Abiotic and microbiotic factors controlling biofilm formation by thermophilic sporeformers.

One of the major concerns in the production of dairy concentrates is the risk of contamination by heat-resistant spores from thermophilic bacteria. In order to acquire more insight in the composition of microbial communities occurring in the dairy concentrate industry, a bar-coded 16S amplicon sequencing analysis was carried out on milk, final products, and fouling samples taken from dairy concentrate production lines. The analysis of these samples revealed the presence of DNA from a broad range of bacterial taxa, including a majority of mesophiles and a minority of (thermophilic) spore-forming bacteria. Enrichments of fouling samples at 55°C showed the accumulation of predominantly Brevibacillus and Bacillus, whereas enrichments at 65°C led to the accumulation of Anoxybacillus and Geobacillus species. Bacterial population analysis of biofilms grown using fouling samples as an inoculum indicated that both Anoxybacillus and Geobacillus preferentially form biofilms on surfaces at air-liquid interfaces rather than on submerged surfaces. Three of the most potent biofilm-forming strains isolated from the dairy factory industrial samples, including Geobacillus thermoglucosidans, Geobacillus stearothermophilus, and Anoxybacillus flavithermus, have been characterized in detail with respect to their growth conditions and spore resistance. Strikingly, Geobacillus thermoglucosidans, which forms the most thermostable spores of these three species, is not able to grow in dairy intermediates as a pure culture but appears to be dependent for growth on other spoilage organisms present, probably as a result of their proteolytic activity. These results underscore the importance of abiotic and microbiotic factors in niche colonization in dairy factories, where the presence of thermophilic sporeformers can affect the quality of end products.

Virulence aspects of Listeria monocytogenes LO28 high pressure-resistant variants.

High pressure treatment is a novel food processing approach for reducing pathogens in foods and food ingredients. However, relatively little is known about the pathogenic potential of organisms that survive the treatment. Twelve previously isolated and characterized variants of Listeria monocytogenes LO28 obtained after a high pressure treatment were assessed for their virulence potential and antibiotic susceptibility. Ten variants showed attenuated virulence while two variants retained full virulence in a mouse model of infection. Seven of the attenuated variants demonstrated a reduction in virulence factor activity. Compared to the wild type, all variants exhibited similar or increased susceptibility to multiple antibiotics commonly used in listeriosis treatment.

A wide variety of Clostridium perfringens type A food-borne isolates that carry a chromosomal cpe gene belong to one multilocus sequence typing cluster.

Of 98 suspected food-borne Clostridium perfringens isolates obtained from a nationwide survey by the Food and Consumer Product Safety Authority in The Netherlands, 59 strains were identified as C. perfringens type A. Using PCR-based techniques, the cpe gene encoding enterotoxin was detected in eight isolates, showing a chromosomal location for seven isolates and a plasmid location for one isolate. Further characterization of these strains by using (GTG)(5) fingerprint repetitive sequence-based PCR analysis distinguished C. perfringens from other sulfite-reducing clostridia but did not allow for differentiation between various types of C. perfringens strains. To characterize the C. perfringens strains further, multilocus sequence typing (MLST) analysis was performed on eight housekeeping genes of both enterotoxic and non-cpe isolates, and the data were combined with a previous global survey covering strains associated with food poisoning, gas gangrene, and isolates from food or healthy individuals. This revealed that the chromosomal cpe strains (food strains and isolates from food poisoning cases) belong to a distinct cluster that is significantly distant from all the other cpe plasmid-carrying and cpe-negative strains. These results suggest that different groups of C. perfringens have undergone niche specialization and that a distinct group of food isolates has specific core genome sequences. Such findings have epidemiological and evolutionary significance. Better understanding of the origin and reservoir of enterotoxic C. perfringens may allow for improved control of this organism in foods.

Diversity in biofilm formation and production of curli fimbriae and cellulose of Salmonella Typhimurium strains of different origin in high and low nutrient medium.

The biofilm forming behavior of 51 Salmonella Typhimurium strains was determined in Tryptone Soya Broth (TSB) and 20 times diluted TSB (1/20TSB) at 25°C and 37°C. The results indicated that biofilm forming behavior is influenced by environmental conditions and associated with the origin of the strains. Clinical, outbreak-associated and retail product isolates showed dense biofilm formation in both media at 25°C, and in TSB also at 37°C. However, industrial isolates only showed dense biofilm formation in 1/20TSB at 25°C. By enumeration of biofilm cells, LIVE/DEAD staining and SEM analysis of biofilms it was found that the ratio of cells and extracellular matrix is affected by environmental conditions. Indeed, the genes involved in curli fimbriae and cellulose production are highly induced during biofilm formation at 25°C in 1/20TSB. This indicates that these are important matrix components during biofilm formation in 1/20TSB at 25°C and that other factors contribute to biofilm formation of clinical, outbreak-associated and retail product isolates at 37°C and/or nutrient-rich conditions.

