Heat resistance of Listeria monocytogenes

The heat resistance data on Listeria monocytogenes in culture media and foods are summarized. Most heat resistance data for foods have been obtained in dairy, meat, poultry, and egg products. Limited data have been published on seafood, fruits, and vegetables. The methodologies employed have evolved over time; hence data from earlier experiments are not directly comparable to more recent studies. Many factors influence the heat resistance of L. monocytogenes. Variation exists among different strains in their ability to withstand heat treatment. In addition, heat resistance is influenced by age of the culture, growth conditions, recovery media, and characteristics of foods such as salt content, a(w), acidity, and the presence of other inhibitors. Listeriae are more heat resistant than most other nonspore-forming foodborne pathogens, and thus, processing recommendations based on data from experiments with Salmonella spp. or pathogenic Escherichia coli may not be sufficient to eliminate similar numbers of L. monocytogenes. The data provided in this review may prove useful for food processors in determining appropriate times and temperatures for producing foods free of vegetative pathogens.     

Heat resistance of Listeria monocytogenes in vegetables: evaluation of blanching processes

The heat resistance of a Listeria monocytogenes composite (serotypes 1/2a, 1/2b, and 4b) was determined in fresh broccoli florets, sweet green peppers, onions, mushrooms, and peas using an end-point procedure in polyester pouches. The heat resistance of L. monocytogenes was higher in peas (D(60 degrees C) = 1.0 min) and mushrooms (D(60 degrees C) = 0.7 min) than in other vegetables tested (D(60 degrees C) in onions = 0.2 min) and was highest when cells were subjected to starvation before the thermal death time experiments (D(60 degrees C) of starved L. monocytogenes in mushrooms = 1.6 min). The results showed that blanching can be used as an antilisterial treatment (inactivation of 5 logs of L. monocytogenes) when the cold spot of vegetables is treated for at least 10 s at 75 degrees C or instantaneously (<1 s) at temperatures above 82 degrees C.     

Heat stability of milk supplemented with calcium chloride

Calcium chloride (0-25 mM) was added to skim milk powder that was reconstituted to 9% total solids. Heat stability was evaluated between 60 and 120°C for different times by observing whether samples had coagulated, and by measuring the amount of sediment and residual protein in the centrifuged supernatant. Milk samples were also dialyzed during their respective heat treatments to recover the soluble phase at different temperatures to measure pH and ionic calcium. The transition conditions between good and poor heat stability were established for different calcium chloride concentrations and temperatures. As temperature increased, coagulation occurred at lower levels of added calcium chloride. The transition was quite distinct at higher temperatures but less so at lower temperatures; it was initiated by an increase in sediment formation before a firm coagulum was formed. Both pH and ionic calcium decreased in dialysates as temperature increased. No coagulation was observed if Ca(2+) was <0.5 mM and pH was >6.3 in dialysates taken at their respective coagulation temperatures. Being able to measure pH and ionic calcium at high temperatures will allow better understanding of factors affecting heat stability. Electrophoresis of the supernatants permitted identification of the protein fractions participating in the coagulation process. When coagulation was observed below 80°C, substantial amounts of undenatured β-lactoglobulin and α-lactalbumin were found in the supernatant, as well as some soluble casein fractions. All the major whey protein and casein fractions were found in the sediment.     

Heat resistance of an outbreak strain of Listeria monocytogenes in hot dog batter

The heat resistance of a strain of Listeria monocytogenes responsible for a listeriosis outbreak in hot dogs was not higher than the heat resistance of other L. monocytogenes strains when tested in tryptic soy broth and in laboratory-prepared hot dog batter. For the thermal death time experiments, the cells were grown to stationary phase or were starved in phosphate-buffered saline, pH 7, for 6 h at 30 degrees C. Starvation increased the heat resistance of L. monocytogenes in broth but not in hot dog batter. D-values in hot dog batter were higher than in broth. For the hot dog formulation used in this study, cooking the hot dog batter for 30 s at 71.1 degrees C (160 degrees F), or its equivalent using a z-value of 6 degrees C (11 degrees F), would inactivate 5 logs of L. monocytogenes.     

