Real-time PCR detection of 16S rRNA genes speeds most-probable-number enumeration of foodborne Listeria monocytogenes

Quantifying foodborne pathogens at concentrations of 0.1 to 1,000 CFU/g of food generally involves most-probable-number (MPN) enumeration, which takes at least 4 days. A real-time PCR assay (RTi-PCR) was developed to accelerate MPN enumeration of foodborne Listeria monocytogenes. Foods were spiked from 70 to 110 CFU/g, and triplicate subportions from 0.0001 to 1 g were selectively enriched for 48 h at 30 degrees C. For standard MPN enumeration, the enrichments were subcultured on Oxford agar (48 h at 35 degrees C) to isolate Listeria. For RTi-PCR MPN, the L. monocytogenes cells from the same enrichments were washed and resuspended in 2 ml of sterile water. DNA was extracted by boiling for 10 min. The DNA in the extract's supernatant was targeted with published oligonucleotide primers for amplifying an Lmo-specific sequence of 16S rRNA genes. Amplification was continuously monitored with SYBR Green. The resulting amplicon was characterized by its melting temperature. The L. monocytogenes specificity of the primers was confirmed by testing L. monocytogenes (15 strains), Listeria innocua (11 strains), and Listeria welshimeri, Listeria seeligeri, Listeria ivanovii, and Listeria grayi (1 strain each). Quantitatively spiked milk, lettuce, smoked salmon, Brie cheese, ice cream, pork paté, salami, ready-to-eat shrimp, raw ground beef, and fresh soft cheese were enumerated by both the standard and the PCR MPN method. The paired results from the two MPN methods agreed well, except for the fresh cheese. For some foods, l-g samples required a decimal dilution for a positive test result, suggesting concentration-dependent food ingredient interference with the RTi-PCR. This RTi-PCR method reduced the time necessary for the MPN enumeration of foodborne L. monocytogenes from 4 to 2 days.     

Comprehensive survey of pasteurized fluid milk produced in the United States reveals a low prevalence of Listeria monocytogenes

A comprehensive survey was undertaken to generate contemporary data on the prevalence of Listeria monocytogenes in pasteurized fluid milk produced in the United States. Samples (5,519) near the sell-by expiration date were purchased at retail outlets over a 5-week period and analyzed for presence of L. monocytogenes. Products consisted of whole milk, nonfat milk, and chocolate milk packaged in gallon, half gallon, quart, pint, and half-pint containers. Samples were collected from both large and small retail stores in urban and suburban locations in four FoodNet cities (Baltimore, Md., Atlanta, Ga., St. Paul/ Minneapolis, Minn., and San Francisco, Calif.). Samples were prescreened for L. monocytogenes by the AOAC-approved rapid Vitek immunodiagnostic assay system, enzyme-linked fluorescent assay method. Positive prescreening samples were cultured according to the Bacteriological Analytical Manual, enumerated for L. monocytogenes with a nine-tube most-probable-number (MPN) procedure, and confirmed by biochemical characterization. The frequency of isolation of L. monocytogenes in these products was 0% (0 of 1,897) in whole milk, 0.05% (1 of 1,846) in nonfat milk, 0% (0 of 1,669) in chocolate milk, and 0% (0 of 107) in other (reduced fat and low fat) milk samples. Overall, L. monocytogenes was confirmed in only 0.018% of pasteurized milk samples (1 of 5,519). Enumeration of the single confirmed positive nonfat milk sample revealed low-level contamination (<0.3 MPN/g), even when sampled 5 days past the expiration of the sell-by date. The results confirm the low frequency of contamination of pasteurized fluid milk products by L. monocytogenes for products sold in the United States and reaffirm the reduction of contamination frequency of fluid milk by L. monocytogenes when compared with earlier estimates from the U.S. Food and Drug Administration Dairy Safety Initiatives Program.     

Behavior of Listeria monocytogenes during processing and storage of experimentally contaminated hot-smoked trout.