Comparative analyses imply that the enigmatic Sigma factor 54 is a central controller of the bacterial exterior.

BACKGROUND: Sigma-54 is a central regulator in many pathogenic bacteria and has been linked to a multitude of cellular processes like nitrogen assimilation and important functional traits such as motility, virulence, and biofilm formation. Until now it has remained obscure whether these phenomena and the control by Sigma-54 share an underlying theme. RESULTS: We have uncovered the commonality by performing a range of comparative genome analyses. A) The presence of Sigma-54 and its associated activators was determined for all sequenced prokaryotes. We observed a phylum-dependent distribution that is suggestive of an evolutionary relationship between Sigma-54 and lipopolysaccharide and flagellar biosynthesis. B) All Sigma-54 activators were identified and annotated. The relation with phosphotransfer-mediated signaling (TCS and PTS) and the transport and assimilation of carboxylates and nitrogen containing metabolites was substantiated. C) The function annotations, that were represented within the genomic context of all genes encoding Sigma-54, its activators and its promoters, were analyzed for intra-phylum representation and inter-phylum conservation. Promoters were localized using a straightforward scoring strategy that was formulated to identify similar motifs. We found clear highly-represented and conserved genetic associations with genes that concern the transport and biosynthesis of the metabolic intermediates of exopolysaccharides, flagella, lipids, lipopolysaccharides, lipoproteins and peptidoglycan. CONCLUSION: Our analyses directly implicate Sigma-54 as a central player in the control over the processes that involve the physical interaction of an organism with its environment like in the colonization of a host (virulence) or the formation of biofilm.

Isolation of highly heat-resistant Listeria monocytogenes variants by use of a kinetic modeling-based sampling scheme.

Stable high-hydrostatic-pressure (HHP)-resistant Listeria monocytogenes LO28 variants were previously isolated and characterized. These HHP variants were also more resistant to heat. In addition, nonlinear heat inactivation kinetics pointed toward the existence of heat-resistant variants, although these could not be isolated so far. In this study, we used kinetic modeling of inactivation curves of two isolated HHP variants and their wild type, and this revealed that the probability of finding resistant variants should depend on the nature of the inactivation treatment and the time of exposure. At specific heat and HHP conditions, resistant LO28 and EGDe variants were indeed isolated. Resistant LO28 variants were even isolated after a heat inactivation at 72°C in milk, and these variants showed high resistance to standard pasteurization conditions. The increased resistance of part of the isolated LO28 and EGDe variants was due to mutations in their ctsR genes. For the variants whose ctsR genes and upstream regions were not altered, the mechanisms leading to increased resistance remain to be elucidated. This research showed the strength of kinetic modeling in unraveling the causes of nonlinear inactivation and facilitating the isolation of heat-resistant L. monocytogenes variants.

Germination and outgrowth of spores of Bacillus cereus group members: diversity and role of germinant receptors.

Bacillus cereus is a gram-positive, facultative anaerobic, endospore-forming toxicogenic human pathogen. Endospores are highly specialized, metabolically dormant cell types that are resistant to extreme environmental conditions, including heat, dehydration and other physical stresses. B. cereus can enter a range of environments, and can in its spore form, survive harsh conditions. If these conditions become favorable, spores can germinate and grow out and reach considerable numbers in a range of environments including processed foods. Certainly the last decade, when consumer preferences have shifted to mildly processed food, new opportunities arose for spore-forming spoilage and pathogenic organisms. Only rigorous methods have been shown to be capable of destroying all spores present in food, thus a shift toward e.g., milder heat preservation strategies, may result in low but significant amounts of viable spores in food products. Hence, the need for a mild spore destruction strategy is eminent including control of spore outgrowth. Consequently, there is a large interest in triggering spore germination in foodstuffs, since germinated spores have lost the extreme resistance of dormant spores and are relatively easy to kill. Another option could be to prevent germination so that no dangerous levels can be reached. This contribution will focus on germination and outgrowth characteristics of B. cereus and other members of the B. cereus group, providing an overview of the niches these spore-formers can occupy, the signals that trigger germination, and how B. cereus copes with these wake-up calls in different environments including foods, during food processing and upon interaction with the human host.

