Two-Component-System Histidine Kinases Involved in Growth of Listeria monocytogenes EGD-e at Low Temperatures

Two-component systems (TCSs) aid bacteria in adapting to a wide variety of stress conditions. While the role of TCS response regulators in the cold tolerance of the psychrotrophic foodborne pathogen Listeria monocytogenes has been demonstrated previously, no comprehensive studies showing the role of TCS histidine kinases of L. monocytogenes at low temperature have been performed. We compared the expression levels of each histidine kinase-encoding gene of L. monocytogenes EGD-e in logarithmic growth phase at 3°C and 37°C, as well as the expression levels 30 min, 3 h, and 7 h after cold shock at 5°C and preceding cold shock (at 37°C). We constructed a deletion mutation in each TCS histidine kinase gene, monitored the growth of the EGD-e wild-type and mutant strains at 3°C and 37°C, and measured the minimum growth temperature of each strain. Two genes, yycG and lisK, proved significant in regard to induced relative expression levels under cold conditions and cold-sensitive mutant phenotypes. Moreover, the ΔresE mutant showed a lower growth rate than that of wild-type EGD-e at 3°C. Eleven other genes showed upregulated gene expression but revealed no cold-sensitive phenotypes. The results show that the histidine kinases encoded by yycG and lisK are important for the growth and adaptation of L. monocytogenes EGD-e at low temperature.     

Changes in Listeria monocytogenes Membrane Fluidity in Response to Temperature Stress

Listeria monocytogenes is a food-borne pathogen that has been implicated in many outbreaks associated with ready-to-eat products. Listeria adjusts to various stresses by adjusting its membrane fluidity, increasing the uptake of osmoprotectants and cryoprotectants, and activating the σ^B stress factor. The present work examines the regulation of membrane fluidity through direct measurement based on fluorescent anisotropy. The membrane fluidities of L. monocytogenes Scott A, NR30, wt10403S, and cld1 cells cultured at 15 and 30°C were measured at 15 and 30°C. The membrane of the cold-sensitive mutant (cld1) was more rigid than the membranes of the other strains when grown at 30°C, but when grown at 15°C, it was able to adjust its membrane to approach the rigidity of the other strains. The difference in rigidities, as determined at 15 and 30°C, was greater in liposomes than in whole cells. The rates of fluidity adjustment and times required for whole cells to adjust to a different temperature were similar among strains but different from those of liposomes. This suggests that the cells had a mechanism for homeoviscous adaptation that was absent in liposomes.     

Surface Attachment of Listeria monocytogenes Is Induced by Sublethal Concentrations of Alcohol at Low Temperatures

Sublethal concentrations of ethanol or isopropanol increased attachment of Listeria monocytogenes at 10, 20, or 30°C; no induction occurred at 37°C. The alcohol induction phenotype was retained in sigB and cesRK mutants; however, the degree of induction was affected. These results suggest that alcohol may contribute to the persistence of L. monocytogenes.     

Control of Listeria monocytogenes in a Biofilm by Competitive-Exclusion Microorganisms

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37°C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10³ CFU of L. monocytogenes/ml and 10⁵ CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37°C for 24 h, 15°C for 14 days, 8°C for 21 days, and 4°C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37°C, two at 15 and 8°C, and three at 4°C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4°C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log₁₀ CFU of L. monocytogenes/cm²). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37°C.     

Enhanced Levels of Cold Shock Proteins in Listeria monocytogenes LO28 upon Exposure to Low Temperature and High Hydrostatic Pressure

Listeria monocytogenes is a psychrotrophic food-borne pathogen that is problematic for the food industry because of its ubiquitous distribution in nature and its ability to grow at low temperatures and in the presence of high salt concentrations. Here we demonstrate that the process of adaptation to low temperature after cold shock includes elevated levels of cold shock proteins (CSPs) and that the levels of CSPs are also elevated after treatment with high hydrostatic pressure (HHP). Two-dimensional gel electrophoresis combined with Western blotting performed with anti-CspB of Bacillus subtilis was used to identify four 7-kDa proteins, designated Csp1, Csp2, Csp3, and Csp4. In addition, Southern blotting revealed four chromosomal DNA fragments that reacted with a csp probe, which also indicated that a CSP family is present in L. monocytogenes LO28. After a cold shock in which the temperature was decreased from 37°C to 10°C the levels of Csp1 and Csp3 increased 10- and 3.5-fold, respectively, but the levels of Csp2 and Csp4 were not elevated. Pressurization of L. monocytogenes LO28 cells resulted in 3.5- and 2-fold increases in the levels of Csp1 and Csp2, respectively. Strikingly, the level of survival after pressurization of cold-shocked cells was 100-fold higher than that of cells growing exponentially at 37°C. These findings imply that cold-shocked cells are protected from HHP treatment, which may affect the efficiency of combined preservation techniques.     