Inactivation of Listeria monocytogenes Scott A 49594 in apple juice supplemented with cinnamon

Normal (pH 3.7) and adjusted (pH 5.0) pasteurized apple juice containing cinnamon (0, 0.1, 0.2, and 0.3%) was inoculated with Listeria monocytogenes Scott A 49594 at 10(4) CFU/ml and stored at 5 and 20 degrees C for 7 days. Counts on tryptic soy agar (TSA), modified Oxford (MOX) medium, and thin agar layer (TAL) were determined at 1 h and 1, 3, and 7 days. The TAL method (MOX medium overlaid with TSA) was used for the recovery of injured cells. In apple juice, both at normal and adjusted pH, with any doses of cinnamon, no L. monocytogenes (a 4.6-log CFU/ml reduction) was detected after 1 h of storage at both temperatures, and no growth occurred at any points of storage. Therefore, cinnamon by itself (regardless of pH) had a pronounced killing effect. A further enrichment step with brain heart infusion agar showed that L monocytogenes was completely inactivated in apple juice stored at 20 degrees C, except in pH 5.0 samples with 0.1% of cinnamon. The TAL method was as effective as TSA in recovering injured cells of L. monocytogenes. Cinnamon considerably inactivates L. monocytogenes in apple juice and thus enhances the safety of this product.     

Heat shock induces barotolerance in Listeria monocytogenes

The aim of this study was to investigate the effect of heat shock on the resistance of Listeria monocytogenes to high pressure processing (HPP). L. monocytogenes ATCC 19115 was grown to stationary phase at 15 degrees C and inoculated into whole ultrahigh-temperature milk at approximately 10(7) CFU/ml. Milk samples (5 ml) were placed into plastic transfer pipettes, which were heat sealed and then heated in a water bath at 48 degrees C for 10 min. Immediately after heat shock, the milk was cooled in water (20 degrees C) for 25 min and then placed on ice. The samples were high pressure processed at ambient temperature (approximately 23 degrees C) at 400 MPa for various times up to 150 s. Following HPP, the samples were spread plated on tryptic soy agar supplemented with yeast extract. Heat shock significantly increased the D400 MPa-value of L. monocytogenes from 35 s in non-heat-shocked cells to 127 s in heat-shocked cells (P < 0.05). Addition of chloramphenicol before heat shock eliminated the protective effect of heat shock (P < 0.05). Heat shock for 5, 10, 15, or 30 min at 48 degrees C resulted in maximal barotolerance (P < 0.05); increasing the time to 60 min significantly decreased survival compared with that at 5, 10, 15, or 30 min (P < 0.05). These results indicate that prior heat shock significantly increases the barotolerance of L. monocytogenes and that de novo protein synthesis during heat shock is required for this enhanced barotolerance.     

Thermal resistance of Listeria monocytogenes in foods

Recent studies on the heat resistance of Listeria monocytogenes in dairy products, the discrepancies in results and some of the difficulties involved in comparing interlaboratory experiments are reviewed. In addition, some current work on the thermal resistance of the organism in meat products is discussed.     

Thermal resistance of Listeria monocytogenes Scott A in ultrafiltered milk as related to the effect of different milk components

Pasteurization parameters for grade A milk are well established and set by regulation. However, as solids levels increase, an increased amount of heat is required to destroy any pathogens present. This effect is not well characterized. In this work, the effect of increased dairy solids levels on the thermal resistance of Listeria monocytogenes was examined through the use of ultrafiltered (UF) milk, reconstituted milk powder, and the milk components lactose and caseinate. From the results obtained, lactose and caseinate did not appear to affect thermal resistance. In addition, the level of milk fat, up to 10% of the total solids in UF whole milk, did not result in statistically significant changes to thermal resistance when compared with UF skim milk. Reconstituted skim milk powder at 27% total solids (D?2-value = 1.16 ± 0.2 [SD] min, z = 5.7) did result in increased thermal resistance, as compared with reconstituted skim milk powder at 17.5% (D?2-value = 0.86 ± 0.02 min, z = 5.57) and UF whole milk at 27% total solids (D?2-value = 0.66 ± 0.07 min, z = 5.16). However, that increase appeared to be due to the increase in salt levels, not to increases in caseinate, fat, or lactose. Consequently, total solids, as a single measure, could not be used to predict increased thermal resistance of L. monocytogenes in concentrated milk.     

