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.     

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 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.     

Mathematically modeling the repair of heat-injured Listeria monocytogenes as affected by temperature, pH, and salt concentration

Heat-injured cells of Listeria monocytogenes were inoculated into Listeria repair broth (LRB) adjusted to various pH levels (4.2, 5.0, 6.6, 8.0 and 9.6) and salt concentrations (0.5%, 2.5%, 5.0%, 7.5% and 10.0% w/v) at controlled temperatures (4, 10, 22, 37 and 43 °C) in a complete factorial manner (5³). Repair of the injured microorganisms was evaluated using selective and non-selective plating media. The Gompertz parameters, which were generated by fitting the equation with the bacterial counts, were used to calculate the repair percentage as a function of time from which the repair time was estimated. All growth curves fit the Gompertz equation well (R² ≥ 0.972). A first-order model described the repair trend closely (R² = 0.989 ± 0.011). Heat-injured Listeria could fully repair in LRB only under 63 of 125 conditions tested during 21 days of incubation. Refrigeration temperature was the most effective means to prevent the repair of heat-injured Listeria. The minimum temperature required for repair increased with an increase in NaCl concentration. The pH ranges at which the repair could occur were narrower at 4 and 10 °C than those at higher temperature. The repair was observed in media containing 10% NaCl between temperatures of 22 and 43 °C at pH 6.6.     

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 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.     

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.     

Behaviour of Listeria monocytogenes in raw sausages (merguez) in presence of a bacteriocin-producing lactococcal strain as a protective culture

The effectiveness of a bacteriocin produced by Lactococcus lactis subsp. lactis M in reducing population level and growth of Listeria monocytogenes ATCC 7644 in fermented merguez sausage was examined. Two different formulas (with or without added nitrites) were assayed and predetermined numbers of Listeria (ca 10⁶ cfu g⁻¹) were added to sausage mixture. The effect of in situ production of the bacteriocin by Lactococcus lactis M on Listeria monocytogenes ATCC 7644 during fermentation and storage of merguez sausages at room (ca 22 °C) or at refrigeration (ca 7 °C) temperature was tested. Results indicated that counts of Listeria monocytogenes were decreased during fermentation of merguez samples fermented with either the bacteriocin-producing Lactococcus lactis M (Bac⁺) or a nonbacteriocin-producing Lactococcus lactis J (Bac⁻). However, reduction in Listeria cfu's was greater in samples fermented with the Bac⁺ than in those fermented with the Bac⁻ starter. In merguez sausage made without nitrites addition, the Bac⁺ starter induced further decrease in Listeria counts by 1.5 log cycles compared with that induced by the Bac⁻ starter. While in merguez samples with added nitrites (0.4%), the effect of the bacteriocin produced in situ was less important than in those made without nitrites addition.     

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.     

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.     

Comparison of the hydrophobic grid-membrane filter DNA probe method and the Health Protection Branch standard method for the detection of Listeria monocytogenes in foods

The standard Health Protection Branch (HPB) method for the detection of L. monocytogenes in foods involves lengthy enrichment, selection and biochemical testing, requiring up to 8 days to complete. A hydrophobic grid-membrane filter (HGMF) method employing a digoxigenin-labelled listeriolysin O probe required 5 days to complete, and included an image-analysis system for electronic data acquisition. A total of 200 food samples encompassing 8 high-risk food groups (soft and semi-soft cheeses, packaged raw vegetables, frozen cooked shrimp, ground poultry, ground pork, ground beef, jellied meats, and pâté) were screened for the presence of L. monocytogenes by the two methods. Overall, 32 (16%) and 30 (15%) of the naturally-contaminated food samples tested positive for L. monocytogenes by the HPB and DNA methods, respectively. The DNA probe method was highly specific in discriminating L. monocytogenes from other Listeria spp. present in 50 of the samples tested. Results showed 94% sensitivity and 100% specificity between the two methods. The HGMF DNA probe method is an efficient and reliable alternative to the HPB standard method for detecting L. monocytogenes in foods.     

Listeria innocua and aerobic mesophiles during chill storage of inoculated mechanically recovered poultry meat treated with high hydrostatic pressure

Mechanically recovered poultry meat (MRPM) was inoculated with Listeria innocua 910 CECT at a level of approximately 10⁸ CFU g⁻¹. Vacuum-packaged samples were treated by combinations of pressure (350, 400, 450 and 500 MPa), time (5, 10, 15 and 30 min) and temperature (2, 10 and 20°C) and later stored at 2°C for 2 months. Counts of L. innocua and aerobic mesophilic bacteria were determined 1, 4, 7, 15, 30 and 60 days after pressurisation. For mesophiles, in most treatments, pressurization at 2°C gave the significantly best results. High pressure caused a marked bactericidal effect on L. innocua: reductions higher than 7.5 log units were achieved in several cases. Some cells were just sublethally injured by pressure. Samples treated at 500 MPa for 30 min at 2°C had counts of only 2.3 log units after 60 days of chill storage. Noninoculated pressurised MRPM did not show Listeria growth throughout storage. These results suggest that high pressure processing can enhance the microbiological quality of MRPM.     

