A model to predict the fate of Listeria monocytogenes in different cheese types - A major role for undissociated lactic acid in addition to pH, water activity, and temperature.

Undissociated lactic acid has been shown to play a major role in complete growth inhibition of Listeria monocytogenes in Gouda cheese. In addition, low water activity conditions may contribute to growth inhibition. In the current study, it was assessed whether the major factors that inhibit growth of L. monocytogenes in Gouda cheese are the factors that determine growth in other types of ready-to-eat cheese as well. Various types of cheeses were selected, some of which had been associated with listeriosis, while others had not. Based on the composition of the different cheese types, the concentrations of undissociated lactic acid were calculated for each type. The ability to support growth of L. monocytogenes was predicted using the Gamma model, based on literature data on total lactic acid content, moisture content, fat content, pH, Aw, and temperature, and optimal growth rates in milk at 30-37 °C. In addition, the actual specific growth rates of L. monocytogenes in the various cheeses were calculated based on available experimental growth data. In 9 out of the 10 RTE cheeses reviewed, the undissociated lactic acid concentrations and aw determined growth/no growth of L. monocytogenes. No growth was correctly predicted for feta, Cheddar and Gouda, and growth was correctly predicted for ricotta, queso fresco, Camembert, high-moisture mozzarella, cottage and blue cheese. Growth of L. monocytogenes was not observed in practice upon inoculation of Emmental, whereas growth in this cheese type was predicted when including the above mentioned factors in the models. Other factors, presumably acetic and propionic acid, are thought to be important to inhibit growth of the pathogen in Emmental. The results from our study show that for cheeses in which lactic acid is a main acid, our model based on undissociated lactic acid, temperature, pH and aw gives a good prediction of potential outgrowth of L. monocytogenes. Implications for L. monocytogenes legislation are discussed per type of RTE cheese reviewed.

How NaCl and water content determine water activity during ripening of Gouda cheese, and the predicted effect on inhibition of Listeria monocytogenes.

This study describes the diffusion of NaCl and water in Gouda cheese during brining and ripening. Furthermore, we established water activity as a function of the NaCl-in-moisture content in Gouda cheese during ripening. We determined NaCl content, water content, and water activity in block-type Gouda cheeses that were brined for 3.8d and foil-ripened for a period of 26 wk, and in wheel-type Gouda cheeses that were brined for 0.33, 2.1, or 8.9d and subsequently nature-ripened for a period of 26 wk. The calculated diffusion coefficients of NaCl during brining were 3.6·10(-10) m(2)s(-1) in the block-type Gouda cheeses and 3.5·10(-10) m(2)s(-1) in the wheel-type Gouda cheeses. Immediately after brining, gradients of NaCl and water were observed throughout both types of cheese. During ripening, these gradients disappeared, except for the water gradient in nature-ripened cheeses. An empirical model was derived for Gouda cheese, in which water activity is expressed as a function of the NaCl-in-moisture content, as established for different brining times, locations and ripening times. Moreover, the effect of reduced water activity on inhibition of growth of Listeria monocytogenes in Gouda cheese was calculated. In addition to the presence of lactate and a pH of 5.2 to 5.3, the reduced water activity as seen in Gouda cheese can substantially contribute to inhibition of microbial growth and even to inactivation when cheeses are brined and ripened for extended times and subjected to nature-ripening.

The growth limits of a large number of Listeria monocytogenes strains at combinations of stresses show serotype--and niche-specific traits.

AIMS: The aim of this study was to associate the growth limits of Listeria monocytogenes during exposure to combined stresses with specific serotypes or origins of isolation, and identify potential genetic markers. METHODS AND RESULTS: The growth of 138 strains was assessed at different temperatures using combinations of low pH, sodium lactate, and high salt concentrations in brain heart infusion broth. None of the strains was able to grow at pH < or = 4.4, a(w) < or = 0.92, or pH < or = 5.0 combined with a(w) < or = 0.94. In addition, none of the strains grew at pH < or = 5.2 and NaLac > or = 2%. At 30 degrees C, the serotype 4b strains showed the highest tolerance to low pH and high NaCl concentrations at both pH neutral (pH 7.4) and mild acidic conditions (pH 5.5). At 7 degrees C, the serotype 1/2b strains showed the highest tolerance to high NaCl concentrations at both pH 7.4 and 5.5. Serotype 1/2b meat isolates showed the highest tolerance to low pH in the presence of 2% sodium lactate at 7 degrees C. ORF2110 and gadD1T1 were identified as potential biomarkers for phenotypic differences. CONCLUSIONS: Differences in growth limits were identified between specific L. monocytogenes strains and serotypes, which could in some cases be associated with specific genetic markers. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data confirm the growth limits of L. monocytogenes as set out by the European Union for ready-to-eat foods and provides an additional criterion. The association of L. monocytogenes serotypes with certain stress responses might explain the abundance of certain serotypes in retail foods while others are common in clinical cases.

