Colonization and spreading dynamics of Lactiplantibacillus plantarum spoilage isolates on wet surfaces.

The role of lactic acid bacteria, including Lactiplantibacillus plantarum, in food spoilage is well recognized, while the behavior of these non-motile bacteria on wet surfaces, such as those encountered in food processing environments has gained relatively little attention. Here, we observed a fast colony spreading of non-motile L. plantarum spoilage isolates on wet surfaces via passive sliding using solid BHI agar media as a model. We investigated the effect of physical properties of agar hydrogel substrate on the surface spreading of six L. plantarum food isolates FBR1-6 and a model strain WCFS1, using increasing concentrations of agar from 0.25 up to 1.5% (w/v). Our results revealed that L. plantarum strain FBR2 spreads significantly on low agar concentration plates compared to the other strains studied here (with a factor of 50-60 folds higher surface coverage), due to the formation of very soft biofilms with high water content that can float on the surface. The fast-spreading of FBR2 colonies is accompanied by an increased number of cells, elongated cell morphology, and a higher amount of extracellular components. Our finding highlights colonization dynamics and the spreading capacity of non-motile bacteria on surfaces that are relatively wet, thereby revealing an additional hitherto unnoticed parameter for non-motile bacteria that may contribute to contamination of foods by fast surface spreading of these bacteria in food processing environments.

Enhanced stress resistance of Bifidobacterium breve NRBB57 by induction of stress proteins at near-zero growth rates.

Bifidobacterium breve is a common habitant of the human gut and is used as probiotic in functional foods. B. breve has to cope with multiple stress conditions encountered during processing and passage through the human gut, including high temperature, low pH and exposure to oxygen. Additionally, during industrial processing and in the gut, B. breve could encounter nutrient limitation resulting in reduced growth rates that can trigger adaptive stress responses. For this reason, it is important to develop culture methods that elicit resistance to multiple stresses (robustness) encountered by the bacteria. To investigate the impact of caloric restriction on robustness of the probiotic B. breve NRBB57, this strain was grown in lactose-limited chemostat cultures and in retentostat for 21 days, at growth rates ranging from 0.4 h-1 to 0.00081 h-1. Proteomes of cells harvested at different growth rates were correlated to acid, hydrogen peroxide and heat stress survival capacity. Comparative proteome analysis showed that retentostat-grown cells had significantly increased abundance of a variety of stress proteins involved in protein quality maintenance and DNA repair (DnaJ, Hsp90, FtsH, ClpB, ClpP1, ClpC, GroES, RuvB, RecA), as well as proteins involved in oxidative stress defence (peroxiredoxin, ferredoxin, thioredoxin peroxidase, glutaredoxin and thioredoxin reductase). Exposure to three different stress conditions, 45 °C, pH 3, and 10 mM H2O2, showed highest stress resistance of retentostat cells sampled at week 2 and week 3 grown at 0.0018 and 0.00081 h-1. Our findings show that cultivation at near-zero growth rates induces higher abundance of stress defence proteins contributing to the robustness of B. breve NRBB57, thereby offering an approach that may support its production and functionality.

Combining enrichment with multiplex real-time PCR leads to faster detection and identification of Campylobacter spp. in food compared to ISO 10272-1:2017.

Conventional protocols for the detection of Campylobacter from foods are laborious and time-consuming. This research describes an alternative procedure (EMRT-PCR) for the detection of Campylobacter from food by combining ISO 10272-1:2017 enrichment in Bolton broth (BB) with a multiplex real-time (MRT-) PCR assay. Species differentiation was done by targeting C. jejuni (mapA), C. coli (ceuE), and both species (cje). The detection limit of the MRT-PCR assay was 4.5 and 5.5 log10 cfu/ml in BB and BB containing chicken skin, respectively. A Monte Carlo simulation was conducted to predict the probability that campylobacters reach the MRT-PCR detection threshold throughout enrichment in BB, and results suggested that cold-stressed campylobacters could reach the detection limit after 40 h of enrichment (p = 0.99). As a proof of principle, 23 naturally contaminated meat products were enriched according to ISO 10272-1:2017 procedure A, and the EMRT-PCR in parallel. After 24 h, 12 and 11 samples already tested positive for Campylobacter with the ISO method and EMRT-PCR, respectively. After 40 h, the 24-h-negative sample was also positive with EMRT-PCR. The EMRT-PCR takes about 2 days to produce reliable results, while results using ISO 10272-1:2017 can take up to 8 days, which demonstrate the potential of the EMRT-PCR method.