Optimizing detection of heat-injured Listeria monocytogenes in pasteurized milk.

Optimal conditions for the detection of heat-injured cells of Listeria monocytogenes in modified Pennsylvania State University (mPSU) broth were determined using a response surface design generated by a computer program, EChip. Different combinations of incubation temperatures and lithium, magnesium, and D-serine concentrations were evaluated to determine the optimum conditions for the detection of heat-injured L. monocytogenes in filter-sterilized whole milk inoculated with selected problematic background microflora. A concentration of 212 mM lithium chloride completely inhibited the growth of Enterococcus faecium while permitting recovery and detection of L. monocytogenes. A concentration of 15.8 mM MgSO4 was found to be optimum for the recovery and detection of L. monocytogenes. A concentration of 140.2 mM D-serine was found to completely inhibit the germination of Bacillus subtilis var. globii spores but not recovery and detection of L. monocytogenes. Under optimum concentrations of LiCl, MgSO4, and D-serine and in the absence of background microflora, the effect of incubation temperature on percentage detection was described by a second-order polynomial model, and 28 degrees C was determined to be optimal. In the presence of background microflora, the effect of incubation temperature on percentage detection of heat-injured cells was described by a third-order polynomial model, and 30 degrees C was found to be optimal. Optimizing the levels of highly specific and selective agents, nutrients, and incubation temperature in one recovery enrichment system dramatically increased the Listeria/background microflora ratio. This resulting medium, optimized PSU (oPSU) broth, greatly improved the detection of heat-injured and nonheat-injured L. monocytogenes by both conventional and molecular methods (Oxoid's Listeria Rapid Test, Gen-Probe's Accuprobe Listeria monocytogenes Culture Identification Test, and Qualicon's BAX for screening Listeria monocytogenes).     

Rapid enumeration of Listeria monocytogenes in milk using competitive PCR

Competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Listeria monocytogenes in milk. Sterile milk was artificially inoculated with L. monocytogenes and DNA was extracted using guanidine thiocyanate/phenol/chloroform, followed by PCR. Several primers for L. monocytogenes hlyA gene were tested for specific detection and DG69/DG74 primer set was selected. The primer set produced a 636-bp band from L. monocytogenes, but no band appeared from the other six Listeria spp. tested. A detection limit was as few as 10(3) colony-forming unit (cfu) per 0.5 ml of milk with this primer set. When the samples were cultured at 25 degrees C for 15 h in a TSBY medium, even a single bacterium could be detected with this primer set by PCR. For the cPCR, hlyA gene segment was cloned in pGem-4Z vector and was modified to produce competitor DNA. The competitor DNA has the same primer binding sites and sequences as the target DNA except EcoRI site. Known amount of competitor DNA was coamplified with L. monocytogenes total DNA isolated from artificially inoculated milk. The target DNA and competitor DNA were distinguished by EcoRI digestion after cPCR. The cell number determined by cPCR was approximately equal to the colony-forming unit from conventional plate counting method. For the whole procedure, it took only 5 h.     

Incidence of Listeria monocytogenes in two milk processing environments, and assessment of Listeria monocytogenes blood agar for isolation

A year-long survey of two Northern Ireland milk processing plants for Listeria monocytogenes was carried out. Sample sites included the milk processing environment (walls, floors, drains, and steps), processing equipment, raw and pasteurised milk. The FDA listeria-selective enrichment procedure was used to process samples and an additional agar medium, L. monocytogenes Blood Agar (LMBA), was utilized as part of the isolation procedure in order to compare its performance to that of the recommended Oxford and Palcam agars. LMBA proved to be a very useful tool and was able to detect L. monocytogenes from 94.1% of sites compared to the 76.5% and 79.4% detection rate displayed by Oxford and Palcam agars, respectively. The overall incidence of listeria on equipment was 18.8% (6.3% L. monocytogenes), in the environment was 54.7% (40.6% L. monocytogenes) and in raw milk 44.4% (22.2% L. monocytogenes). On one occasion, L. welshimeri was isolated from pasteurised milk, probably demonstrating post-pasteurisation contamination of product. The main environmental sources of L. monocytogenes were considered to be a floor drain and stainless steel steps.     

