The in vitro inhibition of growth of intracellular Listeria monocytogenes by lymphocyte products

The listeria-inhibiting activity of culture supernatants from listeria-immune and nonimmune lymphocytes was assessed on listeria-infected macrophage cultures. It was found that supernatant from listeria-immune lymphocyte cultures stimulated in vitro with antigen was markedly inhibitory to the multiplication of intracellular listeria. Some inhibitory activity was also evident in supernatant from antigen-stimulated non-immune lymphocyte cultures. Supernatant from listeria-immune lymphocytes stimulated in vivo with antigen was capable of inhibiting listeria. Some inhibitory activity was still evident upon dilution of active supernatant at 1:100.     

Pathogenicity of Listeria monocytogenes grown on crabmeat

The pathogenicity of Listeria monocytogenes as influenced by growth on crabmeat at 5 and 10 degrees C was studied. Crabmeat was inoculated with L. monocytogenes V7 (ca. 10(4) CFU/g) and incubated for up to 14 days at 5 and 10 degrees C. At selected incubation times, L. monocytogenes was removed from crabmeat by washing with 0.1 M potassium phosphate buffer (pH 7.0), and populations were determined by surface plating on LiCl-phenylethanol-moxalactam agar. Buffered suspensions were then centrifuged, and the resulting pellets were suspended in phosphate buffer containing 10% glycerol and stored at -18 degrees C. Thawed, diluted suspensions of cells were tested for pathogenicity by intraperitoneal injection into immunocompromised and nonimmunocompromised mice. L. monocytogenes cells recovered from crabmeat and then recultured in tryptose phosphate broth (TPB), as well as cells which had not been passed through crabmeat but had been cultured in TPB, were likewise harvested, suspended in buffered 10% glycerol, frozen, thawed, diluted, and tested for pathogenicity by intraperitoneal injection. Growth on crabmeat at 5 and 10 degrees C did not have a significant effect on pathogenicity. The population of L. monocytogenes necessary to kill about 50% of the immunocompromised mice in each test set within 7 days was about 10(4) CFU, and this result was not significantly affected by storage temperature of the crabmeat or type of substrate, i.e., crabmeat or TPB, on which it had grown.     

Petite colony formation by Listeria monocytogenes and Listeria species grown on esculin-containing agar

Several strains of Listeria species formed petite-sized colonies from parent stock cultures when grown on agar media containing 0.2-1% (w/v) esculin. This was observed in Listeria monocytogenes (7/22 strains), L. innocua (1/3), L. grayi (1/1), L. seeligeri (1/3), and L. welshimeri (1/1), but not in L. ivanovii (0/1) and L. murrayi (0/1). This phenomenon was only observed on agar media that contained esculin. All petite isolates had biotyping profiles identical to their larger, normal-sized counterpart isolates. Normal and petite-sized isolates from two L. monocytogenes strains, Scott A and V7, were pathogenic to immunosuppressed white mice. On media containing 0.5% (w/v) esculin + ferric iron, Listeria cultures produced colony diameters intermediate in size between those of normal and petite cultures. When pregrown in glucose broth, all petite isolates demonstrated visible beta-glucosidase (esculinase) activity within 5 min, while the normal-sized isolates showed beta-glucosidase activity only after at least 20-70 min. This evidence suggests that cells forming petite colonies are beta-glucosidase constitutive variants within the parent population, while cells that form normal-sized colonies are inducible for beta-glucosidase (esculinase) activity. A possible role for the esculin hydrolysis product, esculetin, in causing petite colony formation is discussed.     

Pathogenicity of Listeria monocytogenes grown on crabmeat.

The pathogenicity of Listeria monocytogenes as influenced by growth on crabmeat at 5 and 10 degrees C was studied. Crabmeat was inoculated with L. monocytogenes V7 (ca. 10(4) CFU/g) and incubated for up to 14 days at 5 and 10 degrees C. At selected incubation times, L. monocytogenes was removed from crabmeat by washing with 0.1 M potassium phosphate buffer (pH 7.0), and populations were determined by surface plating on LiCl-phenylethanol-moxalactam agar. Buffered suspensions were then centrifuged, and the resulting pellets were suspended in phosphate buffer containing 10% glycerol and stored at -18 degrees C. Thawed, diluted suspensions of cells were tested for pathogenicity by intraperitoneal injection into immunocompromised and nonimmunocompromised mice. L. monocytogenes cells recovered from crabmeat and then recultured in tryptose phosphate broth (TPB), as well as cells which had not been passed through crabmeat but had been cultured in TPB, were likewise harvested, suspended in buffered 10% glycerol, frozen, thawed, diluted, and tested for pathogenicity by intraperitoneal injection. Growth on crabmeat at 5 and 10 degrees C did not have a significant effect on pathogenicity. The population of L. monocytogenes necessary to kill about 50% of the immunocompromised mice in each test set within 7 days was about 10(4) CFU, and this result was not significantly affected by storage temperature of the crabmeat or type of substrate, i.e., crabmeat or TPB, on which it had grown.     

