Development of a rapid, simple paddle-style dipstick dye immunoassay specific for Listeria monocytogenes

We have developed two types of new paddle-style dipstick dye immunoassays. The first is genus Listeria specific and the second is specific to Listeria monocytogenes. They are based respectively, on antisera raised against heat-killed L. monocytogenes cells and against internalin B crude extract, a virulence protein found only in the pathogenic L. monocytogenes. The minimum detectable level for L. monocytogenes is 2×10⁷ CFU ml⁻¹ for strain number 88/049 in pure culture. Detection is unaffected by the presence of high numbers (approximately log 8.0 CFU/ml) of the other microorganisms tested. When the dipsticks were applied to milk samples inoculated with L. monocytogenes reference material (ALM92), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of milk and ice cream food samples.     

In-milk inactivation of Escherichia coli O157:H7 by the environmental lytic bacteriophage ECPS-6

Shiga toxin-producing Escherichia coli is a common foodborne pathogen which transmission includes dairy products. In the search for novel biocontrol methods, bacteriophages have become important candidates for the eradication of foodborne pathogens. The aim of this study was to evaluate the bacteriophage-mediated reduction of E. coli O157:H7 in raw and filtered milk. Laboratory-scale tests showed that the bacteriophage ECPS-6 efficiently adsorbed to E. coli O157: H7 cells. Furthermore, ECPS-6 remained stable when heated at 70°C for 20 min and in a wide pH range from 3.0 to 11.0. The trials on contaminated milk were performed using filtered and unfiltered raw milk contaminated with 1 × 10⁵ CFU × ml⁻¹ of E. coli O157: H7. Bacteriophage was added at multiplicity of infection (MOI) 5 and 50. The ECPS-6 reached the highest lytic activity at MOI = 5 (25°C) which resulted in 4.74 Log₁₀ CFU × ml⁻¹ and 7.3 Log₁₀ CFU × ml⁻¹ reduction after 10 days for both tested strains, respectively. Under refrigerated conditions (4°C) the quantity of E. coli decreased to 1.5 Log₁₀ CFU × ml⁻¹ and 3.04 Log₁₀ CFU × ml⁻¹ for these strains, respectively. Usage of MOI = 50 for the treatment unfiltered milk led to the reduction of E. coli O157:H7 A-2 below the detection limit after 6 hr.     

Microbial competition: effect of Pseudomonas fluorescens on the growth of Listeria monocytogenes

Listeria monocytogenes Scott A was cultured alone and in coculture with Pseudomonas fluorescens ATCC 33231 to characterize quantitatively the effects of microbial competition on the growth of this psychrotrophic pathogen. The bacteria were cultured in brain–heart infusion broth (BHI), using a 3×3×3×2 complete factorial design to assess the impact of temperature (4, 12, 19°C), initial pH (5·0, 6·0, 7·0), and sodium chloride content (5, 25, 45 gl⁻¹) on the interaction between the two micro-organisms. Samples were periodically plated on BHI agar and Vogel Johnson agar to obtain total counts and L. monocytogenes counts, respectively. Growth curves were generated by fitting the data to the Gompertz equation, and the derived growth kinetics were compared. WhenP. fluorescens did influence the growth of L. monocytogenes, the primary effect was a suppression of the maximum population density (MPD) reached by the pathogen. Suppression of L. monocytogenes was generally associated with low incubation temperatures (4°C) and sodium chloride levels (5 and 25 gl⁻¹). Slight increases (<1·0 log cfu ml⁻¹) in the MPD attained by L. monocytogenes were observed when grown in the presence of P. fluorescens at higher temperatures (12 and 19°C) and sodium chloride levels (25 and 45 gl⁻¹) when the pH was 5·0. The current study supports earlier work that indicates that reliance on microbial competition as a barrier to control L. monocytogenes in refrigerated foods will require detailed knowledge of how the interaction between the pathogen and the microflora is affected by environmental and food characteristics such as storage temperature, pH, and water activity.     