Role of substrate availability in the growth of Campylobacter co-cultured with extended spectrum beta-lactamase-producing Escherichia coli in Bolton broth.

It is well-established that Extended-spectrum beta-lactamase-producing (ESBL-) Escherichia coli challenge reliable detection of campylobacters during enrichment in Bolton broth (BB) following ISO 10272-1:2017. The overgrowth of Campylobacter by ESBL-E. coli in the enrichment medium BB can lead to false-negative detection outcomes, but the cause for the growth suppression is yet unknown. A plausible reason could be the competition-induced lack of certain growth substrates. Therefore, this study aimed to investigate whether campylobacters and ESBL-E. coli compete for the same medium components and whether this is the cause for the observed growth repression. The availability of possible growth substrates in BB was determined and changes in their extracellular concentration were measured over time during mono-culture enrichment of C. jejuni, C. coli or ESBL-E. coli as well as in co-culture enrichments of campylobacters and ESBL-E. coli. Comparative analysis showed lactate and fumarate utilization by C. jejuni and C. coli exclusively, whereas ESBL-E. coli rapidly consumed asparagine, glutamine/arginine, lysine, threonine, tryptophan, pyruvate, glycerol, cellobiose, and glucose. Both campylobacters and ESBL-E. coli utilized aspartate, serine, formate, a-ketoglutarate and malate. Trends in compound utilization were similar for C. jejuni and C. coli and trends in compound utilization were rather comparable during enrichment of reference and freeze-stressed campylobacters. Since final cell densities of C. jejuni and C. coli in co-cultures were not enhanced by the addition of surplus l-serine and final cell densities were similar in fresh and spent medium, growth suppression seems not to be caused by a lack of substrates or production of inhibitory compounds. We hypothesized that oxygen availability was limiting growth in co-cultures. Higher oxygen availability increased the competitive fitness of C. jejuni 81-176 in co-culture with ESBL-E. coli in duplicate experiments, as cell concentrations in stationary phase were similar to those without competition. This could indicate the critical role of oxygen availability during the growth of Campylobacter and offers potential for further improvement of Campylobacter spp. enrichment efficacy.

Lactobacillus plantarum strains show diversity in biofilm formation under flow conditions.

In many natural and technological applications, microbial biofilms grow under fluid flow. In this project, we investigated the influence of flow on the formation and growth of biofilms produced by gram-positive Lactobacillus plantarum strains WCFS1 and CIP104448. We used an in-house designed device based on a 48-well plate with culture volumes of 0.8 ml, and quantified total biofilm formation under static and flow conditions with flow rates 0.8, 1.6, 3.2 and 4.8 ml/h (with 1, 2, 4 and 6 volume changes per hour) using crystal violet (CV) staining, and determined the number of viable biofilm cells based on plate counts. The amount of total biofilm under flow conditions increased in the CIP 104448 strain, with significantly increased staining at the wall of the wells. However, in the WCFS1 strain, no significant difference in the amount of biofilm formed under flow and static conditions was observed. Plate counts showed that flow caused an increase in the number of viable biofilm cells for both strains. In addition, using enzyme treatment experiments, we found that for WCFS1 in the static condition, the amount of mature biofilm was declined after DNase I and Proteinase K treatment, while for flow conditions, the decline was only observed for DNase I treatment. The CIP104448 biofilms formed under both static and flow conditions only showed a decline in the CV staining after adding Proteinase K, indicating different contributions of extracellular DNA (eDNA) and proteinaceous matrix components to biofilm formation in the tested strains.

Variability in lag-duration of Campylobacter spp. during enrichment after cold and oxidative stress and its impact on growth kinetics and reliable detection.