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 HCl. Comparative phenotype and transcriptome analyses revealed three acid stress-induced 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.) and peroxynitrite (ONOO(-))-specific fluorescent probe 3'-(p-hydroxyphenyl) 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.

A novel hybrid kinase is essential for regulating the sigma(B)-mediated stress response of Bacillus cereus.

A common bacterial strategy for monitoring environmental challenges is to use two-component systems, which consist of a sensor histidine kinase (HK) and a response regulator (RR). In the food-borne pathogen Bacillus cereus, the alternative sigma factor sigma(B) is activated by the RR RsbY. Here we present strong indications that the PP2C-type phosphatase RsbY receives its input from the multi-sensor hybrid kinase BC1008 (renamed RsbK). Genome analyses revealed that, across bacilli, rsbY and rsbK are located in a conserved gene cluster. A B. cereus rsbK deletion strain was shown to be incapable of inducing sigma(B) upon stress conditions and was impaired in its heat adaptive response. Comparison of the wild-type and rsbK mutant transcriptomes upon heat shock revealed that RsbK was primarily involved in the activation of the sigma(B)-mediated stress response. Truncation of the RsbK RR receiver domain demonstrated the importance of this domain for sigma(B) induction upon stress. The domain architecture of RsbK suggests that in the B. cereus group and in other bacilli, environmental and intracellular stress signalling routes are combined into one single protein. This strategy is markedly different from the sigma(B) activation pathway in other low-GC Gram-positives.

Direct-imaging-based quantification of Bacillus cereus ATCC 14579 population heterogeneity at a low incubation temperature.

Bacillus cereus ATCC 14579 was cultured in microcolonies on Anopore strips near its minimum growth temperature to directly image and quantify its population heterogeneity at an abusive refrigeration temperature. Eleven percent of the microcolonies failed to grow during low-temperature incubation, and this cold-induced population heterogeneity could be partly attributed to the loss of membrane integrity of individual cells.

Comparative transcriptomic and phenotypic analysis of the responses of Bacillus cereus to various disinfectant treatments.

Antimicrobial chemicals are widely applied to clean and disinfect food-contacting surfaces. However, the cellular response of bacteria to various disinfectants is unclear. In this study, the physiological and genome-wide transcriptional responses of Bacillus cereus ATCC 14579 exposed to four different disinfectants (benzalkonium chloride, sodium hypochlorite, hydrogen peroxide, and peracetic acid) were analyzed. For each disinfectant, concentrations leading to the attenuation of growth, growth arrest, and cell death were determined. The transcriptome analysis revealed that B. cereus, upon exposure to the selected concentrations of disinfectants, induced common and specific responses. Notably, the common response included genes involved in the general and oxidative stress responses. Exposure to benzalkonium chloride, a disinfectant known to induce membrane damage, specifically induced genes involved in fatty acid metabolism. Membrane damage induced by benzalkonium chloride was confirmed by fluorescence microscopy, and fatty acid analysis revealed modulation of the fatty acid composition of the cell membrane. Exposure to sodium hypochlorite induced genes involved in metabolism of sulfur and sulfur-containing amino acids, which correlated with the excessive oxidation of sulfhydryl groups observed in sodium hypochlorite-stressed cells. Exposures to hydrogen peroxide and peracetic acid induced highly similar responses, including the upregulation of genes involved in DNA damage repair and SOS response. Notably, hydrogen peroxide- and peracetic acid-treated cells exhibited high mutation rates correlating with the induced SOS response.

Population diversity of Listeria monocytogenes LO28: phenotypic and genotypic characterization of variants resistant to high hydrostatic pressure.

