Inhibition of Listeria monocytogenes by a bacteriocinogenic Lactobacillus sake strain in modified atmosphere-packaged Brazilian sausage

Lactobacillus sake 2a is a bacteriocinogenic strain isolated from “lingüiça frescal”, a Brazilian sausage. The combined effect of modified-atmosphere (MA) packaging (100% CO₂ and 50% CO₂/50% N₂) and addition of L. sake 2a on inhibition of growth of Listeria monocytogenes was evaluated in “lingüiça” stored at 6 °C. By the end of the first week, the inhibition of L. monocytogenes due to MA was significant (P0.05) while the presence of L. sake 2a did not influence significantly the growth of the pathogen. After 14 days, a reduction of 1.3–1.4 log in counts of L. monocytogenes was observed in samples containing L. sake 2a only or MA packaged only, while a reduction of 3.5 log was detected in those submitted to both treatments. Results indicate that inhibition of L. monocytogenes in “lingüiça frescal” by the bacteriocinogenic L. sake 2a is enhanced by the packaging of the product in MA.     

Antilisterial activity of lactic acid bacteria inoculated on cooked ham

This study was conducted to evaluate the ability of Lactobacillus sakei 1, a bacteriocin-producing (bac⁺) lactic acid bacterium (LAB), isolated from Brazilian fresh pork sausage to inhibit two Listeria monocytogenes strains (serotypes 4b and 1/2a) on cooked, sliced vacuum-packaged ham. L. sakei ATCC 15521 was used as a non-bacteriocin producer (bac⁻). L. monocytogenes (ca. 2 log CFU/mL) and LAB (ca. 6 log CFU/ml) were inoculated on the sterilized ham, vacuum-sealed and incubated at 8 °C for 10 days. A treatment with the bacteriocin Chrisin (UI/ml) was included. Both L. monocytogenes strains were significantly inhibited in the presence of either bac⁺ and bac⁻ LAB in comparison to the control (L. monocytogenes alone). Using a bacteriocinogenic strain of LAB did not offer an additional barrier to listerial growth in the studied meat system. The application of Chrisin did not affect at all the growth of L. monocytogenes.     

The occurrence of Listeria monocytogenes in cheese from a manufacturer associated with a case of listeriosis

A case of listeriosis was associated with the consumption of a soft cheese produced in England. Goats cheese and other products from the same food manufacturer were examined for the presence of Listeria over the following 11 months. Listeria monocytogenes was isolated from 16 of 25 cheese samples on retail sale, 12 of 24 cheese samples obtained directly from the factory, and from shelving within the plant. Phage-typing of 68 isolates of L. monocytogenes from cheese samples and the factory showed that 66 (97%) were indistinguishable from the strain isolated from the patient's cerebrospinal fluid and stool. L. monocytogenes was not isolated from seven goats milk or two yoghurt samples. Listeria innocua was isolated from 10 cheese samples, two of which contained no other species of Listeria. Levels of L. monocytogenes shortly after production were low (<10/g), but were higher (10⁵–10⁷ cfu/g) in six of the 16 cheese samples obtained from retail outlets. Multiplication of L. monocytogenes was demonstrated in cheeses contaminated at the factory and held at 4°C in the laboratory.     

Characterization of Listeria monocytogenes and Listeria innocua from a vegetable processing plant by RAPD and REA

The incidence of Listeria monocytogenes in a vegetable processing plant was investigated over a 23-month period. Frozen ready-to-eat vegetable samples, well as the plant environment, were sampled. The molecular subtyping techniques, Random Amplified Polymorphic DNA (RAPD) and Restriction Endonuclease Analyses (REA), were performed to help investigate the origin and routes of Listeria dissemination.

The low and sporadic incidence of L. monocytogenes made it impossible to establish an epidemiological sequence in the processing plant, though a case of cross-contamination between tomato and ratatouille was detected. Listeria innocua subtyping, however, allowed us to determine the prevalence of several strains in vegetables, and their presence on machinery samples suggested the possibility of cross-contamination during processing.

