Development of a simple recovery-enrichment system for enhanced detection of heat-injured Listeria monocytogenes in pasteurized milk

A simple anaerobic recovery-enrichment system, semisolid Penn State University (ssPSU) broth, that enhances recovery of heat-injured Listeria monocytogenes, was rapidly achieved in 10-ml screw-capped tubes by adding Bacto-agar (2.5 g/liter) and L-cysteine (0.5 g/liter) to Penn State University broth. Glucose was removed from the formulation for ssPSU broth to prevent the growth of thermoduric lactobacilli. Ferric ammonium citrate was added to ssPSU broth to detect esculin hydrolysis and to indicate the presumptive presence of L. monocytogenes. Replacement of phosphate buffer with 3-[N-morpholino]propanesulfonic acid (MOPS) buffer and addition of magnesium sulfate (15 mM) enhanced recovery and detection of L. monocytogenes heat treated at 62.8 degrees C for 20 min. D-Serine, at a concentration of 150 mM, was found to inhibit germination of Bacillus spp. spores but did not inhibit severely heat-injured L. monocytogenes. Finally, ssPSU broth was modified (to mPSU broth) to contain the following: (i) Bacto-agar, 2.5 g/liter; (ii) ferric ammonium citrate, 0.5 g/liter; (iii) MOPS buffer, pH 7.0; (iv) D-serine, 13.7 g/liter; (v) D-alanine, 11.6 g/liter; and (iv) magnesium sulfate, 1.81 g/liter. Incubation temperature significantly affected the recovery and detection of severely heat-injured L. monocytogenes. L. monocytogenes that were heat challenged in filter-sterilized whole milk at 62.8 degrees C for 20, 25, and 30 min could not be detected at incubation temperatures > or = 30 degrees C but were consistently detected after incubation at 25 degrees C for 174, 199, and 330 h, respectively. Heat-injured cells of L. monocytogenes that were added to various commercial brands of pasteurized whole milk were also detected using mPSU broth. When clostridial spores (10(4) spores per ml) were added to filter-sterilized milk containing either heat-injured or non-heat-injured L. monocytogenes, only the latter could be detected in mPSU broth. The mPSU broth system requires no purging with nitrogen gas to create anaerobic conditions and permits recovery, growth, and detection of L. monocytogenes in one vessel in the presence of thermoduric background microflora commonly found in pasteurized milk.     

Comparison of different enrichment broths and background flora for detection of heat-injured Listeria monocytogenes in whole milk

Various primary enrichment broths, including University of Vermont medium (UVM), Listeria enrichment broth (LEB), modified LEB, and aerobic and anaerobic L-PALCAMY, were compared with aerobic and anaerobic Pennsylvania State University (PSU) broths for the detection of severely heat-injured (62.8 degrees C for 5, 10, or 15 min; no colony appearance after heat injury on aerobic Trypticase soy agar containing 0.6% yeast extract and modified Oxford medium) Listeria monocytogenes Scott A. Anaerobic conditions were produced by adding L-cysteine and then purging the headspace with N2. The effect of uninjured background flora (10(3) CFU/ml of Enterococcus faecium) on frequency of detection was examined. Anaerobic PSU broth resulted in the lowest false-positive rate and the highest frequency of detection of severely heat-injured L. monocytogenes compared with UVM, LEB, and modified LEB (P < 0.05). The presence of E. faecium significantly enhanced the detection of heat-injured (10 min at 62.8 degrees C) L. monocytogenes in aerobic and anaerobic PSU and aerobic and anaerobic L-PALCAMY broths (P < 0.05). The highest concentration of uninjured E. faecium (>10(6) CFU/ml) inhibited the detection of heat-injured L. monocytogenes (P < 0.05). A heat-resistant, LiCl-tolerant Lactobacillus isolate from raw milk increased the rate of both false-positive and false-negative reactions.     

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.     