A note on the inhibition of Listeria monocytogenes NCTC 11994 in milk by an activated lactoperoxidase system

A lactoperoxidase system activated by glucose oxidase was bacteriostatic to Listeria monocytogenes inoculated into UHT milk supplemented with glucose. The incorporation of urea peroxide as an additional hydrogen peroxide-generating agent did not enhance the inhibitory effect.     

Evidence on inhibition of Listeria monocytogenes by divercin V41 action

AIMS: The aim of this study was to investigate the role of divercin V41 in inhibition and prevention of Listeria monocytogenes. METHODS AND RESULTS: Carnobacterium divergens V41 deficient in bacteriocin production was isolated and characterized by enzyme-liked immunosorbent assay, multiplex polymerase chain reaction and bacteriocin diffusion test. Carnobacterium divergens V41 (divercin+) and Carnobacterium divergens V41C9 (divercin-) were grown in the presence of L. monocytogenes in smoked salmon model medium. Carnobacterium divergens V41, but not C. divergens V41C9, was able to inhibit growth of L. monocytogenes. The results indicate that inhibition of L. monocytogenes in the presence of C. divergens V41 is because of the production of divercin V41 and not to a nutritional advantage. CONCLUSIONS: Carnobacterium divergens V41 may be a promising agent in food safety. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates a potential use of a bacteriocin producing lactic acid bacteria in the area food protection.     

Growth inhibition of Listeria monocytogenes by a nonbacteriocinogenic Carnobacterium piscicola

AIMS: This study elucidates the mechanisms by which a nonbacteriocinogenic Carnobacterium piscicola inhibits growth of Listeria monocytogenes. METHODS AND RESULTS: Listeria monocytogenes was exposed to live cultures of a bacteriocin-negative variant of C. piscicola A9b in co-culture, in a diffusion chamber system, and to a cell-free supernatant. Suppression of maximum cell density (0-3.5 log units) of L. monocytogenes was proportional to initial levels of C. pisciola (10(3)-10(7) CFU ml(-1)). Cell-to-cell contact was not required to cause inhibition. The cell-free C. piscicola supernatant caused a decrease in L. monocytogenes maximum cell density, which was abolished by glucose addition but not by amino acid, vitamin or mineral addition. The fermentate also gave rise to a longer lag phase and a reduction in growth rate. These effects were independent of glucose and may have been caused by acetate production by C. piscicola. 2D gel-electrophoretic patterns of L. monocytogenes exposed to C. piscicola or to L. monocytogenes fermentate did not differ. Treatment with C. piscicola fermentate resulted in down-regulation (twofold) of genes involved in purine- or pyrimidine metabolism, and up-regulation (twofold) of genes from the regulon for vitamin B12 biosynthesis and propanediol and ethanolamine utilization. CONCLUSIONS: A nonbacteriocinogenic C. piscicola reduced growth of L. monocytogenes partly by glucose depletion. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mechanism of microbial interaction enhances prediction of growth in mixed communities as well as use of bioprotective principles for food preservation.     

Sterilization of Listeria monocytogenes by guinea pig peritoneal exudate cell cultures