The low incidence of L. monocytogenes indicated that the risk of listeriosis transmission by vegetable consumption is low. On the other hand, the isolation of the same strain of L. innocua in several surveys pointed out the risk of colonisation on surfaces and machinery. The persistence of Listeria spp. is a cause for concern as can lead to future contamination of vegetables processed in the plant and to a possible increased risk for health. Therefore, periodic controls for the presence of Listeria spp. and a further review of the cleaning and disinfection procedures used in frozen vegetable plants are recommended.     

Detection and Rapid Purification of Internalin B as a Protein Marker in Listeria monocytogenes

Clinical and food strains of Listeria monocytogenes have been found to express InternalinB (InlB) without polymorphism. InlB, which is a 67-kDa surface protein, behaves as an invasion and adhesion protein of the bacterium into cells. Thus, InlB could be a good candidate as a protein marker to detect L. monocytogenes. Three strains of L. monocytogenes (ATCC 19115, serotype 4b, ATCC 19111, serotype 1/2a, ATCC 7644, serotype 1/2c) were tested to detect and purify InlB. L. grayii (ATCC 25400) and L. innocua (isolated from soft cheese) were used as controls that did not express InlB due to the absence of its gene on the chromosome. InlB was quantitatively extracted in a solubilized form by treatment of L. monocytogenes with 1 M Tris-Cl at pH 7.5. Immunoblot analysis using anti-InlB polyclonal antibody revealed that L. monocytogenes 19115 had the lowest expression, which required enrichment of InlB for its detection. Simple spin-ion exchange chromatography with strong acidic cation exchanger was used to enrich the InlB protein that is strongly basic. Most impurities in the column were washed with 25 mM sodium acetate whereas the InlB protein was only protein retained in the column and can be eluted by 1 M NaCl. The data presented here showed that spin-ion exchange chromatography was found to be a simple and rapid method to enrich and purify InlB within 20 min.     

Bacteriocin-like inhibitory activities among various species of Listeria

Three hundred Listeria isolates were examined for inhibitory activities using a deferred antagonism plating assay. Approximately 75% of the surveyed isolates produced inhibitory activity, the majority of which (71%) resulted from the production of bacteriophage or defective bacteriophage particles. Twenty-three isolates (8%) produced inhibitory activities distinct from those resulting from bacteriophage. Four of these isolates (Listeria innocua 743, L. innocua 755, L. innocua 228, L. monocytogenes 538) produced heat-stable, protease sensitive peptides, which demonstrated broad-spectrum inhibitory activities against all L. monocytogenes serotypes tested.     

Antagonistic effect on Listeria monocytogenes and L. innocua of a bacteriocin-like metabolite produced by lactic acid bacteria isolated from sucuk

Two Lactobacilli and four Pediococci strains producing bacteriocin-like metabolities isolated from sucuk were tested with agar spot tests and well diffusion assays for their inhibitory activity against 16 Listeria strains, also isolated from sucuk. The production of organic acids and hydrogen peroxide limited, L. sake Lb 706 (used as a bacteriocin producer strain) and the isolated lactic acid bacteria (LAB) showed inhibitory activity against all of the Listeria strains, while L. sake Lb 706-A (used as a bacteriocin non-producer mutant) had the same effects against only two Listeria monocytogenes strains (51, 52) in agar spot tests. In the well diffusion assays, while L sake Lb 706 and four Pediococci isolates (413, 416, 419, 446) exhibited inhibitory activity against all of the Listeria strains tested, L. sake Lb 706-A and two of the Lactobacilli isolates (77, 116) showed no effect on the Listeria strains tested.     

Characterization of Listeria spp. isolated from ready-to-eat products in Argentina using SDS–PAGE and restriction endonuclease

Listeria spp. are considered of interest in public health since their presence indicates the potencial existence of L. monocytogenes. Total cellular proteins and DNA from four strains of L. monocytogenes serotype 4, four strains of L. monocytogenes belonging to serotype 1, twelve strains of L. innocua, four strains of L. seeligeri and two strains of L. welshimeri isolated from ready–to–eat food were studied by SDS–PAGE and restriction endonuclease digestion. SDS–PAGE protein profiles obtained were species specific and could be evaluated by visual comparison. Enzyme for restriction endonuclease analysis was EcoRI, discriminating L. monocytogenes from other Listeria spp. These methodologies might be a helpful tool and a good alternative for epidemiological tracking of listeriosis in laboratories, where other methods are not available.     