The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus.

The natural antimicrobial compound carvacrol shows a high preference for hydrophobic phases. The partition coefficients of carvacrol in both octanol-water and liposome-buffer phases were determined (3.64 and 3.26, respectively). Addition of carvacrol to a liposomal suspension resulted in an expansion of the liposomal membrane. Maximum expansion was observed after the addition of 0.50 micromol of carvacrol/mg of L-alpha-phosphatidylethanolamine. Cymene, a biological precursor of carvacrol which lacks a hydroxyl group, was found to have a higher preference for liposomal membranes, thereby causing more expansion. The effect of cymene on the membrane potential was less pronounced than the effect of carvacrol. The pH gradient and ATP pools were not affected by cymene. Measurement of the antimicrobial activities of compounds similar to carvacrol (e.g., thymol, cymene, menthol, and carvacrol methyl ester) showed that the hydroxyl group of this compound and the presence of a system of delocalized electrons are important for the antimicrobial activity of carvacrol. Based on this study, we hypothesize that carvacrol destabilizes the cytoplasmic membrane and, in addition, acts as a proton exchanger, thereby reducing the pH gradient across the cytoplasmic membrane. The resulting collapse of the proton motive force and depletion of the ATP pool eventually lead to cell death.

Antioxidative properties of Lactobacillus sake upon exposure to elevated oxygen concentrations.

The ability of bacteria to overcome oxidative stress is related to the levels and types of antioxidative mechanisms which they possess. In this study, the antioxidative properties in Lactobacillus sake strains from different food origins were determined at low temperature (8 degrees C) and upon exposure to oxygen levels between 20 and 90% O(2). The L. sake strains tested grew well at 8 degrees C and in the presence of 20% O(2), however, most of the strains could not grow at O(2) levels as high as 50 and/or 90%. Cell-free extracts of all strains possessed certain levels of hydroxyl radical scavenging, metal chelating and reducing capacities essential for growth of cells at ambient O(2). At elevated O(2) concentrations, a high H(2)O(2) splitting capacity and low specific rates of H(2)O(2) production were demonstrated in the O(2)-insensitive strain L. sake NCFB 2813, which could grow at elevated O(2) conditions. Although H(2)O(2) was generated in the O(2)-sensitive L. sake DSM 6333 at levels which were not directly toxic to the cells (<0.2 mM), we can conclude that its removal is essential for cell protection at elevated O(2) conditions.

Superoxide dismutase plays an important role in the survival of Lactobacillus sake upon exposure to elevated oxygen.

In this study, the responses of two Lactobacillus sake strains to elevated oxygen concentrations at 8 degrees C were investigated. L. sake DSM 6333 (L. sake(sens)), unlike L. sake NCFB 2813 (L. sake(ins)), showed a low growth rate in the presence of 90% O(2) and a rapid loss in viability shortly after entry into stationary phase. The steady-state cytosolic superoxide radical (O(2)(-)) concentration in L. sake(sens) was 0.134 microM and in the oxygen-insensitive mutant LSUV4 it was 0.013 microM. The nine- to ten-fold decrease in the rate of O(2)(-) elimination in L. sake(sens) indicates the significance of the O(2)(-)-scavenging system in protecting against elevated O(2). The superoxide dismutase (SOD) activity was 10- to 20-fold higher in L. sake(ins) than in L. sake(sens), depending on the growth phase. An oxygen-insensitive mutant of L. sake(sens), designated as strain LSUV4, had a ten-fold higher SOD activity than the wild-type strain, which likely restored its oxygen tolerance. Damage to proteins in L. sake(sens) was evidenced by the increased protein carbonyl content and reduced activities of the [Fe-S]-cluster-containing enzymes fumarase and fumarate reductase. This study forms a physiological basis for understanding the significance of elevated oxygen stress as an additional method for inhibition of microbial growth in relation to food preservation.

Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate.