Hot-smoked fish like smoked trout is quite frequently contaminated with Listeria monocytogenes. In order to estimate the potential health hazard for the consumer eating such products, the behavior of L. monocytogenes was studied during processing and storage of artificially-inoculated hot-smoked trout. Four trials were performed; in trials 1 and 3 a wild-type strain was used, while in trials 2 and 4 a serological reference strain, SLCC 2755, was used. In the first two trials, raw trout was surface inoculated with L. monocytogenes, marinated, hot-smoked (core temperature 65 degrees C during 20 min), and stored at 4 and 8-10 degrees C, respectively, for up to 20 days. At different times during processing and storage, samples were taken and, by means of a MPN-method, quantitatively analysed for L. monocytogenes. The initial Listeria levels in the trout were 10(6) MPN/g. Until smoking, the concentrations remained about the same. After the hot-smoking process and during storage, however, L. monocytogenes could no longer be detected. In trials 3 and 4, the trout were inoculated after hot-smoking at a final concentration of 4.5 x 10(1) MPN/g and 3.1 x 10(1) MPN/g, respectively. During storage at 4 degrees C, neither an increase nor a decrease of L. monocytogenes was observed. At 8-10 degrees C, however, a significant increase up to 10(7) MPN/g occurred. By hot-smoking, low level contaminations of raw fish with L. monocytogenes will easily be eliminated. Nevertheless, it is of great importance to prevent postprocessing contamination, because during storage at refrigeration temperatures growth of L. monocytogenes is possible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevalence of Listeria monocytogenes in fresh and fermented Italian sausages and ribotyping of contaminating strains

Listeria monocytogenes has been detected in fresh as well as dry and semidry fermented sausages, rendering preparation and consumption of these products as a potential risk to human health. The aims of this study were (1) to evaluate the L. monocytogenes prevalence in 288 fresh and 237 fermented sausages produced in northern Italy; (2) to quantify the average pathogen Most Probable Number (MPN) per g of sausage; (3) to evaluate the sausage strain genetic diversity by automated PvuII ribotyping; and (4) to predict the pathogenicity lineage of these isolates determining their DuPont Identification Library Codes (DUP-IDs) by EcoRI ribotyping. The overall prevalence of L. monocytogenes in the sampled sausages was 28.2%. The percentage of L. monocytogenes positive fresh sausages was significantly higher than that of fermented sausages (i.e. 38.9 vs 15.2%), which had a pathogen load always lower than 10 MPN/g. In contrast, 16.1% of fresh sausages were contaminated by 10 to 100 MPN/g and 20.5% had more than 100 MPN/g. PvuII successfully discriminated sausage isolates with a Simpson's numerical index of discrimination of 0.637. A total of 12 and 9 different PvuII ribogroups were identified among 47 fresh and 24 fermented randomly selected sausage strains, respectively. Six of those ribogroups were shared between strains contaminating both kinds of sausages. According to the evaluation of the strain DUP-IDs, the majority of the isolates investigated in this study were part of the type II L. monocytogenes pathogenicity lineage, but type I lineage strains were identified among fermented sausage isolates. In conclusion, L. monocytogenes prevalence in Italian sausages was estimated to be around 28.2%. However, 84.2% of the samples were contaminated by less than 100 MPN of L. monocytogenes per g and the majority of L. monocytogenes contaminating strains would be classified in the type II pathogenicity lineage, including serotypes 1/2a, 1/2c and 3a.     