Response of Listeria monocytogenes and Vibrio parahaemolyticus to High Hydrostatic Pressure

Listeria monocytogenes Scott A and CA, were subjected at 23°C to hydrostatic pressures ranging from 2,380 to 3,400 atm and Vibrio parahaemolyticus T-3765-1 from 680 to 1,700 atm. For L. monocytogenes Scott A, pressurization in ultra-high temperature-processed (UHT) milk and raw milk appeared to provide a protective effect and lessened cell death as compared to pressurization in phosphate-buffered saline (100 mM, pH 7.0). A population of about 10⁶ CFU/mL L. monocytogenes was killed by exposure to 3,400 atm within 80 min at 23°C in UHT milk. A population of about 10⁶ CFU/mL V. parahaemolyticus was killed by exposure to 1,700 atm within 10 min at 23°C in clam juice.     

Effects of ultra high hydrostatic pressure on Listeria monocytogenes and natural flora in broth, milk and fruit juices

Listeria monocytogenes was subjected to ultra high hydrostatic pressure (UHHP) treatments from 200 to 700 MPa at 25 °C in broth, raw milk, peach juice and orange juice. Survivor curves showed that cell death increased as pressure increased. After 10 min pressure treatment at 400 MPa reductions of about 2.09 and 2.76 log CFU mL^?1 in aerobic bacteria and L. monocytogenes, respectively, were produced in raw milk, this increased to 5.09 and 6.47 log CFU mL^?1, respectively, at 600 MPa. Death of bacteria at UHHP treatment was greater in orange juice than peach juice, and in peach juice than milk. Listeria monocytogenes was more sensitive to increased pressure than increased pressurization time. Injury of L. monocytogenes occurred from 0 to 100%. Factors effecting the rate of microbial inactivation are: pressure, age of cell, composition of medium, and pressurization time. UHHP inactivation can be used to extend shelf life and increase food quality during storage, and may also contribute to inactivation of L. monocytogenes.     

Growth ofListeria monocytogeneson sliced cooked meat products

During the shelf life (4–6 weeks) of artificially contaminated sliced cooked meat products such as luncheon meat, ham and chicken breast, the growth ofListeria monocytogenesunder vacuum was similar to the growth under modified atmosphere (30% CO₂/70% N₂) packaged products. The presence of competitors (lactobacilli), even in concentrations 100 times those ofL. monocytogenes, only slightly inhibited growth of this pathogen. At the end of the shelf life levels were still 10⁷cfug⁻¹. Due to the lower initial contamination, levels in naturally contaminated products were about 10⁴cfug⁻¹. To prevent outgrowth ofL. monocytogenesto such high levels it is necessary to prevent recontamination during slicing and packaging, and to shorten the rather long shelf life of these products. Due to the low pH of fermented sausage (saveloy) and (raw) Coburger ham the numbers ofL. monocytogenesdecreased below the detection level.     

Effects of curing additives on the control ofListeria monocytogenesby lactocin 705 in meat slurry

Meat slurry inoculated withListeria monocytogenes(4.00 cfu g⁻¹) was mixed with different levels of curing additives and their influence on the inhibitory effect of lactocin 705 (17,000 AU ml⁻¹) was evaluated at 20°C. Inhibition ofL. monocytogeneswas 1.90 and 1.00 log less in meat slurry with 5 and 7% NaCl than in meat slurry without added sodium chloride. When nitrite and bacteriocin were added together, less nitrite (200 μg g⁻¹) was required to obtain the sameListeriapopulation (3.00 log cfu g⁻¹) as when 800 μg g⁻¹NaNO₂was used. However, when compared with lactocin 705 alone, lessListeriainhibition was observed showing also a protective effect of NaNO₂. When ascorbic acid and alginate meat binder were assayed in the presence of the bacteriocin, the inhibition ofL. monocytogeneswas less effective, but when sodium lactate (2%) was added to the meat slurry, almost no protective effect was observed. These results indicated that the use of lactocin 705 to controlL. monocytogeneswas less effective in the presence of curing ingredients.     