Escherichia coli responds to oxidative stress by activating sets of coregulated genes that help the cell to maintain homeostasis. Identified previously by genetic and biochemical approaches, the soxRS system mediates the induction of 18 of these redox-inducible genes (including the soxS gene itself). An overlapping set of genes is activated by an assortment of structurally unrelated molecules with antibiotic activities; many genes in this response are controlled by the marRAB system. The activation of either the soxRS or the marRAB system results in enhanced resistance to both superoxide-generating agents and multiple antibiotics. In order to probe the extent of these regulatory networks, we have measured whole-genome transcriptional profiles of the E. coli response to the superoxide-generating agent paraquat (PQ), an inducer of the soxRS system, and to the weak acid salt sodium salicylate (NaSal), an inducer of the marRA system. A total of 112 genes was modulated in response to PQ, while 134 genes were modulated in response to NaSal. We have also obtained transcriptional profiles of the SoxS and MarA regulons in the absence of global stress, in order to establish the regulatory hierarchies within the global responses. Several previously unrelated genes were shown to be under SoxS or MarA control. The genetic responses to both environmental insults revealed several common themes, including the activation of genes coding for functions that replenish reducing potential; regulate iron transport and storage; and participate in sugar and amino acid transport, detoxification, protein modification, osmotic protection, and peptidoglycan synthesis. A large number of PQ- and NaSal-responsive genes have no known function, suggesting that many adaptive metabolic changes that ensue after stress remain uncharacterized.

The combined action of carvacrol and high hydrostatic pressure on Listeria monocytogenes Scott A.

AIMS: The aim of the study was to investigate the combined antimicrobial action of the plant-derived volatile carvacrol and high hydrostatic pressure (HHP). METHODS AND RESULTS: Combined treatments of carvacrol and HHP have been studied at different temperatures, using exponentially growing cells of Listeria monocytogenes, and showed a synergistic action. The antimicrobial effects were higher at 1 degrees C than at 8 or 20 degrees C. Furthermore, addition of carvacrol to cells exposed to sublethal HHP treatment caused similar reductions in viable numbers as simultaneous treatment with carvacrol and HHP. Synergism was also observed between carvacrol and HHP in semi-skimmed milk that was artificially contaminated with L. monocytogenes. CONCLUSION: Carvacrol and HHP act synergistically and the antimicrobial effects of the combined treatment are greater at lower temperatures. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the synergistic antimicrobial effect of essential oils in combination with HHP and indicates the potential of these combined treatments in food processing.

Combined action of S-carvone and mild heat treatment on Listeria monocytogenes Scott A.

The combined action of the plant-derived volatile, S-carvone, and mild heat treatment on the food-borne pathogen, Listeria monocytogenes, was evaluated. The viability of exponential phase cultures grown at 8 degrees C could be reduced by 1.3 log units after exposure to S-carvone (5 mmol l-1) for 30 min at 45 degrees C, while individual treatment with S-carvone or exposure to 45 degrees C for 30 min did not result in a loss in viability. Other plant-derived volatiles, namely carvacrol, cinnamaldehyde, thymol and decanal, were also found to reduce the viability of L. monocytogenes in combination with the same mild heat treatment at concentrations of 1.75 mmol l-1, 2.5 mmol l-1, 1.5 mmol l-1 and 2 mmol l-1, respectively. These findings show that essential oil compounds can play an important role in minimally processed foods, and can be used in the concept of Hurdle Technology to reduce the intensity of heat treatment or other individual hurdles.

Defining a rob regulon in Escherichia coli by using transposon mutagenesis.

The Rob protein of Escherichia coli is a member of the AraC-XylS family of prokaryotic transcriptional regulators and is expressed constitutively. Deletion of the rob gene increases susceptibility to organic solvents, while overexpression of Rob increases tolerance to organic solvents and resistance to a variety of antibiotics and to the superoxide-generating compound phenazine methosulfate. To determine whether constitutive levels of Rob regulate basal gene expression, we performed a MudJ transposon screen in a rob deletion mutant containing a plasmid that allows for controlled rob gene expression. We identified eight genes and confirmed that seven are transcriptionally activated by normal expression of Rob from the chromosomal rob gene (inaA, marR, aslB, ybaO, mdlA, yfhD, and ybiS). One gene, galT, was repressed by Rob. We also demonstrated by Northern analysis that basal expression of micF is significantly higher in wild-type E. coli than in a rob deletion mutant. Rob binding to the promoter regions of most of these genes was substantiated in electrophoretic mobility shift assays. However, Mu insertions in individual Rob-regulated genes did not affect solvent sensitivity. This phenotype may depend on changes in the expression of several of these Rob-regulated genes or on other genes that were not identified. Rob clearly affects the basal expression of genes with a broad range of functions, including antibiotic resistance, acid adaptation, carbon metabolism, cell wall synthesis, central intermediary metabolism, and transport. The magnitudes of Rob's effects are modest, however, and the protein may thus play a role as a general transcription cofactor.

