The effect of pH and temperature on initiation of growth of Listeria monocytogenes

The ability of 16 strains of Listeria monocytogenes , including two serotype 1 and 14 serotype 4 strains, to grow in a nutrient medium acidified with HC1 to pH values between 4·2 and 7·0 at intervals of 0·2 units has been investigated. The minimum pH values at which growth was detected at 30, 20, 10, 7 and 4°C were, respectively, 4·39, 4·39, 4·62, 4·62 and 5·23.     

The effect of pH, salt concentration and temperature on the survival and growth of Listeria monocytogenes

Factorially designed experiments have been used to study the growth and survival of Listeria monocytogenes in different combinations of pH and salt concentrations at ambient and chill temperatures. Survival at low pH and high salt concentration was strongly temperature dependent. The minimum pH values that allowed survival after 4 weeks from an initial 10⁴ cells were 4·66 at 30†C, 4·36 at 10†C and 4·19 at 5†C. These limits were salt dependent, low (4–6%) salt concentrations improved and higher concentrations reduced survival at limiting pH values. The lowest pH that allowed a 100-fold increase in cell numbers within 60 d was 4·66 at 30†C but this was increased to 4·83 at 10†C. At 5†C growth occurred at pH 7·0 but not at pH 5·13. Simple predictive models describing the effect of hydrogen-ion and salt concentration on the time for at least a 100-fold increase in numbers at 10†C and 30†C were constructed after analysis of the results for a least squares fit to a quadratic model. The interactions between salt and hydrogen-ion concentration on growth were found to be purely additive.     

The effect of pH, salt concentration and temperature on the survival and growth of Listeria monocytogenes

Factorially designed experiments have been used to study the growth and survival of Listeria monocytogenes in different combinations of pH and salt concentrations at ambient and chill temperatures. Survival at low pH and high salt concentration was strongly temperature dependent. The minimum pH values that allowed survival after 4 weeks from an initial 10(4) cells were 4.66 at 30 degrees C, 4.36 at 10 degrees C and 4.19 at 5 degrees C. These limits were salt dependent, low (4-6%) salt concentrations improved and higher concentrations reduced survival at limiting pH values. The lowest pH that allowed a 100-fold increase in cell numbers within 60 d was 4.66 at 30 degrees C but this was increased to 4.83 at 10 degrees C. At 5 degrees C growth occurred at pH 7.0 but not at pH 5.13. Simple predictive models describing the effect of hydrogen-ion and salt concentration on the time for at least a 100-fold increase in numbers at 10 degrees C and 30 degrees C were constructed after analysis of the results for a least squares fit to a quadratic model. The interactions between salt and hydrogen-ion concentration on growth were found to be purely additive.     

RsbV of Listeria monocytogenes contributes to regulation of environmental stress and virulence

SigmaB factor is an important regulatory factor for stress response in Gram-positive bacteria such as Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus and Bacillus subtilis. However, the activity of SigmaB factor is regulated by RsbV factor. Currently, the functional studies of RsbV factor are mostly focused on non-pathogenic B. subtilis, but the roles of RsbV factor in pathogenic L. monocytogenes during the regulation of environmental stress and virulence are still unclear. In the study, a ?RsbV mutant of L. monocytogenes was constructed to explore the regulatory role of RsbV in environmental stress and virulence. The environmental stress experiments indicated that the growth and survival capability of ?RsbV mutant obviously decreased in stress of low temperature, osmotic pressure, alcohol and acid, compared with EGD strain. The macrophage infection experiment indicated that ?RsbV mutant had weaker survival capability than EGD strain, and the expression of PrfA, actA, PlcA and LLO was down-regulated in infected cells. Animal inoculation experiments indicated that RsbV deletion significantly reduced the pathogenicity of L. monocytogenes. Our data demonstrate that, in addition to regulating tolerance under environmental stress conditions, RsbV also contributes to regulation of L. monocytogenes virulence.     

The effect of various acidulants on the growth of Listeria monocytogenes

The ability of four Listeria monocytogenes strains to initiate growth in brain heart infusion broth adjusted to various pH values with either acetic, lactic, citric or hydrochloric acid was investigated. Acetic acid was the most effective inhibitor tested, since in broth adjusted with this acid a higher minimum pH was required for growth of the various strains at both 4 and 30°C, as compared with broth adjusted with the other acidulants. The minimum pH value required for the initiation of growth of L. monocytogenes ranged from 5·0 to 5·7 at 4°C, and from 4·3 to 5·2 at 30°C, depending upon the acidulant used.     

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 sigma(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 degrees C were measured at 15 and 30 degrees C. The membrane of the cold-sensitive mutant (cld1) was more rigid than the membranes of the other strains when grown at 30 degrees C, but when grown at 15 degrees 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 degrees 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.     

Cold stress proteins induced in Listeria monocytogenes in response to temperature downshock and growth at low temperatures.

Listeria monocytogenes is a food-borne pathogen with the ability to grow at refrigerator temperatures. Twelve cold shock proteins (Csps) with apparent M(r)s of 48,600, 41,000, 21,800, 21,100, 19,700, 19,200, 18,800, 18,800, 17,200, 15,500, 14,500, and 14,400 were induced by cold shocking L. monocytogenes 10403S from 37 to 5 degrees C, as revealed by labeling with L-[35S]methionine followed by two-dimensional gel electrophoresis. Strain SLCC53 showed a similar response. Cold acclimation proteins were observed in cultures of strain 10403S growing at 5 degrees C, and four of these proteins, with apparent M(r)s 48,000, 21,100, 19,700, and 18,800, were also Csps. Two cold-sensitive transposon-induced mutants were labeled less efficiently than the parent strain, but the Csp response of the mutant examined was very similar to that of the parent strain.     