Occurrence of Listeria species in raw milk in farms on the outskirts of Mexico city

Listerosis may be transmitted by direct contact with infected animals or by consumption of contaminated vegetables or meat and milk products. In Mexico, raw milk is widely consumed and the incidence of milkborne disease is unknown. A total of 1300 raw milk samples were obtained from 20 l bulk tanks at four different dairy farms in southeast of Mexico City from June 1998 to June 1999. The samples were enriched for 48 h at 30°C and plated onto McBride's Modified Agar (MMA). Suspect colonies were biochemically tested to confirm identity. Overall, 23% of all raw milk samples examined tested positive forListeria species; 13% were positive for L. monocytogenes (45·6% were serotype-4b and 54·4% were serotype 1); 6% for L. ivanovii; 4% for L. seeligeri and 1% forL. innocua. L. monocytogenes contamination was more frequent during the spring and summer months as isolation rates were 12·2% from June to October 1998 and 17% from March to June 1999. Serotype-4b isolates were not pathogenic for the mouse, while for serotype-1, strains DL₅₀ranged from 1·8×10⁶to 4×10⁷CFU ml⁻¹. Additional studies are needed to assess the public health impact of contaminated milk in Mexico.     

The fate of Listeria monocytogenes during the manufacture of Camembert-type cheese: A comparison between raw milk and milk treated with high hydrostatic pressure

Camembert-type cheese was produced from: raw bovine milk; raw milk inoculated with 2 or 4 log CFU/ml Listeria monocytogenes; raw milk inoculated with L. monocytogenes and subsequently pressure-treated at 500 MPa for 10 min at 20 °C; or uninoculated raw milk pressure-treated under these conditions. Cheeses produced from both pressure-treated milk and untreated milk had the typical composition, appearance and aroma of Camembert. Curd and cheese made from inoculated, untreated milk contained large numbers of L. monocytogenes throughout production. An initial inoculum of 1.95 log CFU/ml in milk increased to 4.52 log CFU/g in the curd and remained at a high level during ripening, with 3.85 log CFU/g in the final cheese. Pressure treatment inactivated L. monocytogenes in the raw milk at both inoculum levels and the pathogen was not detected in any of the final cheeses produced from pressure-treated milk. Therefore high pressure may be useful to inactivate L. monocytogenes in raw milk that is to be used for the production of soft, mould-ripened cheese.

Industrial relevance

This paper demonstrates the potential of high pressure (HP) for treatment of raw milk to be used in the manufacture of soft cheeses. HP treatment significantly reduced the level of Listeria monocytogenes in the raw milk and so allowed the production of safer non-thermally processed camembert-like soft cheese.     

Evaluation of sodium dichloroisocyanurate treatment on recovered concentrations of Salmonella enterica,Escherichia coli O157:H7, and Listeria monocytogenes from cattle hide surfaces and culture medium

A major concern of the cattle industry is cross-contamination of meat with pathogens. Cattle are exposed to fecal material, mud, and other contaminants which harbor pathogens that can be shed onto meat and meat processing equipment. Due to increased chances of meat contamination during processing, new antimicrobial formulations for carcass washing before hide removal needs to be identified and tested. Sodium dichloroisocyanurate (SDIC) has biocidal properties and belongs to the N-halamine group of compounds. Disk diffusion assays revealed, 1,000 ppm SDIC effectively reduced pathogen concentrations. SDIC was evaluated for its effects on pathogens in Tryptic Soy Broth and results revealed that 1,000 ppm SDIC had a strong correlation with time and treatment with no bacterial growth in log CFU ml⁻¹ observed at the lowest detection level. Treatment of inoculated hides with 1,000 ppm SDIC for 5 min resulted in reduction of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes at 1.97, 2.02, and 2.84 log CFU cm⁻², respectively.     