Two-step large-volume magnetic separation combined with PCR assay for sensitive detection of Listeria monocytogenes in pasteurized milk

Immunomagnetic separation (IMS) is an effective tool for the preconcentration and purification of food-borne pathogens from complex food samples because of its high capture efficiency (CE). In conventional IMS, antibodies are usually conjugated on the surface of magnetic beads (MB); the random orientation and conformation changes of antibodies on the MB surface can decrease their bioactivity. Moreover, the Brownian motion of immobilized antibodies is weakened, thereby rendering their binding efficiency with bacteria lower than that of free antibodies. Thus, abundant antibodies are commonly required to ensure high CE for IMS, particularly for large volumes. In this study, a 2-step large-volume magnetic separation (10 mL) was proposed to preconcentrate Listeria monocytogenes from pasteurized milk. First, the biotinylated anti-L. monocytogenes monoclonal antibodies (mAb) were bound with L. monocytogenes in 10 mL of diluted milk through an antigen-antibody interaction, and then streptavidin-labeled MB were used to capture biotin-mAb coated with L. monocytogenes by biotin and streptavidin interaction. Under optimal conditions, the CE of 2-step magnetic separation was >90% with L. monocytogenes concentrations ranging from 8 × 10⁰ to 8 × 10⁴ cfu/mL, whereas the amount of biotin-mAb was 14 fold lower than that of the conventional IMS method. Coupled with a PCR assay, the detection limit of the proposed method was 8 × 10⁰ cfu/mL in pure culture and 8 × 10¹ cfu/mL in pasteurized milk without any pre-enrichment process. Moreover, the overall detection time, including sample preparation, large-volume magnetic separation, and PCR, took less than 7 h. In summary, the developed 2-step large-volume IMS combined with PCR was highly sensitive and low cost and, thus, has considerable potential for the rapid screening of food-borne pathogenic bacteria.     

Comparison of different reducing agents for enhanced detection of heat-injured Listeria monocytogenes

The effect of different reducing agents (L-cysteine, Oxyrase, and Enterococcus faecium) and their combinations on the detection of heat-injured (62.8 degrees C, 7.5 min or 10 min) Listeria monocytogenes was examined. The incorporation of L-Cysteine (0.5 g/liter) yielded higher percentage detection than any of the other reducing agents (P < 0.05). The combination of Oxyrase (10 U/ml) and E. faecium (10(3) CFU/ml) synergistically enhanced the detection of L. monocytogenes heat-injured for 10 min at 62.8 degrees C (P < 0.05). Simultaneous addition of L-cysteine (0.5 g/liter) and Oxyrase (10 U/ml) significantly lowered the recovery of heat-injured L. monocytogenes (P < 0.05). Higher activities of Oxyrase (50 U/ml) inhibited the detection of heat-injured L. monocytogenes (P < 0.05). The rates of depletion of relative percentage O2 were in the order: L-cysteine (0.5 g/liter; 6.63%/ min) > Oxyrase (10 U/ml; 5.00%/min) > E. faecium (10(8) CFU/ml; 1.66%/min) > E. faecium (10(3) CFU/ml; 0.20%/min). The final levels of redox potential (Eh) achieved were -110.5 mV, -100 mV, -83.5 mV, and -25 mV for E. faecium (10(8) CFU/ml), L-cysteine, Oxyrase, and E. faecium (10(3) CFU/ml), respectively.     

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.     

Antimicrobial activity of clove and cinnamon essential oils against Listeria monocytogenes in pasteurized milk

The antimicrobial activity of essential oils (EOs) of cinnamon bark, cinnamon leaf, and clove against Listeria monocytogenes Scott A were studied in semiskimmed milk incubated at 7 degrees C for 14 days and at 35 degrees C for 24 h. The MIC was 500 ppm for cinnamon bark EO and 3,000 ppm for the cinnamon leaf and clove EOs. These effective concentrations increased to 1,000 ppm for cinnamon bark EO, 3,500 ppm for clove EO, and 4,000 ppm for cinnamon leaf EO when the semiskimmed milk was incubated at 35 degrees C for 24 h. Partial inhibitory concentrations and partial bactericidal concentrations were obtained for all the assayed EOs. The MBC was 3,000 ppm for the cinnamon bark EO, 10,500 ppm for clove EO, and 11,000 ppm for cinnamon leaf EO. The incubation temperature did not affect the MBC of the EOs but slightly increased the MIC at 35 degrees C. The increased activity at the lower temperature could be attributed to the increased membrane fluidity and to the membrane-perturbing action of EOs. The influence of the fat content of milk on the antimicrobial activity of EOs was tested in whole and skimmed milk. In milk samples with higher fat content, the antimicrobial activity of the EOs was reduced. These results indicate the possibility of using these three EOs in milk beverages as natural antimicrobials, especially because milk beverages flavored with cinnamon and clove are consumed worldwide and have been increasing in popularity in recent years.     