Effect of temperature and growth media on the attachment of Listeria monocytogenes to stainless steel

Listeria monocytogenes ATCC 19111 cultivated in nutrient-rich medium (brain heart infusion, BHI) or starved in minimal medium (10% filter sterilized pond water and 90% sterilized distilled water) were investigated for their initial attachment to austenitic stainless steel No. 4 with satin finish at 4 °C, 20 °C, 30 °C, 37 °C, or 42 °C. A droplet (10 μl) containing  10⁷ CFU/ml of L. monocytogenes suspended in BHI or minimal medium was placed on the stainless steel surface. After holding in saturated humidity for 3 h at the desired temperature the surface was washed and prepared for scanning electron microscopy (SEM). Using SEM, attachment of L. monocytogenes was determined by counting cells remaining on the surface. When L. monocytogenes cultivated in BHI were used, with the exception of the number of attached cells being lower at 42 °C than at 37 °C and 30 °C, the number of attached cells increased with increasing temperature (P < 0.05). When L. monocytogenes starved in minimal medium were used, the number of attached cells also increased with increasing attachment temperature (P < 0.05), but the number of attached cells at 42 °C was lower than that at the other temperatures. The attachment of L. monocytogenes to stainless steel surface was greater when cultivated in rich medium of BHI vs starved in the minimal medium.     

Survival of pathogenic microorganisms in an egg-nog-like product containing 7% ethanol

A liquor consisting of whole egg, saccharose (25% w/v) and ethanol (7.0% w/v) was artificially contaminated with Salmonella enteritidis, S. typhimurium, Staphylococcus aureus (three different strains), Bacillus cereus and Listeria monocytogenes. After 3 weeks of incubation at 22°C the numbers of Salmonella, S. aureus and L. monocytogenes decreased more than 3 log₁₀ units. Under such conditions, however, the total number of microorganisms increased 3 log₁₀ units. At 4°C the decrease of pathogenic microorganisms was much slower and a decrease of 3 log₁₀ units was observed only after 7 weeks of incubation. Egg-nog, without ethanol, incubated at 22°C allowed growth of Salmonella and S. aureus, while the numbers of B. cereus spores remained unchanged. Vegetative cells of B. cereus as well as L. monocytogenes decreased in numbers. However, after prolonged incubation the numbers of L. monocytogenes increased significantly.     

Characterization of the Effect of Sprouting or Solid-State Bioprocessing by Dietary Fungus on the Antibacterial Activity of Soybean Extracts Against Listeria monocytogenes

Listeria monocytogenes is one of the most severe food-borne bacterial infections causing Listeriosis. As L. monocytogenes can survive harsh adverse conditions - such as low pH, high NaCl, and refrigeration temperatures - as well as resist current antimicrobial measures such as the use of disinfectants and antibiotics, there is a need for alternative anti-Listeria strategies. In the search for new antimicrobial agents, much recent research has focused on the potential of dietary phenolic compounds. In this study, soybean extracts enriched for phenolic content via dark-germination sprouting or solid-state bioprocessing by the dietary fungus Rhizopus oligosporus or Lentinus edodes were investigated for in vitro antibacterial activity against L. monocytogenes.L. monocytogenes growth was inhibited most effectively by R. oligosporus bioprocessed soybean extracts, which showed anti-Listeria activity at total phenolic concentrations as low as 10 µg 100 µL^-1. In both sprouted soybean extract and L. edodes-bioprocessed soybean extract the anti-Listeria activity was not observed until at least 200 µg total phenolic content 100 µL^-1 was used. Anti-Listeria activity by soybean extract was associated with phenolic mobilization but not with antioxidant activity. Further, R. oligosporus bioprocessed soybean extracts were shown to inhibit the growth of L. monocytogenes in fish and meat systems at refrigeration temperatures. The potential involvement of mobilization of antimicrobial versus non-antimicrobial phenolics during sprouting and solid-state bioprocessing was hypothesized and discussed.     