Non-glass-adherent cells (lymphocytes) of peritoneal exudates from guinea pigs infected with bacillus of Calmette-Guerin (BCG), stimulated in vitro by specific (tuberculin) or nonspecific phytohemagglutinin P (PHA) mitogens, conferred on glass-adherent cell (macrophage) cultures from BCG-infected, or homologous, noninfected guinea pigs the ability to sterilize Listeria monocytogenes. Lymphocytes from noninfected guinea pigs, stimulated by mitogens, had little or no activity in this test system, although the adherent monolayer cells were seen to be “activated” by morphologic criteria when PHA was employed.Phagocytosis of the bacteria was inhibited in sterilizing macrophage-lymphocyte cultures even after washing of the cultures had eliminated most of the cell clusters seen in activated cultures. However, the monolayers, before and after washing, were found to produce a soluble, filtrable factor(s) which sterilized the listeria. This activity was detectable as early as 17 hr in mixed-cell culture filtrates, and 42-hr monolayers continued to generate this active material after repeated washings with fresh medium up to 72 hr.No listeria-sterilizing activity was found in filtrates of mitogen-stimulated nonadherent lymphocyte cultures without macrophages, and such filtrates were not active in stimulating macrophage monolayers to produce the material although the cells in such monolayers were seen to manifest increased surface adherence and spreading characteristic of “activated” macrophages. Also, such culture filtrates were shown not to antagonize the antibacterial activity of listeria-sterilizing filtrates.Preliminary characterization of the listeria-sterilizing material revealed the following: (a) a molecular weight of 50,000 or more; (b) stable at 56 °C for 30 min in medium containing serum; (c) bound to the bacterial cells at 0 °C; (d) inactivated by the strong reducing agent, dithiothreitol, and partially reactivated by H₂O₂ oxidation; (e) partially antagonized by deoxyribonucleic acid; (f) inactive against two species, of salmonella; (g) not inhibited or enhanced by listeria-agglutinating antibodies.     

Acetoin production as an indicator of growth and metabolic inhibition of Listeria monocytogenes

It has been shown that Listeria monocytogenes produces acetoin from glucose under aerobic conditions. A defined medium with glucose as the sole carbon source was used in an aerobic shake flask culture to reliably produce acetoin. Acetoin, the reactive compound in the Voges-Proskauer test, was assayable in the medium and was used to quantify the metabolic response when inhibitors were added to the medium. Inhibitors such as lactic, acetic, propionic and benzoic acids were used to demonstrate the utility of acetoin production as an indicator of metabolic disruption. With increasing levels of inhibitor, the metabolic and growth responses were measured by acetoin production and optical density change, respectively. Both measurements decreased in a similar manner with increasing inhibitor concentrations. The data also showed the apparent mode of action of the inhibitors. A bacteriostatic effect was observed for the protonated organic acids, acetic (4 mmol l(-1)) and propionic (4 mmol l(-1)), whereas protonated lactic (4 mmol l(-1)) and benzoic (0.16 mmol l(-1)) acids gave an irreversible (apparent bacteriocidal) effect. Lactic, acetic, and propionic acids showed stimulation of metabolic activity at low concentrations, but benzoic did not. Acetoin production is a novel method for quantifying and assessing the mode of action of inhibitors against L. monocytogenes. This system can be used to screen inhibitors for applications in food safety.     

Minimum growth temperatures of Listeria monocytogenes and non-haemolytic Listeria

Minimum growth temperatures and those of decreased growth were determined for 100 strains of listerias. The ability of 78 strains of Listeria monocytogenes isolated from animals and 22 non-haemolytic strains to grow at low temperatures was studied, using a flooding technique, in a plate-type continuous temperature gradient incubator at temperatures between -1.6 and 14.5 degrees C. The mean minimum temperature for L. monocytogenes was +1.7 +/- 0.5 degrees C. The growth of non-haemolytic listerias was unobservable at +1.7 +/- 0.5 degrees C. The L. monocytogenes strains grew at about 0.6 degrees C lower than the non-pathogenic strains. No differences in growth temperatures were observed among L. monocytogenes strains isolated from different sources. The serovars with the OI antigen grew at lower temperatures (+1.0 +/- 0.3 degrees C) than the other common serovar 4b (+1.3 +/- 0.4 degrees C). The results indicate that L. monocytogenes grows better than non-haemolytic strains under cold conditions. The possible role of haemolysins as growth factors is also discussed.     

Acetoin production as an indicator of growth and metabolic inhibition of Listeria monocytogenes

It has been shown that Listeria monocytogenes produces acetoin from glucose under aerobic conditions. A defined medium with glucose as the sole carbon source was used in an aerobic shake flask culture to reliably produce acetoin. Acetoin, the reactive compound in the Voges–Proskauer test, was assayable in the medium and was used to quantify the metabolic response when inhibitors were added to the medium. Inhibitors such as lactic, acetic, propionic and benzoic acids were used to demonstrate the utility of acetoin production as an indicator of metabolic disruption. With increasing levels of inhibitor, the metabolic and growth responses were measured by acetoin production and optical density change, respectively. Both measurements decreased in a similar manner with increasing inhibitor concentrations. The data also showed the apparent mode of action of the inhibitors. A bacteriostatic effect was observed for the protonated organic acids, acetic (4 mmol l⁻¹) and propionic (4 mmol l⁻¹), whereas protonated lactic (4 mmol l⁻¹) and benzoic (0·16 mmol l⁻¹) acids gave an irreversible (apparent bacteriocidal) effect. Lactic, acetic, and propionic acids showed stimulation of metabolic activity at low concentrations, but benzoic did not. Acetoin production is a novel method for quantifying and assessing the mode of action of inhibitors against L. monocytogenes . This system can be used to screen inhibitors for applications in food safety.     