Reduction of Listeria monocytogenes in queso fresco cheese by a combination of listericidal and listeriostatic GRAS antimicrobials

Single and combined effects of three GRAS (generally recognized as safe) antimicrobials including, bacteriophage P100 (phage P100), lauric arginate (LAE), and potassium lactate-sodium diacetate mixture (PL-SD) were evaluated against Listeria monocytogenes cold growth in queso fresco cheese (QFC). The fate of phage P100 when exposed to LAE (200 ppm) or PL-SD (2.8% PL and 0.2% SD) was determined at 4°C and 30°C in a broth model. Phage P100 was found to be stable in the presence of these antimicrobial agents as plaque forming units (PFU) did not vary between control, LAE or PL-SD treatments. When 9 log CFU/ml of stationary phase cells of L. monocytogenes was exposed to these antimicrobials in tryptic soy broth, there was a 3 to 5 log CFU/ml reduction with phage P100 and a complete 9 log CFU/ml reduction with LAE but no measurable reduction with PL-SD after 24h at 4°C or 30°C. In QFC, the L. monocytogenes populations increased from the initial 3.5 log CFU/cm(2) to 7.7 log CFU/cm(2) in 28 days at 4°C. Treatment with 7.8 log PFU/cm(2) of phage P100 or 200 ppm of LAE showed strong listericidal effect initially by reducing L. monocytogenes counts by 2 to 3.5-4 log CFU/cm(2) while there was a subsequent regrowth of L. monocytogenes at 4°C. Treatment with PL-SD showed strong listeriostatic effect without decreasing L. monocytogenes counts but growth was prevented for 28 days at 4°C. Only the combined treatment of listericidal phage P100 or LAE with listeriostatic PL-SD reduced the initial L. monocytogenes counts by 2-4 log CFU/cm(2) and also kept the L. monocytogenes counts at that reduced level in QFC for 28 days at 4°C.     

Behavior of Listeria monocytogenes in avocado pulp and processed guacamole

The potential ability of Listeria monocytogenes to grow or survive in avocado pulp (AP) and processed guacamole (PG) stored at 22, 4 to 7, and -18 degrees C was studied. Both products were obtained from a factory in Michoacan, Mexico. PG consisted of AP mixed with dehydrated vegetables, antioxidants, and preservatives. Populations of L monocytogenes in AP stored at 22 degrees C increased from 2 to 6 and 9 log CFU/g after 24 and 48 h, respectively. At 4 to 7 degrees C, the growth rate of L monocytogenes in AP was greatly decreased; generation time was 8.2 h, in contrast with 1.35 h observed at 22 degrees C. L. monocytogenes populations did not increase in PG either at 22 degrees C for 48 h or at 4 to 7 degrees C for 15 days. The bacteriostatic effect in PG may have resulted from the presence of added substances, especially citric acid and disodium dihydrogen pyrophosphate. Aerobic plate counts and coliforms increased in AP and PG stored at ambient temperature and under refrigeration. However, these increments did not affect the growth of the pathogen. L. monocytogenes (50,000 most probable number [MPN]/g) survived at least 58 weeks in both products stored frozen at -18 degrees C; the final population was 335 MPN/g in AP and 23 MPN/g in PG. Although the composition of avocado fruit differs significantly (high content of lipids and scarcity of simple carbohydrates) from that typical of most fruits, these results underline AP as a potential vehicle of human listeriosis and indicate that freezing should not be used as the sole mechanism to control this pathogen.     

A novel method for the reduction of numbers of Listeria monocytogenes cells by freezing in combination with an essential oil in bacteriological media

The use of multiple freeze (-20 degrees C)-thaw cycles in combination with isoeugenol and polysorbate 80 was investigated as a method for the reduction of numbers of Listeria monocytogenes cells in a bacteriological medium. Three freeze (1 h, -20 degrees C)-thaw cycles in the presence of isoeugenol at concentrations of 0, 100, and 300 ppm resulted in average L. monocytogenes reductions of 0.69, 2.65, and 3.3 log10 MPN (most probable number) per ml, respectively. Increasing the number of freeze-thaw cycles further decreased cell numbers, with reductions of nearly 5 log10 MPN/ml being obtained with six freeze-thaw cycles. Freeze-thaw cycles were effective in reducing cell numbers at isoeugenol concentrations down to 25 ppm. Rapid freezing rates with liquid nitrogen were found to be less effective in reducing numbers of L. monocytogenes cells. Two rapid freeze-thaw cycles in the presence of 100 ppm isoeugenol and polysorbate 80 resulted in a reduction of 1.45 log10 MPN/ml. Two freezing (-20 degrees C) cycles involving slow freezing and thawing rates with samples being held frozen for 6 h for each cycle resulted in reductions larger than those obtained with faster freezing rates. It was found that complete thawing in freeze-thaw cycles was not necessary to achieve bactericidal action. The application of multiple freeze-thaw cycles in combination with low concentrations of isoeugenol could effectively reduce numbers of L. monocytogenes cells in bacteriological media.     