Testing matrix, inoculum size, and incubation temperature affect monolaurin activity againstListeria monocytogenes

Current literature reports conflicting minimal inhibitory concentrations (MIC) (range 10–96 g ml⁻¹) of glycerol monolaurate (monolaurin) againstListeria monocytogenes. To resolve these disagreements, the individual MIC of monolaurin against eightL. monocytogenesstrains was determined using the standard agar dilution technique on three commercial media (trypticase soy agar, plate count agar, and Oxford modified medium), and a catfish-based medium using three inocula sizes and three incubation temperatures. Mean MIC was 16 g ml⁻¹on commercial media at 25 and 35°C. Endpoints at 15°C were twofold lower (8 g ml⁻¹) than at 25 and 35°C. On catfish-based medium, MIC values were four- to eightfold higher (64–128 g ml⁻¹) compared with commercial media and depended on incubation temperature and size of inoculum tested. Poor solubility of monolaurin (85 g ml⁻¹) in aqueous solutions and its reduced activity in the presence of food components limits its application as an antimicrobial agent in foods.     

Cell viability of Listeria monocytogenes biofilms

Several authors have reported biofilm formation by Listeria monocytogenes, and it is suspected that biofilms form a unique niche for extended survival of this foodborne pathogen in food-processing environments. We have evaluated growth of two L. monocytogenes strains (Murray and 7148) in biofilms and analysed the relationship between culturable and viable-but-non-culturable (VBNC) cells. Biofilms were grown on glass slides in static conditions at 37°C for up to 10 days. Culturable cells for L. monocytogenes Murray grew to 10⁵cfu cm⁻²within 2 days, while L. monocytogenes 7148 required 4 days to reach these cell numbers. After 2 days, cell counts of L. monocytogenes Murray decreased, followed by another increase with cell numbers reaching almost 10⁶cfu cm⁻²on day 10. In contrast, cell counts of L. monocytogenes 7148 stayed close to 10⁵cfu cm⁻²until day 10. VBNC cells of L. monocytogenes Murray increased with biofilm age while this was not seen for strain 7148. Also, swabbing removed biofilms of strain Murray more easily than strain 7148. Comparisons of viable counts obtained for swabbed and in situ biofilms indicated that these strain differences are due either to variable composition of extracellular polymeric substances in the two biofilms or to different cell physiology of the two strains.     

Synergistic antimicrobial effect of lactocin AL705 and nisin combined with organic acid salts against Listeria innocua 7 in broth and a hard cheese

The effectiveness of antimicrobial mixtures against Listeria innocua 7, used as a L. monocytogenes surrogate, was investigated in broth and a food system. Synergistic effects were found for nisin (Nis), potassium sorbate (PS), calcium propionate (CP) and sodium lactate (SL), Nis + PS being the most effective binary mixture that exhibited listericidal activity in broth. To assess the effect of adding lactocin AL705 (AL705) to Nis + organic acid salt combinations, tridimensional isobolograms were generated. Sub-MIC combinations of the antimicrobials exerted bactericidal activity against L. innocua 7 after AL705 addition to the binary mixtures. However, when applied on Sardo cheese contaminated with L. innocua 7 (initial inoculum 4.45 ± 0.06 CFU g⁻¹), only Nis + PS + AL705 produced count reductions respect to the control, reaching 3.04 ± 0.35 CFU g⁻¹ counts after 15 days at 15 °C. Ternary combinations containing AL705 showed potential to reduce antimicrobial usages for L. innocua 7 inhibition.     

Development of a loop-mediated isothermal amplification assay for detecting Listeria monocytogenes prfA in milk

A rapid and sensitive loop-mediated isothermal amplification (LAMP) assay was developed for detecting Listeria monocytogenes prfA in milk. The inclusivity of 23 L. monocytogenes and the exclusivity of 16 non-L. monocytogenes strains were both 100% in the assay. The limit of detection (LoD) of the LAMP assay in Listeria enrichment broth (LEB) was 2.22 CFU/mL after 12 h and 24 h of incubation. The LoDs of the LAMP assay in LEB with artificially contaminated milk (LEB-M) incubated for 12 h (2.22×10¹ CFU/mL) and 24 h (2.22 CFU/mL) were lower than those of the PCR and real-time PCR assays. Comparison of the LoDs in LEB with those in LEB-M showed that the LAMP assay was less influenced by the milk compounds than the real-time PCR assay. Our results indicate that the LAMP assay can be utilized as a potential screening tool for L. monocytogenes in milk.     