Occurrence of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella and Campylobacter spp. on beef carcasses in Northern Ireland

A survey of beef carcasses was conducted in all 10 European community approved abattoirs in Northern Ireland to determine the incidence of Escherichia coli O157:H7. Analyses were based on excised samples of neck meat taken less than 48 h post-kill. Overall, 780 carcasses were sampled and all were negative for E. coli O157:H7. A sub-set of samples was analysed for the presence of Listeria monocytogenes (n=200), Salmonella (n=200) and Campylobacter spp.(n=100). L. monocytogenes was not detected but Listeria innocua was found on five carcasses and Listeria seeligeri on one. Three carcasses carried salmonellas; Salmonella Mbandaka was found on two and Salmonella Thompson on one. Campylobacter spp. were not detected on any carcasses. The results indicate that very few beef carcasses in Northern Ireland appear to carry any of the four pathogens sought, and this may help explain the low incidence of E. coli O157:H7 in the Northern Ireland human population, relative to the rest of the UK.     

Growth of Listeria monocytogenes on vacuum-packaged horsemeat for human consumption

In order to investigate the likelihood of Listeria monocytogenes (serotype 4b, ATCC 19115) growth on vacuum-packaged horsemeat at refrigeration temperature, fourteen horsemeat surface/volume homogeneous 150 g weight pieces were superficially inoculated with serotype 4b L. monocytogenes and vacuum packaged. The samples were stored at 4 ± 1 °C. Two pieces (one for pH determination and one for L. monocytogenes counts) were examined at days 0, 7, 14, 21, 28, 35 and 42. Surface pH did not show significant variations during the experiment. The average L. monocytogenes initial contamination level was 1.77log₁₀ CFU/g. A lag phase of 7 days was recorded. The exponential growth rate between day 7 to day 35 was 0.125log₁₀ CFU/day, corresponding to 3.51log₁₀ CFU/g in 28 days. At the end of the experiment the mean L. monocytogenes log₁₀ CFU/g was 5.78.     

Antibacterial activity of extracts from some edible plants commonly consumed in Asia

Extracts of edible plants (26 species) from China, Japan, Thailand and Yemen were screened for their antibacterial activity against Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella infantis. Buffered methanol (80% methanol and 20% PBS) and acetone extracted inhibitory substances against tested bacteria from 16 plants, as revealed by the disc assay. The minimum inhibitory concentrations (MICs) of extracts determined by the agar dilution method ranged from 165 to 2640 mg l⁻¹. The most sensitive microorganism to extracts from Azadirachta indica, Cinnamomum cassia, Rumex nervosus, Ruta graveolens, Thymus serpyllum and Zingiber officinale was B. cereus, with MIC of 165 to 660 mg l⁻¹. E. coli and S. infantis were only inhibited by Cinnamomum cassia extracts at the highest MIC (2640 mg l⁻¹). L. monocytogenes (Tottori) was more resistant than the ATCC 7644 strain to extracts from Ruta chalepensis, Artemisia absinthium and Cissus spp. EDTA (0.85 mM) reduced the MICs of Cinnamomum cassia and Cissus rotundifolia by at least 50% when tested against E. coli, S. infantis, S. aureus and L. monocytogenes.     

Listeria monocytogenes: Occurrence in beef and identification of the main contamination points in processing plants

This study aimed to establish the occurrence of Listeria spp., especially L. monocytogenes and its main serotypes, in beef and processing plants. A total of 443 samples were obtained from equipment, installations and products from 11 meat processing establishments from Paraná state, Brazil. All samples were analyzed using USDA methodology for Listeria spp. detection, followed by species identification. The occurrence of Listeria spp. in the samples was 38.1% of which 51.4% were from equipment, 35.4% from installations and 30.2% from products. The identified species were: L. monocytogenes (12.6%), L. innocua (78.4%), L. seeligeri (1.2%), L. welshimeri (7.2%) and L. grayi (0.6%). The identified serotypes of L. monocytogenes were 1/2a and 4b. The results demonstrate the significance of equipment and installations as sources of contamination by Listeria spp. and L. monocytogenes in the processing of beef and meat products.     