Estimation of temperature dependent growth rate and lag time of Listeria monocytogenes by optical density measurements.

An automated turbidimetric system, Bioscreen C, was used to monitor growth of ten strains of Listeria monocytogenes at different temperatures. Several methods for estimation of maximum specific growth rate (mu(max)) and lag time (lag) from turbidimetric data were compared to values estimated from viable count data. By using a calibration factor, reliable estimations of mu(max) could be obtained from turbidimetric measurements. On the other hand, accurate estimations of lag required some viable count data.     

Surface-associated, PrfA-regulated proteins of Listeria monocytogenes synthesized under stress conditions

The expression of the five clustered genes of Listeria monocytogenes: plcA, hly, mpl, actA and plcB is under the control of the positive regulation factor PrfA. Listeriolysin, encoded by the hly gene, is the only prominent PrfA-controlled gene product observed when L. monocytogenes strain NCTC 7973 is cultured in a rich medium at 37°C to the logarithmic growth phase. Stress conditions such as heat-shock or stationary culture conditions lead to the induction of additional PrfA-dependent proteins (PdPs): ActA (92 kDa), a 38kDa protein of unknown function and a 34kDa protein which probably represents PlcA. Under nutrient-stress conditions PdPs are preferentially synthesized and in addition to the already known PdPs at least five new, not yet functionally identified PdPs are detected. All PdPs are either secreted or are localized at the cell surface. Differences in the amount as well as the sizes of the PdPs are observed in different L. monocytogenes strains.     

Growth rate and growth probability of Listeria monocytogenes in dairy, meat and seafood products in suboptimal conditions

AIMS: To evaluate the performances of models predicting the growth rate or the growth probability of Listeria monocytogenes in food. METHODS AND RESULTS: Cardinal and square root type models including or not interactions between environmental factors and probability models were evaluated for their ability to describe the behaviour of L. monocytogenes in liquid dairy products, cheese, meat and seafood products. Models excluding interactions seemed sufficient to predict the growth rate of L. monocytogenes. However, the accurate prediction of growth/no-growth limits needed to take interactions into account. A complete and a simplified form (preservatives deducted) of a new cardinal model including interactions and parameter values were suggested to predict confidence limits for the growth rate of L. monocytogenes in food. This model could also be used for the growth probability prediction. CONCLUSIONS: The new cardinal model including interactions was efficient to predict confidence limits for the growth rate of L. monocytogenes and its growth probability in liquid dairy products, meat and seafood products. In cheese, the model was efficient to predict the absence of growth of the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: The suggested model can be used for risk assessment and risk management concerning L. monocytogenes in dairy, meat and seafood products.     

Identification of Listeria monocytogenes genes expressed in response to growth at low temperature

Listeria monocytogenes is a food-borne bacterial pathogen that is able to grow at refrigeration temperatures. To investigate microbial gene expression associated with cold acclimation, we used a differential cDNA cloning procedure known as selective capture of transcribed sequences (SCOTS) to identify bacterial RNAs that were expressed at elevated levels in bacteria grown at 10 degrees C compared to those grown at 37 degrees C. A total of 24 different cDNA clones corresponding to open reading frames in the L. monocytogenes strain EGD-e genome were obtained by SCOTS. These included cDNAs for L. monocytogenes genes involved in previously described cold-adaptive responses (flaA and flp), regulatory adaptive responses (rpoN, lhkA, yycJ, bglG, adaB, and psr), general microbial stress responses (groEL, clpP, clpB, flp, and trxB), amino acid metabolism (hisJ, trpG, cysS, and aroA), cell surface alterations (fbp, psr, and flaA), and degradative metabolism (eutB, celD, and mleA). Four additional cDNAs were obtained corresponding to genes potentially unique to L. monocytogenes and showing no significant similarity to any other previously described genes. Northern blot analyses confirmed increased steady-state levels of RNA for all members of a subset of genes examined during growth at a low temperature. These results indicated that L. monocytogenes acclimation to growth at 10 degrees C likely involves amino acid starvation, oxidative stress, aberrant protein synthesis, cell surface remodeling, alterations in degradative metabolism, and induction of global regulatory responses.     

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 H(2)O(2) stresses. The results of these growth assays indicated that strain LDW1 grew at a lower rate than the wild-type strain at 44 degrees C but at a rate similar to that of the wild-type strain when incubated at 4 degrees C. In addition, strain LDW1 was significantly more sensitive to a 52 degrees C heat shock than the wild-type strain. Strain LDW1 was also defective in its response to H(2)O(2) challenge at 37 degrees C, since 100 or 150 micro g of H(2)O(2) 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.     

The effect of culture medium and aeration on growth of Listeria monocytogenes at pH 4.5

Listeria monocytogenes multiplied at 20°C in medium adjusted to pH 4.5 with HCl, and the lag before growth was eliminated when the inoculum was grown to log phase in the same medium. In a tryptone soya medium with yeast extract and added glucose, growth at pH 4.5 was more rapid than in a tryptose phosphate medium, and this difference was greater in air than under nitrogen. The results show that the bacterium was capable of more rapid growth in air than under nitrogen at this pH and suggest that the tryptose phosphate medium was nutritionally limiting for growth.