The effects on vegetative cells and spores of three bacteriocins from lactic acid bacteria

The sensitivities of vegetative cells of strains ofListeria, Clostridium, Staphylococcus, Lactococcus, Lactobacillus, MicrococcusandPediococcus, and of spores ofClostridiumandBacillusto three broad spectrum bacteriocins (nisin A, nisin Z and pediocin) from lactic acid bacteria were determined by a critical dilution micro-assay. The minimal inhibitory concentrations (MIC) of partially purified bacteriocins, prepared by a pH-dependent adsorption/desorption process, were determined and expressed in arbitrary units ml⁻¹and in μ g ml⁻¹of pure bacteriocin. The MICs of bacteriocins varied considerably between species and even between strains of the same species, as clearly shown for nine strains ofListeria monocytogenes. When bacteriocin activity was expressed in μ g ml⁻¹, pediocin was more effective againstListeria monocytogenesthan nisin A or nisin Z. The latter bacteriocins, in concentrations between 23 and 69 μ g ml⁻¹, prevented outgrowth ofClostridiumandBacillusspores for at least 10 days. Although pediocin at 17 μ g ml⁻¹prevented outgrowth ofB. stearothermophilusandC. butyricumspores for up to 7 days, it apparently activated the germination ofB. subtilisspores.     

Impedimetric Characterization of Adsorption of Listeria monocytogenes on the Surface of an Aluminum‐Based Immunosensor

The impedimetric characteristics of an immunosensor depend on the electrical properties of an immunosensor substrate. The impedimetric characteristics of an immunosensor compared with adsorption of Listeria monocytogenes were investigated on an aluminum surface insulated with an electrically resistive aluminum oxide layer. Antibody for L. monocytogenes (anti‐L. monocytogenes) was immobilized on an aluminum surface that was insulated with a native air‐formed aluminum oxide layer. The resistance of impedance (R) value of an aluminum‐based immunosensor decreased, especially at 10⁴ to 10⁶ Hz, where the effect of the reactance of impedance (X) was minimal when L. monocytogenes was adsorbed on the immunosensor surface. The R value of the immunosensor at 81 kHz decreased proportionally to the concentration of L. monocytogenes from 1.3 to 4.3 log CFU mL^?1. The adsorption of L. monocytogenes produced local protrusions on the immunosensor surface, causing physicochemical changes in the ionic layer formed on the immunosensor surface by a sinusoidal electrical signal input, which might help electrical current to flow and cause the R value to decrease.     

Impact of Mechanical Shear on the Survival of Listeria monocytogenes on Surfaces

Abstract: The impact of mechanical surface shear on microbial viability is rarely a subject for exploration in food processing. The objective of this research was to investigate the impact of mechanical shear on the survival of Listeria monocytogenes on surfaces. Mechanical shear created by slicing a model food was explored to investigate the viability of L. monocytogenes. Cell injury/death was readily demonstrated in fluorescence images by confocal microscopy in which the live and dead cells were fluorescently stained green and red, respectively, with a viability dye kit. Images showed that a large percentage of dead cells appeared after slicing, and they were readily transferred from the slicer blade onto the surfaces of sliced agar, indicating that surface shear may cause the lethal effect on L. monocytogenes. Surface transfer results also showed that viable cell counts on agar slices (in a slicing series) followed a consistently decreasing pattern. The cell counts initially at 5 to 6.5 log CFU/slice (slices 1 to 6), decreased to 3 to 4 log CFU/slice (slices 8 to 30), then to 2 to 3 log CFU/slice (slices 31 to 40), and counts would be expected to further decrease if slicing continued. The overall cell recovery (survival) ratio was about 2% to 3% compared to the initial 8.4 log CFU/blade on a 10 cm² edge area. The impact of shear on microbial viability during slicing may contribute 99% of viable cell count reduction. This study provides clear evidence that surface shear can kill foodborne pathogens and reduce cross-contamination. The lethal effects of surface shear may further enhance food safety.     