Viability of Salmonella and Listeria monocytogenes in delicatessen salads and hummus as affected by sodium content and storage temperature

A study was conducted to determine survival and growth behavior of Salmonella and Listeria monocytogenes in commercially prepared mayonnaise-based potato salad, macaroni salad, and coleslaw and in hummus (initial mean pH values were 4.80 to 4.94, 4.18 to 4.31, 3.87, and 4.50 to 4.52, respectively) as affected by sodium concentration (133 to 364, 190 to 336, 146 to 272, and 264 to 728 mg/100 g, respectively) and storage at 4 or 10°C for up to 27 days. Salmonella did not grow in any of the test products. Initial populations (2.02 to 2.38 log CFU/g) decreased in coleslaw to undetectable levels (<1 CFU/25 g) within 13 days and in most formulations of macaroni salad within 20 to 27 days. Salmonella survived in highest numbers in potato salad and hummus. The presence of added sodium in macaroni salad stored at 4°C and hummus stored at 4 or 10°C appeared to protect Salmonella against inactivation. L. monocytogenes, at an initial population of 1.86 to 2.23 log CFU/g, did not grow in test products, but with the exception of coleslaw containing sodium at a concentration used in the standard (control) recipe, this pathogen was detected by direct plating (≥ 1.0 log CFU/g) in all products stored at 4 or 10°C for 27 days. L. monocytogenes populations were significantly (P < 0.05) lower in potato salad and hummus with no added sodium than in test products with added sodium after storage at 4°C. Sodium concentration did not markedly affect aerobic plate counts over the 27-day storage period. Results confirm that the acidic pH of mayonnaise-based salads and hummus is a major factor preventing growth and influencing rates of inactivation of Salmonella and L. monocytogenes. In the absence of added sodium, death of these bacteria may be more rapid. However, in general decreasing or increasing the sodium concentration in selected delicatessen salad and hummus recipes does not markedly affect the behavior of Salmonella and L. monocytogenes when products are stored at 4 or 10°C for up to 27 days.     

Concentrations and tracking of listeria monocytogenes strains in a seafood-processing environment using a most-probable-number enrichment procedure and randomly amplified polymorphic DNA analysis

Concentrations of environmental microflora and Listeria monocytogenes were monitored at multiple environmental locations within a seafood-processing facility over the course of 6 months. Concentrations of L. monocytogenes were determined using a most-probable-number (MPN) enrichment procedure. Two floor drains had persistent low concentrations of L. monocytogenes (0.03 to >1,100 MPN/cm2). In comparison, concentrations of the other organisms in the drain were much higher (heterotrophic plate count range of 10(5) to 10(8) CFU/cm2). Concentrations of environmental organisms (heterotrophic aerobic plate counts and counts of pseudomonads, Shewanella spp., Aeromonas hydrophila, and coliforms) were not correlated with concentrations of L. monocytogenes. The 178 confirmed L. monocytogenes isolates from the MPN procedure were further characterized by randomly amplified polymorphic DNA analysis. Sixteen different banding patterns were identified, and nine of the patterns were identified from samples collected on two or more collection dates. From all locations, banding type A was observed in 98 confirmed isolates (55%). Although present, L. monocytogenes was a relatively minor component in the ecosystem of the floor drains in this seafood-processing facility.     