Campylobacter jejuni and Campylobacter coli continue to be the leading cause of zoonotic gastroenteritis in the European Union, making reliable detection in food important. Low storage temperatures and atmospheric oxygen concentrations during food production can cause sub-lethal damage or transient non-culturability which is why ISO 10272-1:2017 includes an enrichment step to repair cell damage and increase cell concentrations, thereby supporting detection of campylobacters from foods. The aim of this study was to assess the variability in lag-duration of C. jejuni and C. coli during enrichment after different food-relevant stress treatments and evaluate its impact on growth kinetics and reliability of detection outcomes. Therefore, 13 C. jejuni and 10 C. coli strains were subjected to cold stress during refrigerated and frozen storage. Refrigerated storage did not significantly reduce culturability, but frozen storage reduced cell concentrations by 1.6 ± 0.1 log10cfu/ml for both species. Subsequently, cells were enriched following ISO 10272-1:2017-A and cell concentrations were determined over time and lag-duration and growth rate were determined by fitting the Baranyi-model. Without prior stress treatment, mean lag-duration for C. jejuni and C. coli was 2.5 ± 0.2 h and 2.2 ± 0.3 h, respectively. Refrigerated storage increased lag-duration for C. jejuni to 4.6 ± 0.4 h and for C. coli to 5.0 ± 0.4 h and frozen storage increased lag-duration to 5.0 ± 0.3 h and 6.1 ± 0.4 h for C. jejuni and C. coli, respectively. Comparison of strain- and biological variability showed that differences in recovery after cold stress can be attributed mainly to strain variability since strain variability after refrigeration and freeze stress increased respectively 3-fold and 4-fold while biological variability remained constant. A subset of strains was also subjected to oxidative stress that reduced cell concentrations by 0.7 ± 0.2 log10 cfu/ml and comparison of recovery patterns after oxidative and freeze stress indicated that recovery behaviour was also dependent on the stress applied. A scenario analysis was conducted to evaluate the impact of heterogeneity in outgrowth kinetics of single cells on the reliability of detection outcomes following ISO protocol 10272-1:2017. This revealed that a 'worst-case'-scenario for successful detection by a combination of the longest lag-duration of 7.6 h and lowest growth rate of 0.47 h-1 still resulted in positive detection outcomes since the detection limit was reached within 32.5 h. This suggests that other factors such as competitive microbiota can act as a causative factor in false-negative outcomes of tested food samples.

Short communication: Growth of dairy isolates of Geobacillus thermoglucosidans in skim milk depends on lactose degradation products supplied by Anoxybacillus flavithermus as secondary species.

Thermophilic bacilli such as Anoxybacillus and Geobacillus are important contaminants in dairy powder products. Remarkably, one of the common contaminants, Geobacillus thermoglucosidans, showed poor growth in skim milk, whereas significant growth of G. thermoglucosidans was observed in the presence of an Anoxybacillus flavithermus dairy isolate. In the present study, we investigated the underlying reason for this growth dependence of G. thermoglucosidans. Whole-genome sequences of 4 A. flavithermus strains and 4 G. thermoglucosidans strains were acquired, with special attention given to carbohydrate utilization clusters and proteolytic enzymes. Focusing on traits relevant for dairy environments, comparative genomic analysis revealed that all G. thermoglucosidans strains lacked the genes necessary for lactose transport and metabolism, showed poor growth in skim milk, and produced white colonies on X-gal plates, indicating the lack of ß-galactosidase activity. The A. flavithermus isolates scored positive in these tests, consistent with the presence of a putative lactose utilization gene cluster. All tested isolates from both species showed proteolytic activity on milk plate count agar plates. Adding glucose or galactose to liquid skim milk supported growth of G. thermoglucosidans isolates, in line with the presence of the respective monosaccharide utilization gene clusters in the genomes. Analysis by HPLC of A. flavithermus TNO-09.006 culture filtrate indicated that the previously described growth dependence of G. thermoglucosidans in skim milk was based on the supply of glucose and galactose by A. flavithermus TNO-09.006.

Impact of Pathogen Population Heterogeneity and Stress-Resistant Variants on Food Safety.

This review elucidates the state-of-the-art knowledge about pathogen population heterogeneity and describes the genotypic and phenotypic analyses of persister subpopulations and stress-resistant variants. The molecular mechanisms underlying the generation of persister phenotypes and genetic variants are identified. Zooming in on Listeria monocytogenes, a comparative whole-genome sequence analysis of wild types and variants that enabled the identification of mutations in variants obtained after a single exposure to lethal food-relevant stresses is described. Genotypic and phenotypic features are compared to those for persistent strains isolated from food processing environments. Inactivation kinetics, models used for fitting, and the concept of kinetic modeling-based schemes for detection of variants are presented. Furthermore, robustness and fitness parameters of L. monocytogenes wild type and variants are used to model their performance in food chains. Finally, the impact of stress-resistant variants and persistence in food processing environments on food safety is discussed.