Effect of water activity on inactivation of Listeria monocytogenes and lactate dehydrogenase during high pressure processing

The aim of this study was to investigate the effect of water activity (a_w) on the inactivation of Listeria monocytogenes and lactate dehydrogenase (LDH) during high pressure processing (HPP). For microbial inactivation lyophilized cells of L. monocytogenes 19,115 were left dry or were suspended in 10 ml of 0.1% peptone water, 10 ml of glycerol, or mixtures of glycerol and peptone water. All samples of various a_ws were high pressure (HP) processed at ambient temperature at 600 MPa for 300 s. Following HPP, samples were serially diluted in 0.1% peptone and spread-plated on Tryptic Soy agar supplemented with Yeast Extract. For enzyme inactivation, 4.2 mg of lyophilized LDH was suspended in 2 ml of 100 mM phosphate buffer (pH 7.4), 2 ml of peptone water or glycerol, or in 2 ml mixtures of glycerol and peptone water. A lyophilized sample with no added liquid was also included. All enzyme samples were subjected to HPP as described above. After HPP, LDH was diluted to 0.28 μg/ml in 100 mM phosphate buffer (pH 7.4). LDH activity was assessed by measuring the change in concentration of β-NADH as a function of time. Dynamic light scattering analysis (DLS) was performed to examine the size distribution, polydispersity, and hydrodynamic radius of LDH before and after HPP. No significant difference in CFU/g was observed between lyophilized cells not subjected to HPP and lyophilized cells subjected to 600 MPa for 300 s (P < 0.05). However, lyophilized cells that were suspended in 100% to 60% peptone water showed a ~ 7.5-log₁₀ reduction when subjected to HPP. Survival of L. monocytogenes following HPP significantly increased (P < 0.05) when the peptone water concentration was decreased below 60% (a_w ~ 0.8). DLS results revealed that LDH suspended in buffer underwent aggregation following HPP (600 MPa, 300 s). Inactivation rate constants obtained using a first-order kinetic model indicated that untreated and HP processed lyophilized LDH had similar activities. When LDH was subject to HPP in solutions containing glycerol, enzyme activity decreased as the water content increased (r² = 0.95). Lyophilization completely protected L. monocytogenes and LDH from inactivation by high pressure. Furthermore, enzyme activity and cell survival increased as water activity was decreased. We postulate low a_w results in protein stabilization, which prevents protein denaturation and cell death during HPP.     

Inhibition of Listeria monocytogenes by Elite Clonal Extracts of Oregano (Origanum vulgare)

Food safety continues to be a major concern for the food industry in recent years. One of the industry's top priorities has been to find alternative ways to preserve their newly developed foods while satisfying the increasing consumer demand to produce safe, all-natural products. In order to achieve this “clean label”, much research has been devoted to the use of effective plant-based antimicrobials, such as those from herbs and spices, to replace chemical preservatives. However, due to the cross-pollination character of herbs and spices, there is a lot of genetic heterogeneity among different batches of the same plant species. This poses a problem for the routine use of plants, and their extracts, as a barrier towards microbial growth. In order to combat this, a unique tissue-culture-based selection strategy was used to isolate an elite phenolic phytochemical-producing clonal line of oregano (Origanum vulgare). Ethanol extracts of this elite clonal line of oregano were then used to study its inhibitory action against Listeria monocytogenes in both broth and meat systems. Thymol and carvacrol, two of the main phenolic constituents of oregano extracts, were also tested in both systems to evaluate their activity against that of the whole oregano extract.

Results indicate that thymol, carvacrol, and the clonal oregano line were all effective in inhibiting the growth of L. monocytogenes in both systems. Approximately 150-200 ppm of pure carvacrol or thymol was needed in order to significantly inhibit the growth of L. monocytogenes in broth, while at least 1200 ppm (corresponding to 27.8 μg phenolics/ml) of the elite clonal oregano extract was needed to do the same. Inconclusive results were obtained when the clonal line was compared to store-brand samples of oregano. In meat systems, 800 ppm of the oregano extract was able to significantly inhibit the growth of the pathogen more so than 800 ppm of carvacrol. A possible explanation for this is that the oregano extract was able to work more effectively at the interface of the lipid and water-soluble portions of the meat than the carvacrol. These results are promising for the food industry since we have now developed an approach for a highly consistent and reliable natural source of antimicrobial activity for future studies.     