Antilisterial Activity of Peptide AS-48 and Study of Changes Induced in the Cell Envelope Properties of an AS-48-Adapted Strain of Listeria monocytogenes

The peptide AS-48 is highly active on all Listeria species. It has a bactericidal and bacteriolytic mode of action on Listeria monocytogenes CECT 4032, causing depletion of the membrane electrical potential and pH gradient. The producer strain Enterococcus faecalis A-48-32, releases sufficient amounts of AS-48 into the growth medium to suppress L. monocytogenes in cocultures at enterococcus-to-listeria ratios above 1 at 37°C or above 10 at 15°C. As the temperature decreases, the bactericidal effects of AS-48 are less pronounced, but at 2.5 μg/ml it still can inhibit the growth of listeria at 6°C. AS-48 is highly active on liquid cultures, although concentrations above 0.2 μg/ml are required to avoid adaptation of listeria. AS-48-adapted cells can be selected at low (but still inhibitory) concentrations, and they can be inhibited completely by AS-48 at 0.5 μg/ml. The adaptation is lost gradually upon repeated subcultivation. AS48^ad cells are cross-resistant to nisin and show an increased resistance to muramidases. Their fatty acid composition is modified: they show a much higher proportion of branched fatty acids as well as a higher C_{15:0 An}-to-C_{17:0 An} ratio. Resistance to AS-48 is also maintained by protoplasts from AS48^ad cells. Electron microscopy observations show that the cell wall of AS48^ad cells is thicker and less dense. The structure of wild-type cells is severely modified after AS-48 treatment: the cell wall and the cytoplasmic membrane are disorganized, and the cytoplasmic content is lost. Intracytoplasmic membrane vesicles are also observed when the wild-type strain is treated with high AS-48 concentrations.     

Cold Shock Induction of Thermal Sensitivity in Listeria monocytogenes

Cold shock at 0 to 15°C for 1 to 3 h increased the thermal sensitivity of Listeria monocytogenes. In a model broth system, thermal death time at 60°C was reduced by up to 45% after L. monocytogenes Scott A was cold shocked for 3 h. The duration of the cold shock affected thermal tolerance more than did the magnitude of the temperature downshift. The Z values were 8.8°C for controls and 7.7°C for cold-shocked cells. The D values of cold-shocked cells did not return to control levels after incubation for 3 h at 28°C followed by heating at 60°C. Nine L. monocytogenes strains that were cold shocked for 3 h exhibited D₆₀ values that were reduced by 13 to 37%. The D-value reduction was greatest in cold-shocked stationary-phase cells compared to cells from cultures in either the lag or exponential phases of growth. In addition, cold-shocked cells were more likely to be inactivated by a given heat treatment than nonshocked cells, which were more likely to experience sublethal injury. The D values of chloramphenicol-treated control cells and chloramphenicol-treated cold-shocked cells were no different from those of untreated cold-shocked cells, suggesting that cold shock suppresses synthesis of proteins responsible for heat protection. In related experiments, the D values of L. monocytogenes Scott A were decreased 25% on frankfurter skins and 15% in ultra-high temperature milk if the inoculated products were first cold shocked. Induction of increased thermal sensitivity in L. monocytogenes by thermal flux shows potential to become a practical and efficacious preventative control method.     

Cold Shock and Its Effect on Ribosomes and Thermal Tolerance in Listeria monocytogenes