Behavior of Listeria monocytogenes in pasteurized milk during fermentation with lactic acid bacteria

The behavior of Listeria monocytogenes in pasteurized milk during fermentation with starter and nonstarter lactic acid bacteria was investigated. Pasteurized milk was co-inoculated with approximately 10(4) CFU/ml of L. monocytogenes and 10(6) CFU/ml of Lactococcus lactis, Lactococcus cremoris, Lactobacillus plantarum, Lactobacillus bulgaricus, or Streptococcus thermophilus. Inoculated milks were incubated at 30 degrees C or 37 degrees C for 24 to 72 h. Listeria monocytogenes survived and also grew to some extent during incubation in the presence of all starter cultures; however, inhibition ranged from 83 to 100% based on maximum cell populations. During incubation with L. bulgaricus and L. plantarum, L. monocytogenes was completely inactivated after 20 h and 64 h of incubation at 37 degrees C and 30 degrees C, respectively. The pH of the fermenting milks declined steadily throughout the fermentation periods and was approximately 4.2 at the conclusion of the experimental period regardless both of the starter culture and pathogen combination or the temperature of incubation.     

环介导恒温扩增方法快速检测乳中单增李斯特菌

建立了一种快速检测灭菌乳中单增李斯特菌的环介导恒温扩增(Loop-Mediated Isothermal Amplification,LAMP)方法。以hlyA基因作为靶基因,对人工污染乳中单增李斯特菌进行了LAMP方法的灵敏度试验,同时与PCR方法进行比较。并对单增李斯特菌和7种其他乳中常见致病菌进行了LAMP检测,以验证该方法的特异性。结果表明,LAMP检测单增李斯特菌的特异性强,检出限为42 mL-1,其灵敏度比普通PCR高10倍。并且检测时间比PCR更短,在1.5 h内即可完成扩增反应。此方法快速、特异、简单、灵敏,具有较高的推广价值。     

Estimation of low bacterial concentration: Listeria monocytogenes in raw milk

A time-series bacteriological analysis has been carried out on milk collected on farms from 1997 to 2001 by a plant producing raw milk soft cheese, with the purpose of assessing the time course of the presence/absence of Listeria monocytogenes. A standard data collection procedure was used, in which farms were tested on a monthly or biweekly basis and 2-3 days after the detection of milk tanker contamination. This procedure yielded low figures for contamination frequencies. The average value and the median of the monthly prevalence of farms detected positive for L. monocytogenes were 2.4 and 0%, respectively. A seasonal effect (with peaks in winter) was observed. Between 1997 and 2001, there was no significant decrease of contamination rates, in spite of the efforts on the contaminated farms. Over the last year of the study (from March 2000 to February 2001), a new data collection procedure was implemented that allowed much better detection of sporadic occurrences. Milk samples were collected from the bulk tank of each participating farm just before pick-up, then stored and subsequently analysed whenever the milk tanker was found contaminated. The average value and the median of the monthly prevalence of positive farms were found equal to 7.7 and 0%, respectively (for a mean prevalence of L. monocytogenes in the milk tanker of 3.2%). These results confirm that farm milk contamination is, most often, a sporadic event In addition to this prevalence study, contamination levels were quantified by enumerating L. monocytogenes using direct plating of small volumes of farm milk previously tested positive. Most often, these levels were extremely low. A simple simulation model shows that, when milk tankers were found positive, contamination levels in the corresponding bulk-tank milk are themselves very low (typically, below 3 L. monocytogenes per millilitre with most probable concentration 0.1 Colony Forming Unit (CFU)/ml and median ranging from 5.10(-2) to 0.1 CFU/ml). Such low levels are very likely to be due to environmental contamination.     