Physiological properties of Lactobacillus paracasei, L. danicus and L. curvatus strains isolated from Estonian semi-hard cheese

Growth and survival of Lactobacillus paracasei (six strains), L. danicus sp. nov. (four isolates, two strains) and L. curvatus (two strains) from semi-hard Estonian cheeses were comparatively studied in different environmental conditions of relevance for their growth in cheese and survival in gastric environment. Maximum specific growth rates for L. paracasei strains varied between 0.40 and 0.57 h⁻¹, and all strains were tolerant to low water activities, heating at 60 °C for 30 min and pH 3. The newly discovered genetically distinct species L. danicus was characterized by low maximum growth rates (0.26–0.38 h⁻¹) and low temperature optimum (<30 °C). It was acid and heat sensitive and inhibited at salt concentrations from 4% and water activities below 0.93. L. curvatus was characterized by the highest growth rates (0.65–0.70 h⁻¹), tolerance to high NaCl concentrations, but sensitivity to heating, bile salts and low pH. The study showed that genetically different LAB species isolated from cheese could be distinguished by simple cultivation experiments.     

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.     

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.     

The influence of commonly used selective agents on the growth of Listeria monocytogenes

The effects, upon addition of various selective agents to a rich growth medium (BHIEM) on the growth of Listeria monocytogenes and non-Listeria were studied in microtitre wells. A combination of nalidixic acid (0.1 g/l), acriflavine (0.02 g/l) and fosfomycin (0.1 g/l) added to the medium (BHIEM_NAF) was found to be the least inhibitory to Listeria while at the same time, effectively inhibitory to non-Listeria organisms. Recovery of heat injured Listeria monocytogenes in BHIEM, BHIEM_NAF and the two traditional Listeria-media, UVM I and II, was compared. Recovery in BHIEM was obtained within 24 h of incubation at 30°C, whereas recovery in BHIEM_NAF took up to 48 h. Growth and recovery in UVM I and II never reached the same level as in BHIEM or BHIEM_NAF.     

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.     

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.     

Occurrence of Listeria monocytogenes in fresh and processed foods in Navarra (Spain)

The presence of Listeria spp. was investigated in a total of 3685 food samples obtained from different industries and markets of Northern Spain in the last 4 years. The samples analyzed include fresh raw products (meat, milk and poultry) and treated products (cooked and cured meats, frozen vegetables and smoked salmon). Occurrence of Listeria spp. varied from 8.1% in soft cheese to 76.3% in raw poultry samples. The highest incidence of L. monocytogenes also occurred in raw poultry (36.1% positive samples). Despite this high incidence of contamination, these kinds of products carry a low risk of listeriosis transmission because of the heat treatment prior to consumption. On the other hand, the ready-to-eat products (RTE) tested in this study showed incidences that could pose serious health problems, taking into account that the storage conditions may allow for rapid growth of the pathogen. It was also found that up to 75.5% of the L. monocytogenes strains isolated in this study belonged to serogroup 1, mainly serotype 1/2a, while the clinical cases observed in Navarra in the same period of time belonged mainly to serotype 4b/4bx.     

Selective enumeration and survival of bifidobacteria in fresh cheese

Five species of bifidobacteria (15 strains), two strains of Lactococcus lactis ssp. lactis, two strains of L. lactis ssp. cremoris, and one strain of L. lactis ssp. lactis var diacetylactis were included in a study to develop a selective medium for enumeration of bifidobacteria from fresh cheese. Viable counts of bifidobacteria or lactococci on modified Columbia agar base (CAB with 0.05% cysteine-HCl) plus raffinose (0.5%) containing various selective agents were compared with non-selective media. The mCAB plus raffinose with lithium chloride (2 g L⁻¹) and sodium propionate (3 g L⁻¹) with pH adjusted to 5.1 was used successfully as a selective medium for the enumeration of bifidobacteria from fresh cheese. Using this medium, it was determined that bifidobacteria could survive up to 15 days at a level higher than 10⁶ cfu g⁻¹ in a fresh cheese stored at 4 or 12°C. The decrease in the viable counts of bifidobacteria was faster during storage at 4°C than at 12°C.