Crystal structure of the Escherichia coli Rob transcription factor in complex with DNA.

The Escherichia coli Rob protein is a transcription factor belonging to the AraC/XylS protein family that regulates genes involved in resistance to antibiotics, organic solvents and heavy metals. The genes encoding these proteins are activated by the homologous proteins MarA and SoxS, although the level of activation can vary for the different transcription factors. Here we report a 2.7 A crystal structure of Rob in complex with the micF promoter that reveals an unusual mode of binding to DNA. The Rob-DNA complex differs from the previously reported structure of MarA bound to the mar promoter, in that only one of Rob's dual helix-turn-helix (HTH) motifs engages the major groove of the binding site. Biochemical studies show that sequence specific interactions involving only one of Rob's HTH motifs are sufficient for high affinity binding to DNA. The two different modes of DNA binding seen in crystal structures of Rob and MarA also match the distinctive patterns of DNA protection by AraC at several sites within the pBAD promoter. These and other findings suggest that gene activation by AraC/XylS transcription factors might involve two alternative modes of binding to DNA in different promoter contexts.

Biopreservation in modified atmosphere stored mungbean sprouts: the use of vegetable-associated bacteriocinogenic lactic acid bacteria to control the growth of Listeria monocytogenes.

Two bacteriocinogenic strains of Pediococcus parvulus and one bacteriocinogenic Enterococcus mundtii strain were evaluated for their potential to control the growth of Listeria monocytogenes on refrigerated, modified atmosphere (MA) stored mungbean sprouts. These three strains, which were isolated from minimally-processed vegetables, were shown to grow in culture broth at 4, 8, 15 and 30 degrees C. However, only Ent. mundtii was capable of bacteriocin production at 4-8 degrees C. Examination of the growth of these strains on agar under 1.5% O2 in combination with 0, 5, 20 or 50% CO2 revealed significantly higher maximum specific growth rates for Ent. mundtii than for Pediococcus parvulus at CO2 concentrations below 20%, which are relevant for MA-storage of vegetables. Enterococcus mundtii was subsequently evaluated for its ability to control the growth of L. monocytogenes on vegetable agar and fresh mungbean sprouts under 1.5% O2/20% CO2/78.5% N2 at 8 degrees C. The growth of L. monocytogenes was inhibited by bacteriocinogenic Ent. mundtii on sterile vegetable-medium but not on fresh produce. However, mundticin, the bacteriocin produced by Ent. mundtii, was found to have potential as a biopreservative agent for MA-stored mungbean sprouts when used in a washing step or a coating procedure.

A novel bacteriocin with a YGNGV motif from vegetable-associated Enterococcus mundtii: full characterization and interaction with target organisms.

A novel broad-spectrum antimicrobial peptide produced by vegetable-associated Enterococcus mundtii was purified and characterized, and designated mundticin. To our knowledge, this is the first report on bacteriocin production by this organism. The elucidation of the full primary amino acid sequence of mundticin (KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAANLATGGAAGWSK) revealed that this antimicrobial peptide belongs to the class IIa bacteriocins of lactic acid bacteria which share a highly conserved N-terminal 'YGNGV' motif. Data obtained by computer modelling indicated an oblique orientation of the alpha-helical regions of mundticin and homologous class IIa bacteriocins at a hydrophobic-hydrophilic interface, which may play a role in the destabilization of phospholipid bilayers. The average mass of mundticin, as determined by electron spray mass spectrometry, was found to be 4287.21+/-0.59 Da. With respect to its biological activity, mundticin was shown to inhibit the growth of Listeria monocytogenes, Clostridium botulinum and a variety of lactic acid bacteria. Moreover, it was demonstrated to have a bactericidal effect on L. monocytogenes as a result of the dissipation of the membrane potential, and a loss of intracellular ATP in absence of ATP leakage. Its good solubility in water, and its stability over a wide pH and temperature range indicate the potential of this broad spectrum bacteriocin as a natural preservation agent for foods.

Interactions of nisin and pediocin PA-1 with closely related lactic acid bacteria that manifest over 100-fold differences in bacteriocin sensitivity.