Strain diversity and phage resistance in complex dairy starter cultures.

The compositional stability of the complex Gouda cheese starter culture Ur is thought to be influenced by diversity in phage resistance of highly related strains that co-exist together with bacteriophages. To analyze the role of bacteriophages in maintaining culture diversity at the level of genetic lineages, simple blends of Lactococcus lactis strains were made and subsequently propagated for 152 generations in the absence and presence of selected bacteriophages. We first screened 102 single-colony isolates (strains) from the complex cheese starter for resistance to bacteriophages isolated from this starter. The collection of isolates represents all lactococcal genetic lineages present in the culture. Large differences were found in bacteriophage resistance among strains belonging to the same genetic lineage and among strains from different lineages. The blends of strains were designed such that 3 genetic lineages were represented by strains with different levels of phage resistance. The relative abundance of the lineages in blends with phages was not stable throughout propagation, leading to continuous changes in composition up to 152 generations. The individual resistance of strains to phage predation was confirmed as one of the factors influencing starter culture diversity. Furthermore, loss of proteolytic activity of initially proteolytic strains was found. Reconstituted blends with only 4 strains with a variable degree of phage resistance showed complex behavior during prolonged propagation.

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 pH7 and pH5.5, without and with added NaCl or sorbic acid (HSA). At pH7 non-heated and heat-treated germinated spores required 6h to reach the maximum microcolony stage with limited heterogeneity, and these parameters were only slightly affected with both types of spores when incubated at pH7 with added NaCl. Notably, the most pronounced effects were observed during outgrowth of spores at pH5.5 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 6h at pH5.5, which increased to 12h 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 pH5.5, resulting in large heterogeneity and low numbers of fastest growers reaching the maximum microcolony stage after 10, 12 and 24h, 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 pH5.5 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.

Physiological and transcriptional response of Bacillus cereus treated with low-temperature nitrogen gas plasma.

AIMS: This study was conducted to investigate the inactivation kinetics of Bacillus cereus vegetative cells upon exposure to low-temperature nitrogen gas plasma and to reveal the mode of inactivation by transcriptome profiling. METHODS AND RESULTS: Exponentially growing B. cereus cells were filtered and put on agar plates. The plates, carrying the filters with the vegetative cells, were placed into low-temperature nitrogen gas plasma at atmospheric pressure. After different exposure times, the cells were harvested for RNA extraction and enumeration. The RNA was used to perform whole-transcriptome profiling using DNA microarrays. The transcriptome profile showed a large overlap with profiles obtained from conditions generating reactive oxygen species in B. cereus. However, excess radicals such as peroxynitrite, hydroxyl and superoxide could not be detected using radical-specific fluorescence staining. Lack of UV-specific responses including factors involved in DNA damage repair is in line with the absence of UV-specific emission in the afterglow of the nitrogen gas plasma as analysed using optical emission spectroscopy (OES). CONCLUSIONS: Antibacterial activity of nitrogen gas plasma is not based on UV radiation. Exposure to nitrogen gas plasma leads to oxidative stress and inactivation of targeted cells. A secondary oxidative stress with the indicative formation of reactive oxygen species within cells could not be observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first investigation of differential gene expression on a genome-wide scale in B. cereus following nitrogen gas plasma exposure. This study may help to design economically feasible, safe and effective plasma decontamination devices.

Prediction of Bacillus weihenstephanensis acid resistance: the use of gene expression patterns to select potential biomarkers.

Exposure to mild stress conditions can activate stress adaptation mechanisms and provide cross-resistance towards otherwise lethal stresses. In this study, an approach was followed to select molecular biomarkers (quantitative gene expressions) to predict induced acid resistance after exposure to various mild stresses, i.e. exposure to sublethal concentrations of salt, acid and hydrogen peroxide during 5 min to 60 min. Gene expression patterns of unstressed and mildly stressed cells of Bacillus weihenstephanensis were correlated to their acid resistance (3D value) which was estimated after exposure to lethal acid conditions. Among the twenty-nine candidate biomarkers, 12 genes showed expression patterns that were correlated either linearly or non-linearly to acid resistance, while for the 17 other genes the correlation remains to be determined. The selected genes represented two types of biomarkers, (i) four direct biomarker genes (lexA, spxA, narL, bkdR) for which expression patterns upon mild stress treatment were linearly correlated to induced acid resistance; and (ii) nine long-acting biomarker genes (spxA, BcerKBAB4_0325, katA, trxB, codY, lacI, BcerKBAB4_1716, BcerKBAB4_2108, relA) which were transiently up-regulated during mild stress exposure and correlated to increased acid resistance over time. Our results highlight that mild stress induced transcripts can be linearly or non-linearly correlated to induced acid resistance and both approaches can be used to find relevant biomarkers. This quantitative and systematic approach opens avenues to select cellular biomarkers that could be incremented in mathematical models to predict microbial behaviour.

