Synergistic effect of heat and sodium lactate on the thermal resistance of Yersinia enterocolitica and Listeria monocytogenes in minced beef

The effect of sodium lactate (NaL) (0, 2.4 or 4.8%), in heating and recovery media, on Yersinia enterocolitica and Listeria monocytogenes numbers recovered from minced beef heated at 55 degrees C, was examined. Survivors were enumerated on selective media at pH 5.7/7.4 (Y. enterocolitica) or pH 5.7/7.2 (L. monocytogenes). Recovery of the organisms depended on the pH and NaL levels in the recovery medium. The heat resistance of Y. enterocolitica (P < 0.001) and L. monocytogenes (P < 0.01) decreased as the concentration of NaL in the minced beef increased from 0 to 2.4% or 4.8%. The thermal destruction of pathogens in foods processed using mild temperatures may be enhanced by the addition of 2.4% NaL.     

Catalase, superoxide dismutase, and hemolysin activities and heat susceptibility of Listeria monocytogenes after growth in media containing sodium chloride

The activities of catalase, superoxide dismutase, and a thiol-activated hemolysin produced by four strains of Listeria monocytogenes propagated in media containing various concentrations of sodium chloride were examined. L. monocytogenes 7644 showed an increase in catalase, superoxide dismutase, and thiol-activated hemolysin activities when grown in a medium containing 2.5% (wt/vol) NaCl followed by a decrease in activities when propagated in media containing salt concentrations higher than 2.5%. L. monocytogenes LCDC 81-861 demonstrated enhanced catalase activity when grown in media containing NaCl ranging from 1.5 to 4.6% and increased superoxide dismutase activity when propagated in media containing 1.5 to 3.5% NaCl. L. monocytogenes LCDC 81-861 did not exhibit any detectable hemolysin activity under the conditions tested. After growth in various NaCl-containing media, both strains were subjected to sublethal heat injury for 30 min at 55 degrees C. L. monocytogenes LCDC 81-861 showed increased sensitivity to the heat treatment when grown in media containing 4.6 and 6.5% NaCl, whereas L. monocytogenes 7644 did not exhibit enhanced heat lability.     

Limitation in the detection of Listeria monocytogenes in food in the presence of competing Listeria innocua

Aims: Detectability of Listeria monocytogenes at 10⁰ CFU per food sample in the presence of Listeria innocua using standard microbiological detection was evaluated and compared with the real‐time PCR‐based method. Methods and Results: Enrichment in half‐Fraser broth followed by subculture in Fraser broth according to EN ISO 11290‐1 was used. False‐negative detection of 10⁰ CFU L. monocytogenes was obtained in the presence of 10¹ CFU L. innocua per sample using the standard detection method in contrast to more than 10⁵ CFU L. innocua per sample using real‐time PCR. Identification of L. monocytogenes on the chromogenic medium by the standard procedure was impossible if L. innocua was able to overgrow L. monocytogenes by more than three orders of magnitude after the enrichment in model samples. These results were confirmed using naturally contaminated food samples. Conclusions: Standard microbiological method was insufficient for the reliable detection of 10⁰ CFU L. monocytogenes in the presence of more than 10⁰ CFU of L. innocua per sample. On the other hand, if the growth of L. monocytogenes was sufficient to reach the concentration equal to the detection limit of PCR, the amount of the other microflora present in the food sample including L. innocua was not relevant for success of the PCR detection of L. monocytogenes. Significance and Impact of the Study: After the enrichment, the PCR detection is more convenient than the standard one as PCR detection is not compromised by other present microflora.     

Catalase, superoxide dismutase, and hemolysin activities and heat susceptibility of Listeria monocytogenes after growth in media containing sodium chloride.

The activities of catalase, superoxide dismutase, and a thiol-activated hemolysin produced by four strains of Listeria monocytogenes propagated in media containing various concentrations of sodium chloride were examined. L. monocytogenes 7644 showed an increase in catalase, superoxide dismutase, and thiol-activated hemolysin activities when grown in a medium containing 2.5% (wt/vol) NaCl followed by a decrease in activities when propagated in media containing salt concentrations higher than 2.5%. L. monocytogenes LCDC 81-861 demonstrated enhanced catalase activity when grown in media containing NaCl ranging from 1.5 to 4.6% and increased superoxide dismutase activity when propagated in media containing 1.5 to 3.5% NaCl. L. monocytogenes LCDC 81-861 did not exhibit any detectable hemolysin activity under the conditions tested. After growth in various NaCl-containing media, both strains were subjected to sublethal heat injury for 30 min at 55 degrees C. L. monocytogenes LCDC 81-861 showed increased sensitivity to the heat treatment when grown in media containing 4.6 and 6.5% NaCl, whereas L. monocytogenes 7644 did not exhibit enhanced heat lability.     