The natural variation in the susceptibilities of gram-positive bacteria towards the bacteriocins nisin and pediocin PA-1 is considerable. This study addresses the factors associated with this variability for closely related lactic acid bacteria. We compared two sets of nonbacteriocinogenic strains for which the MICs of nisin and pediocin PA-1 differed 100- to 1,000-fold: Lactobacillus sake DSM20017 and L. sake DSM20497 and Pediococcus dextrinicus and Pediococcus pentosaccus. Strikingly, the bacteriocin-sensitive and -insensitive strains showed a similar concentration-dependent dissipation of their membrane potential (delta psi) after exposure to these bacteriocins. The bacteriocin-induced dissipation of delta psi below the MICs for the insensitive strains did not coincide with a reduction of intracellular ATP pools and glycolytic rates. This was not observed with the sensitive strains. Analysis of membrane lipid properties revealed minor differences in the phospho- and glycolipid compositions of both sets of strains. The interactions of the bacteriocins with strain-specific lipids were not significantly different in a lipid monolayer assay. Further lipid analysis revealed higher in situ membrane fluidity of the bacteriocin-sensitive Pediococcus strain compared with that for the insensitive strain, but the opposite was found for the L. sake strains. Our results provide evidence that the association of bacteriocins with the cell membrane and their subsequent insertion take place in a similar way for cells that have a high or a low natural tolerance towards bacteriocins. For insensitive strains, overall membrane constitution rather than mere membrane fluidity may preclude the formation of pores with sufficient diameters and lifetimes to ultimately cause cell death.

Mechanisms of increased linamarin degradation during solid-substrate fermentation of cassava.

Several fungi and bacteria, isolated from Ugandan domestic fermented cassava, released HCN from linamarin in defined growth media. In 72 h, a Bacillus sp. decreased the linamarin to 1% of initial concentrations, Mucor racemosus to 7%, Rhizopus oryzae and R. stolonifer to 30%, but Neurospora sitophila and Geotrichum candidum hardly degraded the linamarin. Adding pectolytic and cellulolytic enzymes, but not linamarase, to root pieces under aseptic conditions, led to root softening and significantly lower linamarin contents. Neurospora sitophila showed no linamarase activity, in contrast to M. racemosus and Bacillus sp., both of which were less effective in root softening and decreasing the root linamarin content. The most important contribution of microorganisms to linamarin decrease in the solid-substrate fermentation of cassava is their cell-wall-degrading activity, which enhances the contact between endogenous linamarase and linamarin.

The role of oxidative metabolism in hexachlorobenzene-induced porphyria and thyroid hormone homeostasis: a comparison with pentachlorobenzene in a 13-week feeding study.

Hexachlorobenzene (HCB) induces a broad spectrum of effects including disturbances in the heme synthesis (porphyria) and in thyroid hormone homeostasis. For most of its effects, biotransformation of the parent compound seems to be a prerequisite. The present study was designed to assess the relevance of the oxidative metabolites in HCB-induced toxicity, with special attention to the role of the reactive tetrachlorobenzoquinone (TCBQ). To this end, toxicity and biotransformation of HCB were compared with those of pentachlorobenzene (PCB), since this chemical is oxidized to the same products as HCB, i.e., pentachlorophenol (PCP) and TCBQ. Female Wistar rats received diets containing different dose levels of HCB or PCB for 13 weeks, with or without cotreatment with triacetyloleandomycin (TAO), a selective inhibitor of cytochrome P450IIIA1/2. Rats treated with HCB (high dose) had significantly elevated levels of urinary porphyrins from the 4th week on and had a significant hepatic accumulation of porphyrins at the end of the study. Both urinary porphyrin excretion and hepatic porphyrin accumulation were greatly inhibited in rats receiving cotreatment with HCB and TAO. However, the inhibition of HCB-induced porphyria by TAO cannot be explained by a diminished formation of the highly reactive TCBQ, since rats treated with a high dose of PCB, which had a several fold higher urinary excretion of PCP and TCHQ compared to a high dose of HCB, did not develop porphyria. Instead, the present study points to the involvement of a putative reactive intermediate in the primary oxidative step in HCB-induced porphyria, since based on paired observations of individual rats, the degree of porphyria was correlated to a high degree with excretion of PCP, whereas correlation of porphyria with early excretion of TCHQ was much weaker. This finding fits well with the fact that the mechanisms of oxidation of HCB to PCP and PCB to PCP are different. Both HCB and PCB were oxidized to PCP and tetrachlorohydroquinone (TCHQ), the reduced analog of TCBQ. Cytochrome P450IIIA1/2 appears to be involved in the conversion of HCB and PCB, since cotreatment of TAO resulted in a strongly diminished urinary excretion of PCP and TCHQ. Treatment with HCB as well as PCB results in disturbances of retinoid and thyroid hormone homeostasis. These effects, which have also been reported after exposure to polychlorinated biphenyls, originate from interference of hydroxylated metabolites (notably PCP) with the plasma thyroxine transport protein, transthyretine, and since this metabolite is formed from both HCB and PCB, this results in the same toxicity for both compounds.