Antilisterial Properties of Marinades during Refrigerated Storage and Microwave Oven Reheating against Post‐Cooking Inoculated Chicken Breast Meat

This study evaluated growth of Listeria monocytogenes inoculated on cooked chicken meat with different marinades and survival of the pathogen as affected by microwave oven reheating. During aerobic storage at 7 °C, on days 0, 1, 2, 4, and 7, samples were reheated by microwave oven (1100 W) for 45 or 90 s and analyzed microbiologically. L. monocytogenes counts on nonmarinated (control) samples increased (P < 0.05) from 2.7 ± 0.1 (day‐0) to 6.9 ± 0.1 (day‐7) log CFU/g during storage. Initial (day‐0) pathogen counts of marinated samples were <0.5 log CFU/g lower than those of the control, irrespective of marinating treatment. At 7 d of storage, pathogen levels on samples marinated with tomato juice were not different (P ≥ 0.05; 6.9 ± 0.1 log CFU/g) from those of the control, whereas for samples treated with the remaining marinades, pathogen counts were 0.7 (soy sauce) to 2.0 (lemon juice) log CFU/g lower (P < 0.05) than those of the control. Microwave oven reheating reduced L. monocytogenes counts by 1.9 to 4.1 (45 s) and >2.4 to 5.0 (90 s) log CFU/g. With similar trends across different marinates, the high levels of L. monocytogenes survivors found after microwave reheating, especially after storage for more than 2 d, indicate that length of storage and reheating time need to be considered for safe consumption of leftover cooked chicken.     

Microbiological quality of fresh fruit and vegetable products in Catalonia (Spain) using normalised plate‐counting methods and real time polymerase chain reaction (QPCR)

BACKGROUND: Commercially available fruits and raw and ready‐to‐eat vegetables (n = 445) were examined for aerobic, coliform, and yeast and mould counts using normalised methods. Listeria spp., Listeria monocytogenes and Salmonella spp. were detected by real time polymerase chain reaction (QPCR) after enrichment. RESULTS: Aerobic plate counts ranged from < 10 to > 10⁹ colony‐forming units (CFU) g^?1, with the lowest and highest counts recorded for fruits and sprouts respectively. The highest incidence level of coliforms was found in ready‐to‐eat vegetables, with up to 65.7% of samples containing from 5 to 9 log₁₀CFU g^?1. Yeasts and moulds showed their highest incidence level between 5 and 6 log₁₀ CFU g^?1, with an overall range from < 2 to 9 log₁₀ CFU g^?1. Salmonella spp., Listeria spp. and L. monocytogenes were detected in 0.67, 2.7 and 0.9% respectively of the total samples examined. CONCLUSION: The samples analysed can be gathered into two main groups, one showing low microbial counts (fruits) and a second group (raw whole leaves and roots and packed ready‐to‐eat vegetables) with higher microbial contamination. Although incidence levels of pathogenic bacteria reported here are in the lower range of those reported elsewhere, positive detections highlight the importance of good hygienic measures throughout the whole food chain. Copyright © 2007 Society of Chemical Industry     

An inhibitory synergistic effect of a nisin–monolaurin combination on Bacillus sp. vegetative cells in milk

The inhibitory activities of nisin and monolaurin, used alone or in combination, were investigated against four Bacillus species vegetative cells in milk at 37°C for 5 days. In the absence of inhibitors, the four strains grew and sporulated at the end of the exponential growth step and throughout the stationary phase. Nisin (100 IU ml⁻¹) induced an immediate reduction in the population level but transient because regrowth appeared and was strain-dependent; cell concentrations reached the control culture level, e.g. 6–7 log₍₁₀₎as well as the spore load (4–5 log₍₁₀₎). On the other hand, monolaurin (250 μ g ml⁻¹) had a durable bacteriostatic effect followed by a regrowth level constantly lower than that of the control culture; sporulation was low (between 13 and 7×10³spl ml⁻¹) and did not occur in the case of B. coagulans. The use of these inhibitors in combination, induced a synergistic bactericidal effect leading to a total inhibition (0 cfu ml⁻¹) until day 5, except in the case of B. cereus where a concentration of 500 cfu ml⁻¹was constant till the end of the experiment; consequently, sporulation was absent.