Differential scanning calorimetry (DSC) and fatty acid analysis were used to determine how cold shocking reduces the thermal stability of Listeria monocytogenes. Additionally, antibiotics that can elicit production of cold or heat shock proteins were used to determine the effect of translation blockage on ribosome thermal stability. Fatty acid profiles showed no significant variations as a result of cold shock, indicating that changes in membrane fatty acids were not responsible for the cold shock-induced reduction in thermal tolerance. Following a 3-h cold shock from 37 to 0°C, the maximum denaturation temperature of the 50S ribosomal subunit and 70S ribosomal particle peak was reduced from 73.4 ± 0.1°C (mean ± standard deviation) to 72.1 ± 0.5°C (P ≤ 0.05), indicating that cold shock induced instability in the associated ribosome structure. The maximum denaturation temperature of the 30S ribosomal subunit peak did not show a significant shift in temperature (from 67.5 ± 0.4°C to 66.8 ± 0.5°C) as a result of cold shock, suggesting that either 50S subunit or 70S particle sensitivity was responsible for the intact ribosome fragility. Antibiotics that elicited changes in maximum denaturation temperature in ribosomal components also elicited reductions in thermotolerance. Together, these data suggest that ribosomal changes resulting from cold shock may be responsible for the decrease in D value observed when L. monocytogenes is cold shocked.     

Effect of Flagella on Initial Attachment of Listeria monocytogenes to Stainless Steel

At 22°C a flagellin mutant of Listeria monocytogenes was found to attach to stainless steel at levels 10-fold lower than wild-type cells, even under conditions preventing active motility. At 37°C, when flagella are not produced, attachment of both strains was identical. Therefore, flagella per se facilitate the early stage of attachment.     

Role of Growth Temperature in Freeze-Thaw Tolerance of Listeria spp.

The food-borne pathogen Listeria monocytogenes can grow in a wide range of temperatures, and several key virulence determinants of the organism are expressed at 37°C but are strongly repressed below 30°C. However, the impact of growth temperature on the ability of the bacteria to tolerate environmental stresses remains poorly understood. In other microorganisms, cold acclimation resulted in enhanced tolerance against freezing and thawing (cryotolerance). In this study, we investigated the impact of growth temperature (4, 25, and 37°C) on the cryotolerance of 14 strains of L. monocytogenes from outbreaks and from food processing plant environments and four strains of nonpathogenic Listeria spp. (L. welshimeri and L. innocua). After growth at different temperatures, cells were frozen at −20°C, and repeated freeze-thaw cycles were applied every 24 h. Pronounced cryotolerance was exhibited by cells grown at 37°C, with a <1-log decrease after 18 cycles of freezing and thawing. In contrast, freeze-thaw tolerance was significantly reduced (P < 0.05) when bacteria were grown at either 4 or 25°C, with log decreases after 18 freeze-thaw cycles ranging from 2 to >4, depending on the strain. These findings suggest that growth at 37°C, a temperature required for expression of virulence determinants of L. monocytogenes, is also required for protection against freeze-thaw stress. The negative impact of growth at low temperature on freeze-thaw stress was unexpected and has not been reported before with this or other psychrotrophic microorganisms.Listeria monocytogenes remains a leading cause of deaths due to food-borne illness in the United States and other industrialized nations. Neonates, pregnant women, and immunocompromised people are at high risk for infection (15, 28, 37). Outbreaks of listeriosis tend to involve a relatively small number of closely related strains (“epidemic clones”), primarily of serotype 4b. Several major outbreaks have been attributed to epidemic clone I (ECI) and epidemic clone II (ECII), both of serotype 4b (5, 21).Unlike most other human food-borne pathogens, L. monocytogenes grows over a wide temperature range (1 to 45°C), with optimal growth at approximately 37°C (33). It has been known for some time that expression of several key virulence genes, including hly, encoding the hemolysin listeriolysin O and actA, encoding a protein that mediates actin polymerization required for intracellular pathogenesis, is optimal at 37°C but severely repressed at temperatures below 30°C (22). In contrast, flagellin, motility, and chemotaxis genes are repressed at 37°C but optimally expressed at temperatures below 25°C (9, 10).For an organism such as L. monocytogenes, which is commonly found in the environment but can also colonize and infect warm-blooded animals, temperature is likely to serve as a major signal differentiating environmental from vertebrate host-associated habitats (19). However, with the exception of the thermoregulated phenotypes described above (virulence factor production, motility, and chemotaxis), the impact of growth at different temperatures on specific responses and adaptations of the pathogen remains poorly understood.L. monocytogenes may be exposed to freezing as well as thawing in the course of its existence in natural environments (e.g., soils and water in temperate or cold regions), as well as during the storage and preservation of foods. The organism has been repeatedly isolated from frozen foods (e.g., ice cream) (7). After freezing (−18°C) in laboratory media or in foods and a single thawing cycle, survival depended on strain, freezing medium, and the presence of glycerol as cryoprotectant (13, 14). The freezing and thawing of bacteria grown at 30°C, in combination with essential oil, has been explored as one means to reduce the pathogen in foods (8). The possible role of the general stress sigma factor (sigma B) in survival of bacteria grown at 30°C and exposed to repeated freezing and thawing was also investigated (43). However, there is a surprising dearth of information on the possible impact of growth temperature on tolerance of L. monocytogenes to repeated freezing and thawing (cryotolerance).Studies with another gram-positive psychrotrophic bacterium (Exiguobacterium sibiricum and other Exiguobacterium spp.) revealed that growth in the cold (4°C), or growth on solid media regardless of temperature, resulted in increased tolerance of the bacteria against repeated freezing and thawing (40). It is not known whether low temperature or surface-associated growth may exert similar impacts on the cryotolerance of L. monocytogenes. The objective of the present study was to investigate the impact of growth temperature (4, 25, and 37°C) and of planktonic versus agar growth of L. monocytogenes on protection of the bacteria against repeated freezing and thawing.     

