Impact of sorbic acid on germinant receptor-dependent and -independent germination pathways in Bacillus cereus

Amino acid- and inosine-induced germination of Bacillus cereus ATCC 14579 spores was reversibly inhibited in the presence of 3 mM undissociated sorbic acid. Exposure to high hydrostatic pressure, Ca-dipicolinic acid (DPA), and bryostatin, an activator of PrkC kinase, negated this inhibition, pointing to specific blockage of signal transduction in germinant receptor-mediated germination

Impact of sorbic acid and other mild preservation stresses on germination and outgrowth of Bacillus cereus spores

Weak organic acids such as sorbic acid, lactate, and acetic acid are widely used by the food industry as preservatives to control growth of micro-organisms. With the current trend towards milder processing of food products, opportunities arise for spore-forming spoilage and pathogenic microorganisms such as Bacillus cereus, that may survive the use of milder heating regimes. Dormant spores produced by B. cereus can survive processing conditions and their subsequent outgrowth increases the risk of premature spoilage and food safety issues. As a consequence, the use of additional preservation hurdles, such as acidification with weak organic acid additives to ensure the quality and safety of a product is important. Sorbic acid is widely used as an antimicrobial compound because of its strong inhibitory properties against bacteria and other spoilage organisms. Its effectivity has also been ascribed to its hydrophobic character, resulting in an additional mode of action not observed with other less lipophilic organic acids such as lactic acid and acetic acid. In this project the impact of sorbic acid on spore germination and outgrowth was studied at transcriptome level and was linked to the distinct phenotypic responses observed for spores exposed to different levels of sorbic acid. The various stages of spore germination and outgrowth could be recognized by distinct gene expression profiles representing either the germination phase, transition state between germination and outgrowth or outgrowing cells, respectively. A subset of genes was specifically expressed in sorbic acid-exposed germinating spores and included functions related to cell envelope, (multi) drug transporters and amino acid metabolism. At high concentrations of sorbic acid (3mM of the undisssociated form, HSA), nutrient-induced germination of B. cereus ATCC 14579 spores was completely blocked. This blockage was shown to be reversible and could be bypassed by known non-nutrient triggers that activate spores in a receptor-independent way, pointing to a possible interference of HSA with the signaling event between germinant receptors and proposed SpoVA-channels, possibly by accumulation into the spore’s inner membrane. Additional experiments with other inhibiting compounds, including organic acids and their structurally similar alcohol counterparts, showed that the lipophilic properties are an important determinant of its efficacy to block germination. Building on current knowledge on the interaction of germination-relevant protein clusters, we discuss a hypothetical model on the mode of action of sorbic acid and other short-chain lipophilic compounds in germination inhibition of B. cereus spores. In addition to the interference or even blockage of germination, sorbic acid may increase outgrowth heterogeneity when applied at lower concentrations (0.25-1.5 mM) that still allow outgrowth. The first stages of outgrowth were shown to specifically occur heterogeneously when spores were exposed to multiple stresses simultaneously. Heterogeneity effects were most pronounced for combined stress-effects where heat-treated spores were also exposed to low pH stress. Under these conditions, a large subpopulation of spores was delayed between initial germination and swelling and further outgrowth. For the food producing industry, it would be desirable to have reliable parameters to predict the behavior of surviving spores in a food product. Data presented in this thesis show that germination rate is not a good predictor for outgrowth heterogeneity when applied as a single indicator. In conclusion, the work described in this thesis strive to obtain a better understanding on the impact that preservation stresses, including (sorbic) acid stress, have on the germination and outgrowth (heterogeneity) of B. cereus spores. The results obtained in this project may contribute to the rational design of new concepts for improved food preservation and safety.</p

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

Impact of sorbic acid on germination and outgrowth heterogeneity of Bacillus cereus ATCC 14579 spores

Population heterogeneity complicates the predictability of the outgrowth kinetics of individual spores. Flow cytometry sorting and monitoring of the germination and outgrowth of single dormant spores allowed the quantification of acid-induced spore population heterogeneity at pH 5.5 and in the presence of sorbic acid. This showed that germination efficiency was not a good predictor for heterogeneity in final outgrowth

Impact of sorbic acid on germinant receptor-dependent and -independent germination pathways in Bacillus cereus

Amino acid- and inosine-induced germination of Bacillus cereus ATCC 14579 spores was reversibly inhibited in the presence of 3 mM undissociated sorbic acid. Exposure to high hydrostatic pressure, Ca-dipicolinic acid (DPA), and bryostatin, an activator of PrkC kinase, negated this inhibition, pointing to specific blockage of signal transduction in germinant receptor-mediated germination

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

Quantification of the impact of single and multiple mild stresses on outgrowth heterogeneity of Bacillus cereus spores