Use of commercially available antimicrobial compounds for prevention of Listeria monocytogenes growth in ready-to-eat minced tuna and salmon roe during shelf life

Listeria monocytogenes found in minced tuna and fish roe can cause listeriosis. These products are classified in category B according to the Codex Alimentarius Commission, i.e., ready-to-eat foods in which L. monocytogenes growth can occur. We investigated the effectiveness of nisin and other commercially available antimicrobial compounds (lysozyme, ε-polylysine, and chitosan) for prevention of L. monocytogenes growth during the expected shelf life of raw minced tuna and salmon roe products. Food samples inoculated with L. monocytogenes were incubated with each antimicrobial at 10°C for 7 days or at 25°C for 12 h. Nisaplin (an antimicrobial containing nisin) effectively inhibited L. monocytogenes growth in minced tuna at 500 ppm and in salmon roe at 250 ppm within their standard shelf lives. The effective concentration of each antimicrobial was determined: 2,000 ppm for ART FRESH 50/50 (containing lysozyme) and SAN KEEPER No. 381 (containing ε-polylysine) and 10,000 ppm for SAN KEEPER K-3 (containing chitosan).     

Prevalence and contamination levels of Listeria monocytogenes in retail foods in Japan

Retail foods in Japan were surveyed for the presence and contamination levels of L. monocytogenes. It was isolated from 12.2, 20.6, 37.0 and 25.0% of 41 minced beef, 34 minced pork, 46 minced chicken and 16 minced pork-beef mixture samples, respectively. MPN values were higher than 100/g in five (10.9%) minced chicken samples, but lower than 100/g in all minced beef, pork and pork-beef mixture samples. The organism was also isolated from 5.4% of the 92 smoked salmon samples at MPN values lower than 10/g, and from 3.3% of 213 ready-to-eat raw seafood samples at MPN values from lower than 0.3 to higher than 100/g. None of the 285 vegetable samples were contaminated with L. monocytogenes. These findings indicate that ready-to-eat raw seafoods are relatively high risk among the foods surveyed in this study.     

The fate of two Listeria monocytogenes serotypes in "cig kofte" at different storage temperatures

Cig kofte is a traditional Turkish food prepared from minced beef, bulgur, onions, garlic and varieties of spices. It is generally consumed within a few hours. However, leftovers can be kept in refrigerator or in room temperature up to 24h until they are consumed. In this study, survival and growth of two Listeria monocytogenes serotypes were investigated in cig kofte during the storage. For this purpose, the prepared samples were separately contaminated with serotypes 1/2b or 4b of L. monocytogenes at the level of 10(4)CFU/g and stored at 4°C and 21°C. L. monocytogenes colonies were counted at the beginning, 3rd, 6th, 12th and 24th hours of the storage. At 4°C, L. monocytogenes 4b significantly increased (P<0.05) from 4.12 to 5.49log(10)CFU/g but L. monocytogenes 1/2b remained constant (P>0.05) during the storage period. At 21°C, both L. monocytogenes 1/2b and 4b increased significantly (P<0.05) from 4.56 to 5.16log(10)CFU/g and from 4.23 to 5.65log(10)CFU/g, respectively. The physicochemical and microbiological characteristics of the cig kofte did not inhibit the growths of L. monocytogenes serotypes during the storage. These results indicated that L. monocytogenes was able to survive and grow in cig kofte at the both storage temperatures of 4°C and 21°C and cig kofte seemed to be a suitable medium for this pathogen.     