Bacterial Resistance to Ultrasonic Waves under Pressure at Nonlethal (Manosonication) and Lethal (Manothermosonication) Temperatures

The decimal reduction times of Streptococcus faecium, Listeria monocytogenes, Salmonella enteritidis, and Aeromonas hydrophila corresponding to heat treatment at 62°C were 7.1, 0.34, 0.024, and 0.0096 min, and those corresponding to manosonication treatment (40°C, 200 kPa, 117 μm) were 4.0, 1.5, 0.86, and 0.90 min, respectively. The manosonication decimal reduction times of the four species investigated decreased sixfold when the amplitude was increased from 62 to 150 μm and fivefold when the relative pressure was raised from 0 to 400 kPa. In L. monocytogenes, S. enteritidis, and A. hydrophila, the lethal effect of manothermosonication was the result of the addition of the lethal effects of heat and manosonication, whereas in S. faecium it was a synergistic effect.     

Influence of Temperature and Growth Phase on Expression of a 104-Kilodalton Listeria Adhesion Protein in Listeria monocytogenes

Interaction of Listeria monocytogenes with mammalian intestinal cells is believed to be an important first step in Listeria pathogenesis. Transposon (Tn916) mutagenesis provided strong evidence that a 104-kDa surface protein, designated the Listeria adhesion protein (LAP), was involved in adherence of L. monocytogenes to a human enterocyte-like Caco-2 cell line (V. Pandiripally, D. Westbrook, G. Sunki, and A. Bhunia, J. Med. Microbiol. 48:117–124, 1999). In this study, expression of LAP in L. monocytogenes at various growth temperatures (25, 37, and 42°C) and in various growth phases was determined by performing an enzyme-linked immunoassay (ELISA) and Western blotting with a specific monoclonal antibody (monoclonal antibody H7). The ELISA and Western blot results indicated that there was a significant increase in LAP expression over time only at 37 and 42°C and that the level of LAP expression was low during the exponential phase and high during the stationary phase. In contrast, there were not significant differences in LAP expression between the exponential and stationary phases at 25°C. Examination of the adhesion of L. monocytogenes cells from exponential-phase (12-h) or stationary-phase (24-h) cultures grown at 37°C to Caco-2 cells revealed that there were not significant differences in adhesion. Although expression of L. monocytogenes LAP was different at different growth temperatures and in different growth phases, enhanced expression did not result in increased adhesion, possibly because only a few LAP molecules were sufficient to initiate binding to Caco-2 cells.     

Listeria monocytogenes LO28: Surface Physicochemical Properties and Ability To Form Biofilms at Different Temperatures and Growth Phases

The surface physicochemical properties of Listeria monocytogenes LO28 under different conditions (temperature and growth phase) were determined by use of microelectrophoresis and microbial adhesion to solvents. The effect of these parameters on adhesion and biofilm formation by L. monocytogenes LO28 on hydrophilic (stainless steel) and hydrophobic (polytetrafluoroethylene [PTFE]) surfaces was assessed. The bacterial cells were always negatively charged and possessed hydrophilic surface properties, which were negatively correlated with growth temperature. The colonization of the two surfaces, monitored by scanning electron microscopy, epifluorescence microscopy, and cell enumeration, showed that the strain had a great capacity to colonize both surfaces whatever the incubation temperature. However, biofilm formation was faster on the hydrophilic substratum. After 5 days at 37 or 20°C, the biofilm structure was composed of aggregates with a three-dimensional shape, but significant detachment took place on PTFE at 37°C. At 8°C, only a bacterial monolayer was visible on stainless steel, while no growth was observed on PTFE. The growth phase of bacteria used to inoculate surfaces had a significant effect only in some cases during the first steps of biofilm formation. The surface physicochemical properties of the strain are correlated with adhesion and surface colonization.     