Staphylococcus aureus and Listeria monocytogenes in Norwegian raw milk cheese production

The aim of this study was to survey the presence of Staphylococcus aureus and Listeria monocytogenes during the cheese making process in small-scale raw milk cheese production in Norway.The prevalence of S. aureus in bovine and caprine raw milk samples was 47.3% and 98.8%, respectively. An increase in contamination during the first 2-3 h resulted in a 73.6% prevalence of contamination in the bovine curd, and 23 out of 38 S. aureus-negative bovine milk samples gave rise to S. aureus-positive curds. The highest contamination levels of S. aureus were reached in both caprine and bovine cheese after 5-6 h (after the first pressing). There was no contamination of L. monocytogenes in caprine cheeses and only one (1.4%) contaminated bovine cheese.This work has increased our knowledge about S. aureus and L. monocytogenes contamination during the process of raw milk cheese production and gives an account of the hygiene status during the manufacture of Norwegian raw milk cheeses.     

Dynamic kinetic analysis of growth of Listeria monocytogenes in pasteurized cow milk

The objective of this study was to develop a dynamic model for predicting the growth of Listeria monocytogenes in pasteurized cow milk under fluctuating temperature conditions during storage and temperature abuse. Six dynamic temperature profiles that simulated random fluctuation patterns were designed to change arbitrarily between 4 and 30°C. The growth data collected from 3 independent temperature profiles were used to determine the kinetic parameters and construct a growth model combining the primary and secondary models using a 1-step dynamic analysis method. The results showed that the estimated minimum growth temperature and maximum cell concentration were 0.6 ± 0.2°C and 7.8 ± 0.1 log cfu/mL (mean ± standard error), with the root mean square error (RMSE) only 0.3 log cfu/mL for model development. The model and the associated kinetic parameters were validated using the data collected under both dynamic and isothermal conditions, which were not used for model development, to verify the accuracy of prediction. The RMSE of prediction was approximately 0.3 log cfu/mL for fluctuating temperature profiles, and it was between 0.2 and 1.1 log cfu/mL under certain isothermal temperatures (2–30°C). The resulting model and kinetic parameters were further validated using 3 growth curves at 4, 7, and 10°C arbitrarily selected from ComBase (www.combase.cc). The RMSE of prediction was 0.8, 0.4, and 0.5 log cfu/mL, respectively, for these curves. The validation results indicated the predictive model was reasonably accurate, with relatively small RMSE. The model was then used to simulate the growth of L. monocytogenes under a variety of continuous and square-wave temperature profiles to demonstrate its potential application. The results of this study showed that the model developed in this study can be used to predict the growth of L. monocytogenes in contaminated milk during storage.     

Prevalence of Listeria monocytogenes subtypes in bulk milk of the Pacific Northwest

The prevalence of Listeria monocytogenes in bulk milk from three Pacific Northwest states was assessed for 474 herds at three time points. For samples collected in November 2000 and June 2001, the L. monocytogenes prevalence levels were 4.9 and 7.0%, respectively. All isolates were subtyped by serotyping and by pulsed-field gel electrophoresis (PFGE). Forty-nine of the 55 isolates belonged to serogroup 1/2a, while 6 belonged to serogroup 4. Subtyping by PFGE revealed that isolates from 31 herds shared 10 patterns; there was a weak but significant association between PFGE subtype and geographical distance. Six herds were positive for L. monocytogenes at both time points. Of these six herds, four had indistinguishable PFGE patterns at both time points. Twenty-five of the 33 herds that were positive in June 2001 were sampled again in June 2002. L. monocytogenes was recovered from 17 of these 25 herds (68%), with the ApaI restriction enzyme digestion profiles (REDP) for 8 herds being identical to those of isolates recovered from these herds the previous year. The ApaI REDP for the bulk milk isolates were compared with those for isolates recovered from environmental and human samples that were collected by the Washington Department of Health (n = 23). Analysis of ApaI digestion profiles revealed that only two of the Washington Department of Health isolates had digestion profiles similar to those for isolates from bulk milk; however, further analysis with the use of a second enzyme (AscI) was capable of discriminating between isolates from the two sources. Thus, we found no direct REDP matches between bulk milk and clinical isolates.     