Comparison of swab transport media for recovery of Listeria monocytogenes from environmental samples

Environmental monitoring is recognized as an important strategy for controlling Listeria monocytogenes in food processing facilities. Samples are taken by swabbing environmental surfaces, and the swabs are immersed in a medium for transport to the laboratory. In this study, buffered peptone water (BPW), Dey-Engley neutralizing broth (DE), neutralizing buffer (NB), Letheen broth (LE), and newly described MCC buffer (MCC) were evaluated as transport media for recovery of sanitizer-stressed L. monocytogenes from inoculated swabs. After storage at 4°C, the media performed similarly, but at 25°C relative recovery efficiency from the inoculated sponges was DE > LE > BPW > MCC > NB. Recoveries from stainless steel surfaces followed similar trends. MCC, DE, and NB were compared for L. monocytogenes recovery in the presence of Escherichia coli, Enterococcus faecalis, Lactobacillus plantarum, Pseudomonas fluorescens, and Listeria innocua. After 4°C storage, all population levels changed little; after 25°C storage, DE allowed the best growth of L. monocytogenes regardless of other species present. MCC, DE, and NB performed similarly for recovery of L. monocytogenes from an artificial milk biofilm and for recovery of Listeria spp. from swabs obtained from a meat processing facility. Transport medium formulation, time and temperature of swab storage, and coexistence of other species affect recovery of sanitizer-stressed L. monocytogenes from environmental swabs. The study confirms the need to maintain 4°C storage conditions during swab transport.     

Prevalence of Listeria monocytogenes and Salmonella in ready-to-eat food in Catalonia, Spain

Listeria monocytogenes and Salmonella are pathogenic bacteria that can contaminate food products during or after processing. Ready-to-eat (RTE) food does not undergo any treatment to ensure its safety before consumption, and therefore risk of foodborne disease must be considered if these pathogens are present in the food. To evaluate the prevalence of these pathogens in RTE food, 140 RTE fish product samples, 501 RTE meat product samples, 462 RTE dairy samples, and 123 RTE dishes and desserts, providing a total of 1,226 samples, were collected from retail stores and food industry and analyzed for the presence of L. monocytogenes. A total of 1,379 samples consisting of 187 RTE fish products and 569 RTE meat products, 484 RTE dairy products, and 139 RTE dishes and desserts were collected and analyzed for the presence of Salmonella. L. monocytogenes was isolated from 20% of frozen Atlantic bonito small pies, 7.9% of smoked salmon samples, 11.1% of the pork luncheon meat samples, 6.2% of frozen chicken croquettes, 16.9% of cured dried sausage samples, 12.5% of cooked ham samples, and 20% of cooked turkey breast samples. L. monocytogenes was also found to be present in 1.3% of fresh salty cheese samples and 15.1% of frozen cannelloni samples. Salmonella was isolated from 1.2% of smoked salmon samples, 1.5% of frozen chicken croquettes, 2% of cooked ham samples, and 11.1% of cured dried sausage samples. Overall, occurrence of these pathogens in RTE foods was similar to that previously reported in the literature.     

High hydrostatic pressure and biopreservation of dry-cured ham to meet the Food Safety Objectives for Listeria monocytogenes

This work aimed to evaluate the effect of nisin application (biopreservation) combined with high hydrostatic pressure processing (HHP) on the behavior of Listeria monocytogenes CTC1034 intentionally inoculated (at ca. 10(7)cells/g) onto the surface of ready-to-eat (RTE) sliced dry-cured ham. Two types of dry-cured ham, which had different water activities and fat contents were studied (a(w) of 0.92 and 14.25% fat and a(w) of 0.88 and 33.26% fat). Three batches were prepared for each type of product: (C) control, without nisin; (N) nisin directly applied (200 AU/cm(2)) and (F) nisin applied through active packaging, polyvinyl alcohol films with 200 AU/cm(2). Half of the samples were pressurized at 600 MPa for 5min. Counts of L. monocytogenes were periodically monitored throughout 60 days of storage at 8°C. The physico-chemical characteristics of the products enabled the survival of L. monocytogenes, but it was significantly reduced by the presence of nisin. The effect of biopreservation was greater when applied directly to the surface and in the product with lower water activity in comparison with the active packaging and the high water activity products, respectively. The immediate inactivation of L. monocytogenes by HHP ranged from 1.82 to 3.85 Log units, depending on the type of dry-cured ham. The lower the water activity, the less was the inactivation induced by HHP, both immediately and during storage. The reduction of L. monocytogenes immediately after HHP and during storage was more evident in batches with nisin applied directly to the surface of the product. The pathogen was not detected in some samples from day 5 of storage in the product with higher water activity. The effect of nisin applied through active packaging was lower than the direct application. The results of the present study indicated that HHP, as post-processing listericidal treatment, is more effective (both immediately and long term) than the use of nisin as an antimicrobial measure. However, the both hurdles combined (i.e. biopreservation and HHP) provided a wider margin of safety in the control of L. monocytogenes during the storage of RTE cured meat products.     