Sporulation environment of emetic toxin-producing Bacillus cereus strains determines spore size, heat resistance and germination capacity.

AIM: Heat resistance, germination and outgrowth capacity of Bacillus cereus spores in processed foods are major factors in causing the emetic type of gastrointestinal disease. In this study, we aim to identify the impact of different sporulation conditions on spore properties of emetic toxin-producing B. cereus strains. METHODS AND RESULTS: Spore properties of eight different emetic toxin-producing strains were tested, with spores produced in five different sporulation conditions: aerated liquid cultures, air-liquid biofilms, 1.5% agar plates, 0.75% agar plates and swarming colonies. Model food studies revealed spores from emetic toxin-producing strains to germinate efficiently on meat broth- and milk-based agar plates, whereas germination on rice-based agar plates was far less efficient. Notably, spores of all strains germinated efficiently when 0.1% meat broth was added to the rice plates. Analysis of spores derived from different environments revealed large diversity and showed biofilm spores for the strains tested to be the largest in size, the most heat resistant and with the lowest germination capacity. CONCLUSIONS: Sporulation in complex conditions such as biofilms and surface swarming colonies increases heat resistance and dormancy of spores. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained imply the importance of sporulation conditions on spore properties of emetic toxin-producing B. cereus strains, as occur for instance in food processing.

Germination inhibition of Bacillus cereus spores: impact of the lipophilic character of inhibiting compounds.

In this study, the impact of a range of organic acids and structurally similar alcohols with three to six carbon backbones and increasing lipophilic character, were tested on the germination behavior of B. cereus ATCC 14579 spores. This approach allowed substantiating whether the effectivity of the various compounds was largely dictated by membrane interference or a classic weak acid acidification effect. The octanol-water partition coefficient (log P(oct/water)) ranges from 0.25/0.33 to 2.03/1.96 for propanol/undissociated propionic acid and hexanol/undissociated hexanoic acid, respectively. Performance of germination assays at neutral (pH7) and acidic conditions (pH5.5) allowed for a comparative analysis of the action of dissociated versus undissociated acids, and the presumed pH-independent effect of the corresponding alcohols. Germination assays, based on both continuously measured optical density and time-based plating experiments, and microscopic observations demonstrated the correlation between the lipophilic character of the selected compounds and their inhibiting effect on spore germination. Real-time fluorescence based assays showed that membrane integrity in dormant spores was maintained in the presence of the tested inhibitors. Lowering the critical concentration of inhibitors by a one-step washing procedure resulted in the onset of nutrient-induced germination, indicating the reversible nature of the inhibition process. Furthermore, blocking of nutrient-induced germination in the presence of inhibitory concentrations of selected lipophilic acids and corresponding alcohols was by-passed upon addition of Ca-dipicolinic acid, pointing to loss of signaling capacity in germinant receptor-mediated germination activity. These findings show that lipophilicity is an important determinant for the ability of the selected acids and corresponding alcohols to accumulate in the spore inner membrane and their ability to act as a germination-inhibitor.

Arginine metabolism in sugar deprived Lactococcus lactis enhances survival and cellular activity, while supporting flavour production.

Flavour development in cheese is affected by the integrity of Lactococcus lactis cells. Disintegrated cells enhance for instance the enzymatic degradation of casein to free amino acids, while integer cells are needed to produce specific flavour compounds from amino acids. The impact of the cellular activity of these integer cells on flavour production remains to be elucidated. In this study we investigated whether lactose-deprived L. lactis cells that use arginine as an alternative energy source can extend cellular activity and produce more specific flavours. In cheese experiments we demonstrated that arginine metabolising cells survived about 3 times longer than non-arginine metabolising cells, which suggests prolonged cellular activity. Cellular activity and flavour production of L. lactis was further studied in vitro to enable controlled arginine supplementation. Comparable with the results found in cheese, the survival rates of in vitro incubated cells improved when arginine was metabolised. Furthermore, elongated cellular activity was reflected in 3-4-fold increased activity of flavour generating enzymes. The observed prolonged cellular activity resulted in about 2-fold higher concentrations of typical Gouda cheese flavours. These findings provide new leads for composing starter cultures that will produce specific flavour compounds.