Selection and Identification of a Listeria monocytogenes Target Strain for Pulsed Electric Field Process Optimization

Nine Listeria monocytogenes strains were treated individually with a continuous pulsed electric field (PEF) apparatus, and their sensitivities to the treatment were compared at 25 kV/cm. When cell suspensions of these strains in 0.1% NaCl (pH 7.0) were treated at 23°C for 144 μs, inactivation ranged from 0.7 to 3.7 log₁₀ CFU/ml. Inactivation by 72-μs PEF treatments at 37°C ranged from 0.3 to 2.5 log₁₀ CFU/ml. L. monocytogenes OSY-8578 was substantially more resistant than other strains when cells were PEF treated in 0.1% NaCl, whereas Scott A was one of the most sensitive strains. The superiority of OSY-8578's resistance to that of Scott A was confirmed in 50% diluted acid whey (pH 4.2). Changes in sensitivity to PEF during phases of growth were minimal in OSY-8578 and substantial in Scott A. Use of L. monocytogenes OSY-8578, therefore, is recommended in studies to optimize PEF processes that target L. monocytogenes. The nine L. monocytogenes strains were genotyped with pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) techniques. These strains were better differentiated with PFGE than with AP-PCR. The target strain (OSY-8578) was characterized by both molecular typing techniques, but resistance to PEF, in general, was not associated with a particular genotype group.     

Listeria monocytogenes Scott A: Cell Surface Charge, Hydrophobicity, and Electron Donor and Acceptor Characteristics under Different Environmental Growth Conditions

We determined the variations in the surface physicochemical properties of Listeria monocytogenes Scott A cells that occurred under various environmental conditions. The surface charges, the hydrophobicities, and the electron donor and acceptor characteristics of L. monocytogenes Scott A cells were compared after the organism was grown in different growth media and at different temperatures; to do this, we used microelectrophoresis and the microbial adhesion to solvents method. Supplementing the growth media with glucose or lactic acid affected the electrical, hydrophobic, and electron donor and acceptor properties of the cells, whereas the growth temperature (37, 20, 15, or 8°C) primarily affected the electrical and electron donor and acceptor properties. The nonlinear effects of the growth temperature on the physicochemical properties of the cells were similar for cells cultivated in two different growth media, but bacteria cultivated in Trypticase soy broth supplemented with 6 g of yeast extract per liter (TSYE) were slightly more hydrophobic than cells cultivated in brain heart infusion medium (P < 0.05). Adhesion experiments conducted with L. monocytogenes Scott A cells cultivated in TSYE at 37, 20, 15, and 8°C and then suspended in a sodium chloride solution (1.5 × 10⁻¹ or 1.5 × 10⁻³ M NaCl) confirmed that the cell surface charge and the electron donor and acceptor properties of the cells had an influence on their attachment to stainless steel.     

The htrA (degP) Gene of Listeria monocytogenes 10403S Is Essential for Optimal Growth under Stress Conditions

This report describes a mutant of Listeria monocytogenes strain 10403S (serotype 1/2a) with a defective response to conditions of high osmolarity, an environment that L. monocytogenes encounters in some ready-to-eat foods. A library of L. monocytogenes clones mutagenized with Tn917 was generated and scored for sensitivity to 4% NaCl in order to identify genes responsible for growth or survival in elevated-NaCl environments. One of the L. monocytogenes Tn917 mutants, designated strain OSM1, was selected, and the gene interrupted by the transposon was sequenced. A BLAST search with the putative translated amino acid sequence indicated that the interrupted gene product was a homolog of htrA (degP), a gene coding for a serine protease identified as a stress response protein in several gram-positive and gram-negative bacteria. An htrA deletion strain, strain LDW1, was constructed, and the salt-sensitive phenotype of this strain was complemented by introduction of a plasmid carrying the wild-type htrA gene, demonstrating that htrA is necessary for optimal growth under conditions of osmotic stress. Additionally, strain LDW1 was tested for its response to temperature and H₂O₂ stresses. The results of these growth assays indicated that strain LDW1 grew at a lower rate than the wild-type strain at 44°C but at a rate similar to that of the wild-type strain when incubated at 4°C. In addition, strain LDW1 was significantly more sensitive to a 52°C heat shock than the wild-type strain. Strain LDW1 was also defective in its response to H₂O₂ challenge at 37°C, since 100 or 150 μg of H₂O₂ was more inhibitory for the growth of strain LDW1 than for that of the parent strain. The stress response phenotype observed for strain LDW1 is similar to that observed for other HtrA⁻ organisms, which suggests that L. monocytogenes HtrA may play a role in degrading misfolded proteins that accumulate under stress conditions.     