Purification, partial characterisation and mode of action of enterococcin EFS2, an antilisterial bacteriocin produced by a strain of Enterococcus faecalis isolated from a cheese

Enterococcus faecalis strain EFS2, isolated from the surface of a traditional cheese, produced a bacteriocin active against Gram-positive bacteria including Listeria spp. and some Staphylococcus aureus strains. The bacteriocin, named enterococcin EFS2, has been purified to homogeneity by ammonium sulphate precipitation and reversed-phase high performance liquid chromatography (RP-HPLC). The molecular weight was determined by mass spectrometry to be 7149.6. The amino acid composition of enterococcin EFS2 revealed that it contained 67 amino acid residues and had a blocked amino-terminal end. Enterococcin EFS2 induced viability loss, efflux of K⁺ ions and ATP, and cell lysis. Kinetic study of bactericidal activity of enterococcin EFS2 on Listeria innocua strain LIN 11 indicated slower cell destruction than by nisin. At pH 7.0, the activity of enterococcin EFS2 was the highest at 35 °C and was lost at 15 °C. The bacteriocin was more active against L. innocua strain LIN11 in broth adjusted to pH 6.0, 7.0 and 8.0 than to pH 4.5 at 30 °C.     

Characterization of Staphylococcus xylosus and Staphylococcus carnosus isolated from Slovak meat products

The aims of this study were to isolate, identify and characterize the population of coagulase-negative staphylococci in different types of Slovak traditional sausages and to determine the metabolic properties of selected Staphylococcus xylosus and S. carnosus strains for the selection of potential starter cultures to use in the processing of sausages. The strains were tested for lactic acid production, survival in the presence of bile and sensitivity to antibiotics. Bacteriocin production, adhesion ability as well as biogenic amine (BA) production by isolates were also analysed. Most of the isolates were identified as S. xylosus and S. carnosus. Lactic acid values ranged from 0.40 to 1.03 mmol/l and strains survived in the presence of 1% bile. Most of the strains studied were sensitive to all antibiotics. Two strains, S. xylosus SO3/1M/1/2 and S. carnosus SO2/F/2/5 inhibited Listeria innocua and Pseudomonas sp. S. xylosus strains did not produce any BA, while S. carnosus SO2/F/2/5 did. S. xylosus SO3/1M/1/2 and S. carnosus SO2/F/2/5 appeared as the most adhesive strains. S. xylosus SO3/1M/1/2 with antimicrobial activity against Enterococcus avium EA5, L. innocua LMG13568 and Pseudomonas sp. SO1/1M/1/4, adhesion ability and free BA production could be used as starter culture in sausage manufacture.     

Comparing predicting models for heat inactivation of Listeria monocytogenes and Pseudomonas aeruginosa at different pH

Under the same experimental conditions it has been demonstrated that whereas survival curves of Listeria monocytogenes in the range of temperatures from 54 to 62 °C followed a first-order kinetic, those of Pseudomonas aeruginosa in the range of temperatures from 50 to 56 °C were not linear showing a shoulder followed by a linear region. The first order kinetic model did not describe survival curves of P. aeruginosa. A model based on the Weibull distribution (Log₁₀(N_t/N₀)=(1/−2.303)*(t/b)ⁿ)) accurately described the inactivation kinetics of both microorganisms at the three pHs of 4, 5.5, 7.4 investigated. For both microorganisms, the b value depended on the treatment temperature and the pH of the treatment medium. Whereas for L. monocytogenes the n value was independent of the treatment conditions, for P. aeruginosa the n value depended on the pH of the treatment medium.

The model based on the Weibull distribution was capable of accurately predicting the treatment time to inactivate five Log₁₀ cycles of both microorganisms at the three pHs investigated.