In vitro inhibition of bacterial growth by iron chelators

The antimicrobial activity of the iron(III)-selective 3-hydroxypyridin-4-one chelators, CP251(1) and CP252(2), was evaluated in comparison with that of diethylenetriamine-penta acetic acid (3). CP251 was found to exhibit an inhibitory effect on the growth of both Gram-positive and Gram-negative bacteria. CP251 may find application in the treatment of external infections such as those associated with wounds.     

In vivo and in vitro assessment of the virulence of Listeria monocytogenes strains

To evaluate whether the in vitro model (invasion and intracellular growth in Caco-2 cells) for determining virulence is a suitable alternative to the in vivo model (50% lethal dose), we compared the levels of virulence obtained with the two models. We tested L. monocytogenes strains isolated from food and clinical samples during three episodes of listeriosis occurring in Italy in the period 1993-1995. We also tested L. monocytogenes strains isolated from food during official control activities. The results obtained from the tested strains varied according to the experimental method adopted: the L. monocytogenes strains featuring the same genetic pattern showed a greater uniformity of response in vivo than in vitro. We can conclude that the in vitro model may be used as an alternative to the animal model to determine Listeria spp pathogenicity, though it cannot distinguish levels of virulence within the L. monocytogenes species.     

In vitro studies of haemolysis by some staphylococci grown in chemically defined media

Staphylococcus aureus (five strains) and Staph. epidermidis (one strain) have been evaluated for comparative growth and haemolysin titre in both brain heart infusion (BHI) and in developed, nutritionally adequate, chemically defined media (CDMs) varying only in amino acid composition. The ability to show a particular haemolytic profile was strain-dependent and the haemolytic titre (HU₅₀/ml) was both strain- and medium-dependent. Highest titres of both alpha and beta type haemolysins were obtained in BHI. Maximum titres were in general detected in the late exponential phase in both CDMs and BHI. Titres declined during the stationary phase in CDMs. Staphylococcus epidermidis produced a delta-type haemolysis profile on BHI-based blood agars, but only rabbit blood was sensitive in agars based on a developed, chemically defined medium (CDM/A; 13 amino acids) in which all six staphylococci grew. The addition of yeast extract to CDM/A increased alpha haemolysin titre, but suppressed beta haemolysin formation; beta haemolysin was, however, detected in yeast extract/phosphate-buffered saline. Strain Wood 46 degraded haemoglobin, but only in (initially) whole blood; red blood cell-free haemoglobin-rich plates (BHI) were unaffected during growth. A novel haemolytic profile is described for Staph. aureus NCTC 8532 growing on blood agars based on CDM/A and may relate to the production of methaemoglobin during haemolysis.     

In vitro studies of haemolysis by some staphylococci grown in chemically defined media

Staphylococcus aureus (five strains) and Staph. epidermidis (one strain) have been evaluated for comparative growth and haemolysin titre in both brain heart infusion (BHI) and in developed, nutritionally adequate, chemically defined media (CDMs) varying only in amino acid composition. The ability to show a particular haemolytic profile was strain-dependent and the haemolytic titre (HU50/ml) was both strain- and medium-dependent. Highest titres of both alpha and beta type haemolysins were obtained in BHI. Maximum titres were in general detected in the late exponential phase in both CDMs and BHI. Titres declined during the stationary phase in CDMs. Staphylococcus epidermidis produced a delta-type haemolysis profile on BHI-based blood agars, but only rabbit blood was sensitive in agars based on a developed, chemically defined medium (CDM/A; 13 amino acids) in which all six staphylococci grew. The addition of yeast extract to CDM/A increased alpha haemolysin titre, but suppressed beta haemolysin formation; beta haemolysin was, however, detected in yeast extract/phosphate-buffered saline. Strain Wood 46 degraded haemoglobin, but only in (initially) whole blood; red blood cell-free haemoglobin-rich plates (BHI) were unaffected during growth. A novel haemolytic profile is described for Staph. aureus NCTC 8532 growing on blood agars based on CDM/A and may relate to the production of methaemoglobin during haemolysis.