Survival and Heat Resistance of Listeria monocytogenes after Exposure to Alkali and Chlorine

A strain of Listeria monocytogenes isolated from a drain in a food-processing plant was demonstrated, by determination of D values, to be more resistant to the lethal effect of heat at 56 or 59°C following incubation for 45 min in tryptose phosphate broth (TPB) at pH 12.0 than to that of incubation for the same time in TPB at pH 7.3. Cells survived for at least 6 days when they were suspended in TPB at pHs 9.0, 10.0, and 11.0 and stored at 4 or 21°C. Cells of L. monocytogenes incubated at 37°C for 45 min and then stored for 48 or 144 h in TPB at pH 10.0 were more resistant to heat treatment at 56°C than were cells stored in TPB at pH 7.3. The alkaline-stress response in L. monocytogenes may induce resistance to otherwise lethal thermal-processing conditions. Treatment of cells in 0.05 M potassium phosphate buffer (pH 7.00 ± 0.05) containing 2.0 or 2.4 mg of free chlorine per liter reduced populations by as much as 1.3 log₁₀ CFU/ml, while treatment with 6.0 mg of free chlorine per liter reduced populations by as much as 4.02 log₁₀ CFU/ml. Remaining subpopulations of chlorine-treated cells exhibited some injury, and cells treated with chlorine for 10 min were more sensitive to heating at 56°C than cells treated for 5 min. Contamination of foods by L. monocytogenes cells that have survived exposure to processing environments ineffectively cleaned or sanitized with alkaline detergents or disinfectants may have more severe implications than previously recognized. Alkaline-pH-induced cross-protection of L. monocytogenes against heat has the potential to enhance survival in minimally processed as well as in heat-and-serve foods and in foods on holding tables, in food service facilities, and in the home. Cells surviving exposure to chlorine, in contrast, are more sensitive to heat; thus, the effectiveness of thermal processing in achieving desired log₁₀-unit reductions is not compromised in these cells.     

Exposure to Salt and Organic Acids Increases the Ability of Listeria monocytogenes To Invade Caco-2 Cells but Decreases Its Ability To Survive Gastric Stress

The effects of environmental stress exposure on Listeria monocytogenes growth and virulence-associated characteristics were investigated. Specifically, we measured the effects of temperature (7 or 37°C), pH (5.5 or 7.4), the presence of salt and organic acids (375 mM NaCl, 8.45 mM sodium diacetate [SD], 275 mM sodium lactate [SL], or a combination of NaCl, SD, and SL), and deletion of sigB, which encodes a key stress response regulator, on the ability of L. monocytogenes to grow, invade Caco-2 cells, and survive exposure to synthetic gastric fluid (pH 2.5 or 4.5). Our results indicate that (i) L. monocytogenes log-phase generation times and maximum cell numbers are not dependent on the alternative sigma factor σ^B in the presence of NaCl and organic acids at concentrations typically found in foods; (ii) growth inhibition of L. monocytogenes through the addition of organic acids is pH dependent; (iii) the ability of L. monocytogenes to invade Caco-2 cells is affected by growth phase, temperature, and the presence of salt and organic acids, with the highest relative invasion capabilities observed for cells grown with SL or NaCl at 37°C and pH 7.4; (iv) growth of L. monocytogenes in the presence of NaCl, SD, or SL reduces its ability to survive exposure to gastric fluid; and (v) exposure of L. monocytogenes to gastric fluid reduces the enhanced invasiveness caused by growth in the presence of NaCl or SL. These findings suggest that virulence-associated characteristics that determine the L. monocytogenes infectious dose are likely to be affected by food-specific properties (e.g., pH or the presence of salt or organic acid).