A comparative phenotype analysis of 24 Listeria monocytogenes LO28 stress-resistant variants obtained after high-pressure treatment was performed to assess their robustness and growth performance under a range of food-relevant conditions. In addition, genetic analysis was conducted to characterize the promoter regions and open reading frames of the class I and III transcriptional repressors CtsR and HrcA, which control production of specific sets of stress proteins. Analysis of stress survival capacity, motility, biofilm formation, and growth under various conditions showed all variants to be more resistant to pressure and heat than the wild type; however, differences among variants were observed in acid resistance, growth rate, motility, and biofilm-forming capacity. Genetic analysis revealed no variation in the genetic make-up of hrcA and its upstream region, but two variants had deletions in the upstream region of ctsR and seven variants had mutations in the ctsR gene itself. The results of the characterization were cluster analyzed to obtain insight into the diversity of variants. Ten unique variants and three clusters with specific features could be identified: one cluster consisting of seven variants having a mutation in the CtsR regulator gene, one cluster containing two variants with an aerobic biofilm formation capacity similar to that of the wild type, and a cluster composed of five immotile variants. The large population diversity of L. monocytogenes stress-resistant variants signifies the organism's genetic flexibility, which in turn may contribute to the survival and persistence of this human pathogen in food-processing environments.

Phenotypic and transcriptomic analyses of mildly and severely salt-stressed Bacillus cereus ATCC 14579 cells.

Bacteria are able to cope with the challenges of a sudden increase in salinity by activating adaptation mechanisms. In this study, exponentially growing cells of the pathogen Bacillus cereus ATCC 14579 were exposed to both mild (2.5% [wt/vol] NaCl) and severe (5% [wt/vol] NaCl) salt stress conditions. B. cereus continued to grow at a slightly reduced growth rate when it was shifted to mild salt stress conditions. Exposure to severe salt stress resulted in a lag period, and after 60 min growth had resumed, with cells displaying a filamentous morphology. Whole-genome expression analyses of cells exposed to 2.5% salt stress revealed that the expression of these cells overlapped with the expression of cells exposed to 5% salt stress, suggesting that the corresponding genes were involved in a general salt stress response. Upregulation of osmoprotectant, Na(+)/H(+), and di- and tripeptide transporters and activation of an oxidative stress response were noticeable aspects of the general salt stress transcriptome response. Activation of this response may confer cross-protection against other stresses, and indeed, increased resistance to heat and hydrogen peroxide could be demonstrated after preexposure to salt. A temporal shift between the transcriptome response and several phenotypic responses of severely salt-stressed cells was observed. After resumption of growth, these cells showed cellular filamentation, reduced chemotaxis, increased catalase activity, and optimal oxidative stress resistance, which corresponded to the transcriptome response displayed in the initial lag period. The linkage of transcriptomes and phenotypic characteristics can contribute to a better understanding of cellular stress adaptation strategies and possible cross-protection mechanisms.

Inactivation kinetics of three Listeria monocytogenes strains under high hydrostatic pressure.

High hydrostatic pressure (HHP) inactivation of three Listeria monocytogenes strains (EGDe, LO28, and Scott A) subjected to 350 MPa at 20 degrees C in ACES buffer resulted in survival curves with significant tailing for all three strains. A biphasic linear model could be fitted to the inactivation data, indicating the presence of an HHP-sensitive and an HHP-resistant fraction, which both showed inactivation according to first-order kinetics. Inactivation parameters of these subpopulations of the three strains were quantified in detail. EGDe showed the highest D-values for the sensitive and resistant fraction, whereas LO28 and Scott A showed lower HHP resistance for both fractions. Survivors isolated from the tail of LO28 and EGDe were analyzed, and it was revealed that the higher resistance of LO28 was a stable feature for 24% (24 of 102) of the resistant fraction. These HHP-resistant variants were 10 to 600,000 times more resistant than wild type when exposed to 350 MPa at 20 degrees C for 20 min. Contrary to these results, no stable HHP-resistant isolates were found for EGDe (0 of 102). The possible effect of HHP survival capacity of stress-resistant genotypic and phenotypic variants of L. monocytogenes on the safety of HHP-processed foods is discussed.

Surface behaviour of S. Typhimurium, S. Derby, S. Brandenburg and S. Infantis.

Cross-contamination due to Salmonella on the surface of processing equipment greatly contributes to contamination of pork products. Therefore, a clear understanding of surface and survival behaviour of relevant Salmonella serovars in pork processing environments is needed to develop better strategies for Salmonella control. Within this study the biofilm forming behaviour of S. Typhimurium, S. Derby, S. Brandenburg and S. Infantis isolates was analysed using the crystal violet assay. This assay, commonly used to analyse total biofilm formation, revealed variation in biofilm forming capacity between and within serovars. This has not been shown before for S. Derby, S. Brandenburg and S. Infantis. From each serovar, isolates with different biofilm forming capacity were selected to analyse biofilm formation on stainless steel. This revealed no significant differences between biofilm formation on polystyrene compared to stainless steel. Furthermore a relation was observed between biofilm forming capacity of an isolate and survival on stainless steel surfaces. On such surfaces, biofilms showed greater and longer survival than planktonic cells, and they were less susceptible to peracetic acid disinfection treatments. However, the latter effect was marginal and only observed in the presence of organic material, which drastically decreased the activity of peracetic acid. With the obtained results a hierarchical cluster was also performed to identify differences and similarities between the four different serovars. This indicated that the surface behaviour of S. Typhimurium was more comparable to S. Infantis than to S. Derby or S. Brandenburg.