Aerosol studies with Listeria innocua and Listeria monocytogenes

Aerosol studies of Listeria monocytogenes in food processing plants have been limited by lack of a suitable surrogate microorganism. The objective of this study was to investigate the potential of using green fluorescent protein-labeled strains of Listeria innocua as a surrogate for L. monocytogenes for aerosol studies. These studies were conducted in a laboratory bioaerosol chamber and a pilot food-processing facility. Four strains of L. innocua and five strains of L. monocytogenes were used. In the laboratory chamber study, Listeria cells were released into the environment at two different cell numbers and under two airflow conditions. Trypticase soy agar (TSA) plates and oven-roasted breasts of chicken and turkey were placed in the chamber to monitor Listeria cell numbers deposited from aerosols. A similar experimental design was used in the pilot plant study; however, only L. innocua was used. Results showed that L. monocytogenes and L. innocua survived equally well on chicken and turkey breast meats and TSA plates. No-fan and continuous fan applications, which affected airflow, had no significant effect on settling rates of aerosolized L. monocytogenes and L. innocua in the bioaerosol chamber or L. innocua in the pilot plant study. Listeriae cell numbers in the air decreased rapidly during the first 1.5 h following release, with few to no listeriae detected in the air at 3 h. Aerosol particles with diameters of 1 and 2 microM correlated directly with the number of Listeria cells in the aerosol but not with particles that were 0.3, 0.5, and 5 microM in diameter. Results indicate that L. innocua can be used as a surrogate for L. monocytogenes in an aerosol study.     

Isolation and lytic activity of the Listeria bacteriophage endolysin LysZ5 against Listeria monocytogenes in soya milk

The endolysin gene (lysZ5) from the genome of the Listeria monocytogenes phage FWLLm3 was cloned in Escherichia coli and characterized. Comparative sequence analysis revealed that lysZ5 resembled the murein hydrolase ply511 encoded by L. monocytogenes phage A511. The encoded protein LysZ5 had a predicted molecular mass of 35.8 kDa and was expressed in E. coli as an N-terminal fusion protein of 41.5 kDa. Addition of purified fusion protein to lawns of indicator bacteria showed that LysZ5 could lyse L. monocytogenes, Listeria innocua and Listeria welshimeri, but not Staphylococcus aureus or Enterococcus faecalis. The purified protein was able to kill L. monocytogenes growing in soya milk, with the pathogen concentration reduced by more than 4 log₁₀ CFU ml⁻¹ after 3 h incubation at 4 °C. As far as we know, this is the first report of a Listeria phage endolysin to control pathogens in soya milk and to demonstrate endolysin activity in foods at refrigeration temperatures. Moreover, LysZ5 may also be useful for biocontrol in other ready-to-eat foods.     

Efficacy of UV light for the reduction of Listeria monocytogenes in goat's milk

Certain types of goat's cheeses are produced using unpasteurized milk, which increases the food safety concerns for these types of products. Popularity and consumption of goat's milk products have increased, and the niche market includes gourmet goat's cheeses. The U.S. Code of Federal Regulations and the Pasteurized Milk Ordinance both address the possibility for processing alternatives to heat treatment, and the use of UV light treatment may be a viable alternative that still ensures the safety of the product. Fresh goat's milk was inoculated with Listeria monocytogenes (L-2289) at 10(7) CFU/ml and exposed to UV light using the CiderSure 3500 apparatus (FPE Inc., Macedon, NY). Inoculated milk was exposed to a UV dose range between 0 and 20 mJ/cm2 to determine the optimal UV dose. A greater than 5-log reduction was achieved (P < 0.0001) when the milk received a cumulative UV dose of 15.8 +/- 1.6 mJ/cm2. The results of this study indicate that UV irradiation could be used for the reduction of L. monocytogenes in goat's milk.