Listeriolysin O secretion by Listeria monocytogenes in the presence of cysteine and sorbate

The influence of cysteine or cysteine-HCl and their combination with potassium sorbate on growth of Listeria monocytogenes and listeriolysin O (LLO) secretion, and on activation of LLO in the haemolysin assay system was studied. Both cysteine and cysteine-HCl (10 and 20 mmol 1^-1) enhanced LLO secretion in tryptic soy broth supplemented with yeast extract during 24 h incubation at 35°C. While sorbate did not affect growth, it suppressed both LLO secretion and LLO activation by cysteine in the haemolysin activity assay. These findings provide further evidence that sulphydryl-containing enzymes are implicated in the mechanism of microbial inhibition of sorbate. Addition of sorbate to foods has the potential of inactivating listeriolysin and other thiol-dependent toxins.     

Enhanced inactivation of Listeria monocytogenes by nisin in the presence of ethanol

AIMS: The effect of combinations of nisin and ethanol on the survival of Listeria monocytogenes was investigated. METHODS AND RESULTS: Killing by nisin was enhanced during simultaneous exposure to ethanol (2-7% v/v). For example, while 10 IU ml(-1) nisin reduced viability by 1 log unit in 20 min, a combination of this antimicrobial peptide and 5% ethanol, reduced numbers of surviving cells by 3 log units. Increasing the concentrations of either ethanol (2-7%) or nisin (10-50 IU ml(-1)) led to increased cell death with synergy being demonstrated for all combinations tested and at a range of temperatures from 5 to 37 degrees C. CONCLUSIONS: Ethanol can act synergistically with nisin to reduce the survival of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Combinations of ethanol and nisin may be feasible as an effective way of controlling this pathogen in the food processing environment.     

Inhibition of the growth of acid tolerant yeasts by acetate, lactate and propionate and their synergistic mixtures

Saccharomyces uvarum, Geotrichum candidum, Endomycopsis burtonii and Hansemula canadensis have been isolated from deteriorated silages and/or spoiled lactic acid fermented foods. All yeasts could grow in relatively high (100 mmol/l) concentrations of acetate or lactate in Wickerham's assimilation broth but were inhibited by increasing concentrations of these acids. Propionate was much more inhibitory than acetate or lactate. Growth rate, but not final cell yield, was adversely affected by increasing concentrations of these acids. The three acids used in combination were synergistically inhibitory to H. canadensis and G. candidum. Saccharomyces uvarum was inhibited by synergistic mixtures of acetate/propionate. Endomycopsis burtonii and H. canadensis were inhibited by synergistic mixtures of acetate/lactate, acetate/propionate and lactate/propionate. Lactate and propionate had a synergistic inhibitory effect on G. candidum. The mechanism of inhibition of growth of S. uvarum by acetate and propionate was further investigated. Acetate and propionate each caused a reduction in cellular efficiency (Y, μg cells/μmole glucose consumed). Mixtures caused a drastic reduction in growth rate and Y. These results suggest that the cells may expend energy in reducing intracellular concentrations of acetate while propionate probably acts in a different manner.     

Catalase and superoxide dismutase activities after heat injury of Listeria monocytogenes

Four strains of Listeria monocytogenes were examined for catalase (CA) and superoxide dismutase (SOD) activities. The two strains having the highest CA activities (LCDC and Scott A) also possessed the highest SOD activities. The CA activity of heated cell extracts of all four strains examined decreased sharply between 55 and 60 degrees C. SOD was more heat labile than CA. Two L. monocytogenes strains demonstrated a decline in SOD activity after heat treatment at 45 degrees C, whereas the other two strains demonstrated a decline at 50 degrees C. Sublethal heating of the cells at 55 degrees C resulted in increased sensitivity to 5.5% NaCl. Exogenous hydrogen peroxide was added to suspensions of L. monocytogenes; strains producing the highest CA levels showed the greatest H2O2 resistance.     

Monoclonal antibody specific for Listeria monocytogenes, Listeria innocua, and Listeria welshimeri

Eight hundred fifty-nine murine hybridomas were produced from eight fusions, and 27 were characterized for secretion of antibodies reactive to Listeria monocytogenes. One monoclonal antibody (MAb), P5C9, reacted with all test strains of L. monocytogenes (31 of 31), L. innocua (3 of 3), and L. welshimeri (1 of 1) but not with any strains of the other four Listeria species or with any of 22 gram-positive or 11 gram-negative species of bacteria when tested in microtiter and dot blot enzyme immunoassays. Of the other 26 antibodies, 20 reacted with either L. monocytogenes Scott A or V7 and with some or all of the other six Listeria species but also cross-reacted with some or all of the non-Listeria bacteria tested. MAb P5C9 is of the immunoglobulin G1 murine subclass. In Western blot (immunoblot) analyses, this MAb reacted with a single antigen with a molecular weight of 18,500, and it is shared in common with all three reactive species, L. monocytogenes, L. innocua, and L. welshimeri. This antigen was extracted with detergent and appeared to be cell bound.     