Isolation and quantification of highly acid resistant variants of Listeria monocytogenes.

Heterogeneity in stress response of bacteria is one of the biggest challenges posed by minimal processing, which aims at finding the balance between microbiologically stable foods while maintaining the characteristics of fresh products. In this study, exposure of Listeria monocytogenes LO28 to acid stress, which can be encountered in the food processing environment as well as in the human body upon ingestion, led to inactivation kinetics showing considerable tailing, which was described by a biphasic inactivation model. Stable acid resistant variants of L. monocytogenes LO28 were isolated after exposure of late-exponential phase cells to pH 3.5 for 90 min. The resulting 23 stable resistant isolates could be divided in three groups: (a) highly increased acid resistance (<1 log10 reduction, n=16), (b) slightly increased acid resistance (1-3 log10 reduction, n=6), and (c) one isolate showing a variable acid stress response. The highly acid resistant group showed increased resistance to the tested pH range of 2.5 to 3.5 in both late-exponential and stationary phase. Increased acid resistance showed to be significantly correlated to reduced growth rate. The Weibull model was reparameterized, resulting in improved parameter estimation, and was used to estimate the inactivation kinetics at mild pH. Studying the growth boundaries of the wild type and a representative set of variants indicated that the increased resistance of the variants was only related to survival of severe pH stress but did not allow for better growth or survival at mild pH stress. This study shows that acid exposure of late-exponential phase cells reveals the presence of acid resistant subpopulations and that there is a phenotypic diversity amongst them. The occurrence of heterogeneity and stress resistant subpopulations may lead to a higher number of surviving microorganisms than expected. Also, stress resistant subpopulations can become part of the domestic flora in a food production line. The currently isolated acid resistant variants are a new group of stress resistant variants and underline the importance of gaining more insight in the mechanisms underlying this heterogeneity and increased resistance.

Diversity assessment of Listeria monocytogenes biofilm formation: impact of growth condition, serotype and strain origin.

The foodborne pathogen Listeria monocytogenes has the ability to produce biofilms in food-processing environments and then contaminate food products, which is a major concern for food safety. The biofilm forming behavior of 143 L. monocytogenes strains was determined in four different media that were rich, moderate or poor in nutrients at 12°C, 20°C, 30°C and 37°C. The biofilm formation was mostly influenced by temperature, resulting in decreased biofilm formation with decreasing temperature. Biofilm formation was enhanced in nutrient-poor medium rather than in nutrient-rich medium, and especially in nutrient-poor medium significantly enhanced biofilm production was observed early in biofilm maturation underlining the effect of medium on biofilm formation rate. Also serotype had a significant effect on biofilm formation and was influenced by medium used because strains from both serotype 1/2b and 1/2a formed more biofilm than serotype 4b strains in nutrient-rich medium at 20°C, 30°C and 37°C, whereas in nutrient-poor medium the biofilm production levels of serotype 1/2a and 4b strains were rather similar and lower than serotype 1/2b strains. The strains used originated from various origins, including dairy, meat, industrial environment, human and animal, and the level of biofilm formation was not significantly affected by the origin of isolation, irrespective of medium used and temperature tested. A linear model was used to correlate crystal violet staining of biofilm production to the number of viable cells within the biofilm. This showed that crystal violet staining was poorly correlated to the number of viable cells in nutrient-poor medium, and LIVE/DEAD staining and DNase I treatment revealed that this could be attributed to the presence of non-viable cells and extracellular DNA in the biofilm matrix. The significant impact of intrinsic and extrinsic factors on biofilm production of L. monocytogenes underlined that niche-specific features determine the levels of biofilm produced, and insights in biofilm formation characteristics will allow us to further optimize strategies to control the biofilm formation of L. monocytogenes.

Germinant receptor diversity and germination responses of four strains of the Bacillus cereus group.