Outgrowth heterogeneity of bacterial spore populations complicates both prediction and efficient control of spore outgrowth. In this study, the impact of mild preservation stresses on outgrowth of Bacillus cereus ATCC 14579 spores was quantified during the first stages of outgrowth. Heterogeneity in outgrowth of heat-treated (90 °C for 10 min) and non-heat-treated germinated single spores to the maximum micro-colony stage of 256 cells was assessed by direct imaging on Anopore strips, placed on BHI plates at pH 7 and pH 5.5, without and with added NaCl or sorbic acid (HSA). At pH 7 non-heated and heat-treated germinated spores required 6 h to reach the maximum microcolony stage with limited heterogeneity, and these parameters were only slightly affected with both types of spores when incubated at pH 7 with added NaCl. Notably, the most pronounced effects were observed during outgrowth of spores at pH 5.5 without and with added NaCl or HSA. Non-heat-treated germinated spores showed again efficient outgrowth with limited heterogeneity reaching the maximum microcolony stage after 6 h at pH 5.5, which increased to 12 h and 16 h with added NaCl and HSA, respectively. In contrast, heat-treated spores displayed a strong delay between initial germination and swelling and further outgrowth at pH 5.5, resulting in large heterogeneity and low numbers of fastest growers reaching the maximum microcolony stage after 10, 12 and 24 h, without and with added NaCl or HSA, respectively. This work shows that Anopore technology provides quantitative information on the impact of combined preservation stresses on outgrowth of single spores, showing that outgrowth of germinated heat-treated spores is significantly affected at pH 5.5 with a large fraction of spores arrested in the early outgrowth stage, and with outgrowing cells showing large heterogeneity with only a small fraction committed to relatively fast outgrowth. Keywords Bacillus cereus; Spores; Outgrowth; Heterogeneity; Anopore; Stres

Quantification of the impact of single and multiple mild stresses on outgrowth heterogeneity of Bacillus cereus spores

Outgrowth heterogeneity of bacterial spore populations complicates both prediction and efficient control of spore outgrowth. In this study, the impact of mild preservation stresses on outgrowth of Bacillus cereus ATCC 14579 spores was quantified during the first stages of outgrowth. Heterogeneity in outgrowth of heat-treated (90 °C for 10 min) and non-heat-treated germinated single spores to the maximum micro-colony stage of 256 cells was assessed by direct imaging on Anopore strips, placed on BHI plates at pH 7 and pH 5.5, without and with added NaCl or sorbic acid (HSA). At pH 7 non-heated and heat-treated germinated spores required 6 h to reach the maximum microcolony stage with limited heterogeneity, and these parameters were only slightly affected with both types of spores when incubated at pH 7 with added NaCl. Notably, the most pronounced effects were observed during outgrowth of spores at pH 5.5 without and with added NaCl or HSA. Non-heat-treated germinated spores showed again efficient outgrowth with limited heterogeneity reaching the maximum microcolony stage after 6 h at pH 5.5, which increased to 12 h and 16 h with added NaCl and HSA, respectively. In contrast, heat-treated spores displayed a strong delay between initial germination and swelling and further outgrowth at pH 5.5, resulting in large heterogeneity and low numbers of fastest growers reaching the maximum microcolony stage after 10, 12 and 24 h, without and with added NaCl or HSA, respectively. This work shows that Anopore technology provides quantitative information on the impact of combined preservation stresses on outgrowth of single spores, showing that outgrowth of germinated heat-treated spores is significantly affected at pH 5.5 with a large fraction of spores arrested in the early outgrowth stage, and with outgrowing cells showing large heterogeneity with only a small fraction committed to relatively fast outgrowth. Keywords Bacillus cereus; Spores; Outgrowth; Heterogeneity; Anopore; Stres

Characterization of germination and outgrowth of sorbic acid-stressed Bacillus cereus ATCC 14579 spores: Phenotype and transcriptome analysis

Sorbic acid (SA) is widely used as a preservative, but the effect of SA on spore germination and outgrowth has gained limited attention up to now. Therefore, the effect of sorbic acid on germination of spores of Bacillus cereus strain ATCC 14579 was analyzed both at phenotype and transcriptome level. Spore germination and outgrowth were assessed at pH 5.5 without and with 0.75, 1.5 and 3.0 mM (final concentrations) undissociated sorbic acid (HSA). This resulted in distinct HSA concentration-dependent phenotypes, varying from reduced germination and outgrowth rates to complete blockage of germination at 3.0 mM HSA. The phenotypes reflecting different stages in the germination process could be confirmed using flow cytometry and could be recognized at transcriptome level by distinct expression profiles. In the absence and presence of 0.75 and 1.5 mM HSA, similar cellular ATP levels were found up to the initial stage of outgrowth, suggesting that HSA-induced inhibition of outgrowth is not caused by depletion of ATP. Transcriptome analysis revealed the presence of a limited number of transcripts in dormant spores, outgrowth related expression, and genes specifically associated with sorbic acid stress, including alterations in cell envelope and multidrug resistance. The potential role of these HSA-stress associated genes in spore outgrowth is discussed

Characterization of germination and outgrowth of sorbic acid-stressed Bacillus cereus ATCC 14579 spores: Phenotype and transcriptome analysis

Sorbic acid (SA) is widely used as a preservative, but the effect of SA on spore germination and outgrowth has gained limited attention up to now. Therefore, the effect of sorbic acid on germination of spores of Bacillus cereus strain ATCC 14579 was analyzed both at phenotype and transcriptome level. Spore germination and outgrowth were assessed at pH 5.5 without and with 0.75, 1.5 and 3.0 mM (final concentrations) undissociated sorbic acid (HSA). This resulted in distinct HSA concentration-dependent phenotypes, varying from reduced germination and outgrowth rates to complete blockage of germination at 3.0 mM HSA. The phenotypes reflecting different stages in the germination process could be confirmed using flow cytometry and could be recognized at transcriptome level by distinct expression profiles. In the absence and presence of 0.75 and 1.5 mM HSA, similar cellular ATP levels were found up to the initial stage of outgrowth, suggesting that HSA-induced inhibition of outgrowth is not caused by depletion of ATP. Transcriptome analysis revealed the presence of a limited number of transcripts in dormant spores, outgrowth related expression, and genes specifically associated with sorbic acid stress, including alterations in cell envelope and multidrug resistance. The potential role of these HSA-stress associated genes in spore outgrowth is discussed