The survival of E. coli O157:H7, S. Typhimurium and L. monocytogenes in black carrot (Daucus carota) juice

In this study the survival and growth patterns of Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes in various concentrations of black carrot juice were investigated during incubation period at 4 °C and 37 °C for 7 days. Several parameters, such as juice concentration (%), pH, incubation temperature (°C) and time (days) were found effective on the survival of pathogens tested. Although L. monocytogenes has been found to be the less resistant microorganism to the variable conditions, there were only ca. 1 and 2 log reductions in the number of the cells in the juice samples incubated at 4 °C for 2 and 7 days, respectively. Incubating at low temperature (4 °C) enhanced the survival of test microorganisms.     

Adaptive growth responses of Listeria monocytogenes to acid and osmotic shifts above and across the growth boundaries

The effect of acid and osmotic shifts on the growth of Listeria monocytogenes was evaluated at 10°C. Two types of shifts were tested: (i) within the range of pH and water activity (a(w)) levels that allow growth of L. monocytogenes and (ii) after habituation at no-growth conditions back to growth-permitting conditions. A L. monocytogenes cheese isolate, with high survival capacity during cheesemaking, was inoculated (10(2) CFU/ml) in tryptic soy broth supplemented with 0.6% yeast extract at six pH levels (5.1 to 7.2; adjusted with lactic acid) and 0.5% NaCl (a(w) 0.995), or four a(w) levels (0.995 to 0.93, adjusted with 0.5 to 10.5% NaCl) at pH 7.2 and grown to early stationary phase. L. monocytogenes was then shifted (at 10(2) CFU/ml) to each of the aforementioned growth-permitting pH and a(w) levels and incubated at 10°C. Shifts from no-growth to growth-permitting conditions were carried out by transferring L. monocytogenes habituated at pH 4.9 or a(w) 0.90 (12.5% NaCl) for 1, 5, and 10 days to all pH and a(w) levels permitting growth. Reducing a(w) or pH at different levels in the range of 0.995 to 0.93 and 7.2 to 5.1, respectively, decreased the maximum specific growth rate of L. monocytogenes. The lag time of the organism increased with all osmotic downshifts, as well as by the reduction of pH to 5.1. Conversely, any type of shift within pH 5.5 to 7.2 did not markedly affect the lag times of L. monocytogenes. The longer the cells were incubated at no-growth a(w) (0.90), the faster they initiated growth subsequently, suggesting adaptation to osmotic stress. Conversely, extended habituation at pH 4.9 had the opposite effect on subsequent growth of L. monocytogenes, possibly due to cell injury. These results suggest that there is an adaptation or injury rate induced at conditions inhibiting the growth of the pathogen. Thus, quantifying adaptation phenomena under growth-limiting environments, such as in fermented dairy and meat products or products preserved in brine, is essential for reliable growth simulations of L monocytogenes during transportation and storage of foods.     