Four strains of the Bacillus cereus group were compared for their germinant receptor composition and spore germination capacity. Phylogenetic analysis of the germinant receptor encoding operons of the enterotoxic strains B. cereus ATCC 14579 and ATCC 10987, the emetic strain AH187, and the psychrotolerant strain Bacillus weihenstephanensis KBAB4, indicated a core group of five germinant receptor operons to be present in the four strains, with each strain containing one to three additional receptors. Using quantitative PCR, induction of expression during sporulation was confirmed for all identified germinant receptor operons in these strains. Despite the large overlap in receptors, diversity in amino acid-induced germination capacity was observed, with six out of 20 amino acids, serving as germinants for spores of all four strains. Each strain showed unique features: efficient germination of strain KBAB4 spores required non-inducing amounts of inosine as the co-germinant, strain ATCC 10987 spores germinated only efficiently after heat activation. Furthermore, strain ATCC 14579 and AH187 spores germinated without heat activation or inosine, with strain ATCC 14579 spores being triggered by all amino acids except phenylalanine and strain AH187 spores being specifically triggered efficiently only by phenylalanine. Analysis of all germination data did not reveal strict linkages between specific germinants and germinant receptors. Finally, the diversity in nutrient-induced germination capacity was also reflected in the diverse germination responses of heat-activated spores of the four B. cereus strains in food matrices, such as milk, rice water and meat bouillon, indicating that amino acid composition and/or availability of inosine are important germination determinants in foods.

Short- and long-term biomarkers for bacterial robustness: a framework for quantifying correlations between cellular indicators and adaptive behavior.

The ability of microorganisms to adapt to changing environments challenges the prediction of their history-dependent behavior. Cellular biomarkers that are quantitatively correlated to stress adaptive behavior will facilitate our ability to predict the impact of these adaptive traits. Here, we present a framework for identifying cellular biomarkers for mild stress induced enhanced microbial robustness towards lethal stresses. Several candidate-biomarkers were selected by comparing the genome-wide transcriptome profiles of our model-organism Bacillus cereus upon exposure to four mild stress conditions (mild heat, acid, salt and oxidative stress). These candidate-biomarkers--a transcriptional regulator (activating general stress responses), enzymes (removing reactive oxygen species), and chaperones and proteases (maintaining protein quality)--were quantitatively determined at transcript, protein and/or activity level upon exposure to mild heat, acid, salt and oxidative stress for various time intervals. Both unstressed and mild stress treated cells were also exposed to lethal stress conditions (severe heat, acid and oxidative stress) to quantify the robustness advantage provided by mild stress pretreatment. To evaluate whether the candidate-biomarkers could predict the robustness enhancement towards lethal stress elicited by mild stress pretreatment, the biomarker responses upon mild stress treatment were correlated to mild stress induced robustness towards lethal stress. Both short- and long-term biomarkers could be identified of which their induction levels were correlated to mild stress induced enhanced robustness towards lethal heat, acid and/or oxidative stress, respectively, and are therefore predictive cellular indicators for mild stress induced enhanced robustness. The identified biomarkers are among the most consistently induced cellular components in stress responses and ubiquitous in biology, supporting extrapolation to other microorganisms than B. cereus. Our quantitative, systematic approach provides a framework to search for these biomarkers and to evaluate their predictive quality in order to select promising biomarkers that can serve to early detect and predict adaptive traits.

Comparative analysis of transcriptional and physiological responses of Bacillus cereus to organic and inorganic acid shocks.

Comparative phenotype and transcriptome analyses were performed with Bacillus cereus ATCC 14579 exposed to pH 5.5 set with different acidulants including hydrochloric acid (HCl), lactic acid (HL) and acetic acid (HAc). Phenotypes observed included a decreased growth rate (with HCl), bacteriostatic and bactericidal conditions, with 2mM undissociated HAc or HL, and 15mM undissociated HAc, respectively. In the latter condition a concomitant decrease in intracellular ATP levels was observed. The transcriptome analyses revealed general and specific responses to the acidulants used. The general acid stress response includes modulation of pyruvate metabolism with activation of the butanediol fermentation pathway, and an oxidative stress response that was, however, more extensive in the bacteriostatic and bactericidal conditions. HL-specific and HAc-specific responses include modulation of metabolic pathways for amino acid metabolism. Activation of lactate, formate, and ethanol fermentation pathways, alternative electron-transport chain components and fatty acid biosynthesis genes was noted in the presence of 15mM undissociated HAc. In conclusion, our study has provided insights in phenotype-associated, and general and acidulant-specific responses in B. cereus.