Effect of temperature, sodium chloride, and pH on growth of Listeria monocytogenes in cabbage juice

Human illness and death have resulted from the consumption of milk, cheese, and cole slaw contaminated with Listeria monocytogenes. Since the effects of temperature, NaCl, and pH on the growth of the organism in cabbage were unknown, a series of experiments was designed to investigate these factors. Two strains (LCDC 81-861 and Scott A, both serotype 4b) were examined. At 30 degrees C, the viable population of the LCDC 81-861 strain increased in sterile unclarified cabbage juice (CJ) containing 0 to 1.5% NaCl; a decrease in the population of both strains occurred in juice containing greater than or equal to 2% NaCl. At 5 degrees C, the population of the Scott A strain in CJ containing up to 5% NaCl was reduced by about 90% over a 70-day period; the LCDC 81-861 strain was more sensitive to refrigeration but remained viable in CJ containing less than or equal to 3.5% NaCl for 70 days. Growth in CJ at 30 degrees C resulted in a decrease in pH from 5.6 to 4.1 within 8 days. Death of L. monocytogenes occurred at 30 degrees C when the organism was inoculated into sterile CJ adjusted to pH less than or equal to 4.6 with lactic acid. No viable cells were detected after 3 days at pH less than or equal to 4.2. At 5 degrees C, the rate of death at pH less than or equal to 4.8 was slower than at 30 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacy of high sodium chloride concentrations for the destruction of Listeria monocytogenes

The effect of sodium chloride concentrations (6, 16 and 26% (w/v) NaCl) on the survival of Listeria monocytogenes at low temperatures (10°C and under refrigeration, average 2°C) and frozen (- 18°C) was investigated. All salt concentrations tested were ineffective in reducing numbers over 6 h incubation at low temperatures. Over a longer time (33 d) at low temperatures, the organism grew in 6% NaCl, numbers remained the same in 16% NaCl and numbers declined in 26% NaCl. Although L. monocytogenes was destroyed in 26% NaCl, numbers declined too slowly for immersion in cold brine at this concentration to be a useful bacteriocidal treatment. Storage at - 18°C for 33 d caused no significant reduction in numbers at any of the NaCl concentrations tested.     

Structures and biological activities of peptidoglycans of Listeria monocytogenes and Propionibacterium acnes

The cell-wall skeletons of Listeria monocytogenes strain EGD and Propionibacterium acnes strain C7, which have the ability to induce macrophage activation, were analyzed, and the structures of the peptidoglycans were investigated. The analytical data indicate that both peptidoglycans have glucosamine residues with free amino groups, which are responsible for the resistance to lysozyme. Possible structures of these peptidoglycans were deduced from the composition and the results of determination of N- and C-terminal amino acids, together with the characterization of fragments obtained by enzymatic treatment and partial acid hydrolysis of both peptidoglycans. The results suggested that the peptidoglycan of L. monocytogenes contains a cross-linkage region of peptide chains with meso-diaminopimelic acid and D-alanine, which belongs to the A1 gamma type (Schleifer, K.H. & Kandler, O. (1972) Bacteriol. Rev. 36, 407-477), whereas the peptidoglycan of P. acnes contains a cross-linkage region of peptide chains with L,L-diaminopimelic acid and D-alanine, in which two glycine residues combine with amino and carboxyl groups of two L,L-diaminopimelic acid residues. The latter type should be classified as a new type. These cell-wall skeletons and peptidoglycans were shown to have immunoadjuvant activity on the induction of delayed-type hypersensitivity and suppressive activity on the growth of 3-methylcholanthrene-induced fibrosarcoma in BALB/c mice, and the peptidoglycans were shown to be an immunological-active principle of these cell-wall skeletons.     

Production of formate,acetate, and succinate by anaerobic fermentation of Lactobacillus pentosus in the presence of citrate

Summary The formation of acetate, formate and succinate was studied in Lactobacillus pentosus. These compounds were produced in addition to lactic acid when cells were exposed to anaerobic growth conditions with limited carbohydrates and in the presence of citrate. Citrate was metabolised via oxalacetate serving as an H-acceptor in a joint process together with lactate. The metabolism of citrate resulted in stoichiometric amounts of succinate and acetate. Lactate was degraded to formate and acetate in a reaction catalysed by pyruvate formate lyase. These fermentation products can potentially affect the flavour of fermented food but ecological factors in fermenting meat, e.g. the presence of glucose, nitrate or nitrite prevent this reaction. Offprint requests to: G. Wolf     

Catalase and superoxide dismutase activities after heat injury of Listeria monocytogenes.