A predictive model for the inactivation of Listeria innocua in cooked poultry products during postpackage pasteurization

Contamination of Listeria monocytogenes in ready-to-eat poultry products poses potential risk of listeriosis to the public. To control the level of Listeria contamination, attention has been focused on the postpackage pasteurization of fully cooked poultry products. In this study, we sought to develop a model to predict the thermal inactivation of L. monocytogenes in chicken drumettes during postpackage hot water pasteurization. Fully cooked chicken drumettes were inoculated with Listeria innocua as a surrogate microorganism for Listeria monocytogenes, vacuum packaged, and treated in hot water baths at 60, 70, 80, and 90°C for different heating times. Experimental results showed that a 7-log CFU/g reduction of L. innocua occurred at 54, 28, 18, and 10 min at 60, 70, 80, and 90°C, respectively. The Weibull model was used to fit the survival curves of L. innocua at each heating temperature. The root mean square errors and residual plots indicated good agreements between the predicted and observed values. The predictive model was further validated by predicting a new data set generated in the pilot-plant tests. Model performance was evaluated by the acceptable prediction zone method, and the results indicated that the percentages of acceptable prediction errors were 100, 100, 82.4, and 87.5% at 60, 70, 80 and 90°C, respectively, which were all greater than the threshold acceptable value of 70% , indicating good performance of the model. The developed predictive model can be used as a tool to predict thermal inactivation behaviors of L. monocytogenes in ready-to-eat chicken drumettes products.     

Occurrence and characterization of Listeria spp. in ready-to-eat retail foods from Vancouver, British Columbia

The occurrence of Listeria spp. and Listeria monocytogenes in retail RTE meat and fish products in Vancouver, British Columbia (B.C.) was investigated. To assess potential consumer health risk, recovered L. monocytogenes isolates were subjected to genotypic and phenotypic characterization. Conventional methods were used to recover Listeria spp. from deli meat (n = 40) and fish (n = 40) samples collected from 17 stores. Listeria spp. were recovered only from fish samples (20%); 5% harboured Listeria innocua, 5% had L. monocytogenes and 10% contained Listeria welshimeri. L. monocytogenes isolates serotyped as 1/2a and 1/2b, possessed dissimilar PFGE patterns, and had full-length InlA. Three 1/2a clonal isolates encoded the 50 kb genomic island, LGI1. Antimicrobial resistance (AMR) profiling showed all Listeria spp. possessed resistance to cefoxitin and nalidixic acid. L. monocytogenes were resistant to clindamycin, two were resistant to streptomycin, and one to amikacin. Reduced susceptibility to ciprofloxacin was seen in all L. monocytogenes, L. innocua and three L. welshimeri isolates. Reduced susceptibility to amikacin and chloramphenicol was also observed in one L. monocytogenes and three L. welshimeri isolates, respectively. Recovery of L. monocytogenes in fish samples possessing AMR, full-length InlA, LGI1, and serotypes frequently associated with listeriosis suggest B.C. consumers are exposed to high-risk strains.     

Heat resistance of Listeria monocytogenes in semi-skim milk supplemented with vanillin

The kinetics of destruction of Listeria monocytogenes Scott A in semi-skim milk heated at 55, 58, 60 and 62°C without and with addition of 900, 1400 and 1800 ppm of vanillin was studied. Survival curves displayed an initial shoulder phase followed by an accelerating killing phase. Addition of vanillin to semi-skim milk heated between 55 and 62°C reduced the heat resistance of L. monocytogenes, effect that was more evident at the lowest temperatures. Two kinetic inactivation models were used to fit the data: the shoulder+log-linear model and the Weibull model. The presence of vanillin increased the death rate and reduced the shoulder length of L. monocytogenes in milk when working at low temperatures, while at the highest temperatures, this effect was less evident. Weibull model also showed that at lower temperatures 55°C-58°C, the population was inactivated at different treatment times, leaving a larger proportion of resistant microorganisms. Increasing the heating temperature to 60°C and 62°C, the biggest proportion of the population was destroyed in a very short time, while a very little proportion with higher resistance remained viable. Results suggest that the use of subinhibitory concentrations of vanillin added in combination with mild heat treatment could be used to enhance the inactivation of L. monocytogenes in semi-skim milk.