledge for the control of spoilage and pathogenic bacteria is discussed

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. <br/

Analysis of acid-stressed Bacillus cereus reveals a major oxidative response and inactivation-associated radical formation

Acid stress resistance of the food-borne human pathogen Bacillus cereus may contribute to its survival in acidic environments, such as encountered in soil, food and the human gastrointestinal tract. The acid stress responses of B. cereus strains ATCC 14579 and ATCC 10987 were analysed in aerobically grown cultures acidified to pH values ranging from pH 5.4 to pH 4.4 with HCI. Comparative phenotype and transcriptome analyses revealed three acid stressinduced responses in this pH range: growth rate reduction, growth arrest and loss of viability. These physiological responses showed to be associated with metabolic shifts and the induction of general stress response mechanisms with a major oxidative component, including upregulation of catalases and superoxide dismutases. Flow cytometry analysis in combination with the hydroxyl (OH center dot) and peroxynitrite (ONOO-)-specific fluorescent probe 3'-(phydroxyphenyl) fluorescein (HPF) showed excessive radicals to be formed in both B. cereus strains in bactericidal conditions only. Our study shows that radicals can indicate acid-induced malfunctioning of cellular processes that lead to cell death

Comparative transcriptome and phenotype analysis of acid-stressed Bacillus cereus strains

This SuperSeries is composed of the SubSeries listed below

Comparative transcriptome and phenotype analysis of acid-stressed Bacillus cereus strains

This SuperSeries is composed of the SubSeries listed below

Comparative transcriptome and phenotype analysis of acid-stressed Bacillus cereus strains

This SuperSeries is composed of the SubSeries listed below

Heat stress leads to superoxide formation in Bacillus cereus detected using the fluorescent probe MitoSOX

Bacillus cereus is a food-borne human pathogen and food spoilage organism. Spores and vegetative cells of B. cereus can be found almost everywhere and therefore often end up in food processing equipment and food products. To remove spores and vegetative cells from food or equipment, harsh treatments such as high temperatures are applied. The heat stress response of B. cereus and other organisms has been studied and it has been shown that reactive oxygen species may be involved in inactivating the bacterial cells. Using a novel approach with the fluorescent probe MitoSOX, the formation of superoxide in B. cereus cells upon exposure to heat has been confirmed. MitoSOX can be used in combination with other probes, including, SYTOX green, CYTO 9, and CFDA, showing superoxide formation in combination with damaged cell membranes, intact cell membranes, and esterase activity in cells with intact membranes, respectively. MitoSOX in combination with flow cytometry-assisted sorting showed three distinct populations, a low fluorescent population that was still viable, a highly fluorescent population that could not be recovered on agar plates, and a low fluorescent non-viable population that appeared after prolonged exposure to heat. This third population may include dead cells where MitoSOX binds to DNA without reacting with superoxide. Superoxide formation during exposure to lethal temperatures by B. cereus shows that superoxide plays a role in bacterial cell death and its generation may thus contribute to the efficiency of food preservation conditions