Four strains of Listeria monocytogenes were examined for catalase (CA) and superoxide dismutase (SOD) activities. The two strains having the highest CA activities (LCDC and Scott A) also possessed the highest SOD activities. The CA activity of heated cell extracts of all four strains examined decreased sharply between 55 and 60 degrees C. SOD was more heat labile than CA. Two L. monocytogenes strains demonstrated a decline in SOD activity after heat treatment at 45 degrees C, whereas the other two strains demonstrated a decline at 50 degrees C. Sublethal heating of the cells at 55 degrees C resulted in increased sensitivity to 5.5% NaCl. Exogenous hydrogen peroxide was added to suspensions of L. monocytogenes; strains producing the highest CA levels showed the greatest H2O2 resistance.     

Effect of temperature, sodium chloride, and pH on growth of Listeria monocytogenes in cabbage juice.

Human illness and death have resulted from the consumption of milk, cheese, and cole slaw contaminated with Listeria monocytogenes. Since the effects of temperature, NaCl, and pH on the growth of the organism in cabbage were unknown, a series of experiments was designed to investigate these factors. Two strains (LCDC 81-861 and Scott A, both serotype 4b) were examined. At 30 degrees C, the viable population of the LCDC 81-861 strain increased in sterile unclarified cabbage juice (CJ) containing 0 to 1.5% NaCl; a decrease in the population of both strains occurred in juice containing greater than or equal to 2% NaCl. At 5 degrees C, the population of the Scott A strain in CJ containing up to 5% NaCl was reduced by about 90% over a 70-day period; the LCDC 81-861 strain was more sensitive to refrigeration but remained viable in CJ containing less than or equal to 3.5% NaCl for 70 days. Growth in CJ at 30 degrees C resulted in a decrease in pH from 5.6 to 4.1 within 8 days. Death of L. monocytogenes occurred at 30 degrees C when the organism was inoculated into sterile CJ adjusted to pH less than or equal to 4.6 with lactic acid. No viable cells were detected after 3 days at pH less than or equal to 4.2. At 5 degrees C, the rate of death at pH less than or equal to 4.8 was slower than at 30 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biodiversity of the species Listeria monocytogenes and the genus Listeria

This review describes the Listeria monocytogenes genome sequences available today and their comparison with that of Listeria innocua and Listeria welshimeri by highlighting their characteristic features and common traits. The diversity present among them is analysed with emphasis on putative virulence and host-pathogen interaction related functions. Then large-scale studies comparing gene content of Listeria and how these studies contributed to typing applications will be discussed. Finally, evolutionary conclusions and future perspectives in Listeria genomics are presented.     

Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes.

The concentrations of intracellular solutes in Listeria monocytogenes were examined in cells grown at various concentrations of NaCl. At 5% NaCl, cells contained elevated concentrations of potassium and glycine betaine compared with concentrations in cells grown without NaCl. At 7.5% NaCl, cells contained increased concentrations of K+, glycine betaine, glycine, alanine, and proline. Only glycine betaine, choline, or glycine promoted growth on a solidified defined medium containing 4% NaCl; there was no growth at higher concentrations of NaCl in the defined medium.     

Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes.

The concentrations of intracellular solutes in Listeria monocytogenes were examined in cells grown at various concentrations of NaCl. At 5% NaCl, cells contained elevated concentrations of potassium and glycine betaine compared with concentrations in cells grown without NaCl. At 7.5% NaCl, cells contained increased concentrations of K+, glycine betaine, glycine, alanine, and proline. Only glycine betaine, choline, or glycine promoted growth on a solidified defined medium containing 4% NaCl; there was no growth at higher concentrations of NaCl in the defined medium.     

Differentiation of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, and Listeria seeligeri by pulsed-field gel electrophoresis.

Clamped homogeneous electric field analysis of Listeria DNA with ApaI, AscI, SmaI, or NotI revealed species- and serotype-specific differences in genomic fingerprints. Clamped homogeneous electric field analysis may prove useful, therefore, in epidemiologic studies. Also, the summation of individually sized AscI fragments of genomic DNA from L. monocytogenes serotype 4b 101M and Scott A indicated genome lengths of 2,925 and 3,046 kb, respectively.     

Differentiation of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, and Listeria seeligeri by pulsed-field gel electrophoresis.

Clamped homogeneous electric field analysis of Listeria DNA with ApaI, AscI, SmaI, or NotI revealed species- and serotype-specific differences in genomic fingerprints. Clamped homogeneous electric field analysis may prove useful, therefore, in epidemiologic studies. Also, the summation of individually sized AscI fragments of genomic DNA from L. monocytogenes serotype 4b 101M and Scott A indicated genome lengths of 2,925 and 3,046 kb, respectively.