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.

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.

Soya bean tempe extracts show antibacterial activity against Bacillus cereus cells and spores.

AIMS: Tempe, a Rhizopus ssp.-fermented soya bean food product, was investigated for bacteriostatic and/or bactericidal effects against cells and spores of the food-borne pathogen Bacillus cereus. METHODS AND RESULTS: Tempe extract showed a high antibacterial activity against B. cereus ATCC 14579 based on optical density and viable count measurements. This growth inhibition was manifested by a 4 log CFU ml(-1) reduction, within the first 15 min of exposure. Tempe extracts also rapidly inactivated B. cereus spores upon germination. Viability and membrane permeability assessments using fluorescence probes showed rapid inactivation and permeabilization of the cytoplasmic membrane confirming the bactericidal mode of action. Cooked beans and Rhizopus grown on different media did not show antibacterial activity, indicating the unique association of the antibacterial activity with tempe. Subsequent characterization of the antibacterial activity revealed that heat treatment and protease addition nullified the bactericidal effect, indicating the proteinaceous nature of the bioactive compound. CONCLUSIONS: During fermentation of soya beans with Rhizopus, compounds are released with extensive antibacterial activity against B. cereus cells and spores. SIGNIFICANCE AND IMPACT OF STUDY: The results show the potential of producing natural antibacterial compounds that could be used as ingredients in food preservation and pathogen control.

Transcriptional regulation of metabolic pathways, alternative respiration and enterotoxin genes in anaerobic growth of Bacillus cereus ATCC 14579.

AIMS: To assess genes specifically activated during anaerobic growth that are involved in metabolism and pathogenesis of the foodborne pathogen Bacillus cereus. METHODS AND RESULTS: Growth under anaerobic conditions in Brain Heart Infusion (BHI) broth revealed a reduced growth rate and lower yield as compared to growth under aerobic conditions. Subsequently, comparative transcriptome analysis showed specific genes induced under anaerobic conditions. These included novel genes identified for anaerobic growth of B. cereus, encoding metabolic pathways, such as the arginine deiminase pathway (ArcABDC), formate dehydrogenase (FdhF) and pyruvate formate lyase (Pfl), and alternative respiratory proteins, such as arsenate reductases. Notably, haemolytic enzyme encoding genes were induced during anaerobic growth, and enterotoxin genes were induced in high cell density transition and stationary phases of aerobic cultures. CONCLUSIONS: These data point to induction of stress adaptation and pathogenicity factors and rearrangements of expression of metabolic pathways in response to oxygen limitations in B. cereus. SIGNIFICANCE AND IMPACT OF THE STUDY: The reported changes in gene expression show that the foodborne pathogen B. cereus can adjust to anaerobic conditions, such as encountered in the human GI-tract.

Activity of ethanol-stressed Oenococcus oeni cells: a flow cytometric approach.

AIMS: To study the effect of ethanol on Oenococcus oeni activity at the single cell level. METHODS AND RESULTS: The active extrusion of the fluorescent probe carboxy fluorescein (cF) was used to assess the metabolic activity of ethanol-stressed O. oeni cells. Subsequent flow cytometric analysis revealed that O. oeni cells extrude the accumulated cF upon energizing with l-malic acid. However, O. oeni cells exposed to 12% (v/v) ethanol for 1 h showed a decreased capacity for active extrusion of cF. Moreover, two subpopulations could be distinguished, one of which being able to extrude cF and the other one remaining cF fluorescent. Growing cells in the presence of 8% (v/v) ethanol resulted in robust cells that maintained the capacity to actively extrude cF after being exposed to 12% (v/v) ethanol, which in turn correlated with the high levels of ATP observed in these ethanol stressed, malolactic fermentation (MLF) performing cells. CONCLUSION: From our results, it becomes evident that active extrusion of cF can be used to assess malolactic activity in O. oeni. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides information for the development of a rapid method to assess the malolactic activity of individual O. oeni cells performing MLF during wine production.