Thin agar layer method for recovery of heat-injured Listeria monocytogenes

A thin agar layer (TAL) method was developed to recover heat-injured Listeria monocytogenes. Modified Oxford medium (MOX), a selective plating medium, inhibits heat-injured L. monocytogenes from growing, whereas tryptic soy agar (TSA), a nonselective medium, does not. In order to facilitate recovery of heat-injured L. monocytogenes cells while providing selectivity of isolation of L. monocytogenes from other bacteria in the sample, a unique TAL procedure was developed by overlaying 5 ml of nonselective medium (TSA) onto prepoured and solidified MOX medium in an 8.5-cm-diameter petri dish. The injured L. monocytogenes repaired and started to grow in the TSA during the first few hours after incubation of the plate. During the resuscitation of injured cells, the selective agents from MOX diffused to the TSA top layer to inhibit other microorganisms. L. monocytogenes showed a typical reaction (black colonies) on TAL after 24 h of incubation at 37 degrees C. The recovery rate for heat-injured L. monocytogenes with the TAL method was compared with those rates associated with TSA, MOX, and the traditional overlay method (OV; pouring selective agar on top of resuscitated cells on TSA agar after 3 h incubation). Milk and 0.1% peptone water that were inoculated with L. monocytogenes (4 to 5 log CFU/ml) were heated for 15 min at 55 degrees C. L. monocytogenes was enumerated on TSA, MOX, OV, and TAL media and procedures. No significant difference occurred among TSA, OV, and TAL (P > 0.05) in terms of enumeration of heat-injured L. monocytogenes, but these media recovered significantly higher numbers than did MOX agar (P < 0.05)-in both samples. The TAL method involves only one step, whereas OV is a more cumbersome two-step procedure.     

Inhibition of Listeria monocytogenes in pH-adjusted pasteurized liquid whole egg

Although the transmission of L. monocytogenes to humans via pasteurized egg products has not been documented, L. monocytogenes and other Listeria species have been isolated from commercially broken raw liquid whole egg (LWE) in both the United States and Ireland. Recent Listeria thermal inactivation studies indicate that conventional minimal egg pasteurization processes would effect only a 2.1- to 2.7-order-of-magnitude inactivation of L. monocytogenes in LWE; thus, the margin of safety provided by conventional pasteurization processes is substantially smaller for L. monocytogenes than for Salmonella species (a 9-order-of-magnitude process). The objective of this study was to evaluate the inhibitory effects of nisin on the survival and growth of L. monocytogenes in refrigerated and pH-adjusted (pH 6.6 versus pH 7.5) ultrapasteurized LWE and in a liquid model system. The addition of nisin (1,000 IU/ml) to pH-adjusted ultrapasteurized LWE reduced L. monocytogenes populations by 1.6 to > 3.3 log CFU/ml and delayed (pH 7.5) or prevented (pH 6.6) the growth of the pathogen for 8 to 12 weeks at 4 and 10 degrees C. Bioactive nisin was detected in LWE at both pH values for 12 weeks at 4 degrees C. In subsequent experiments, Listeria reductions of > 3.0 log CFU/ml were achieved within 24 h in both LWE and broth plus nisin (500 IU/ml) at pH 6.6 but not at pH 7.5, and antilisterial activity was enhanced when nisin was added as a solution rather than in dry form.     

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。     

Modeling the growth of Listeria monocytogenes in pasteurized white asparagus

Growth of Listeria monocytogenes in pasteurized white asparagus was monitored at different storage temperatures (4, 10, 20, and 30 degrees C). Among the main microbial kinetic parameters, growth rate (mu) per hour was calculated at each temperature using the Baranyi-Roberts model. L. monocytogenes was able to grow at all temperatures, although at 4 degrees C only a slight increment of the microbial population was observed (approximately 1 log CFU/g) after 300 h of storage. Subsequently, two different secondary modeling approaches were proposed to study the relationship between mu and storage temperature: the Arrhenius and Ratkowsky models. Although both models properly described the data observed, smaller values of root mean square error (RMSE) and standard error of prediction (SEP) were obtained with the Ratkowsky model, providing a better goodness of fit (Ratkowsky model: RMSE = 0.010, SEP = 21.23%; Arrhenius model: RMSE = 0.026, SEP = 54.37%). The maximum population density (MPD) was calculated at each temperature studied. A clear dependence between MPD and temperature was found; lower temperatures produced lower values of MPD. This finding confirmed the Jameson effect, indicating that multiple hurdles in the food-processing chain plus lower temperatures reduced L. monocytogenes growth. Predicting the growth of L. monocytogenes along the food chain will help to reduce microbial risks associated with consumption of pasteurized white asparagus.     

A solid agar overlay method for recovery of heat-injured Listeria monocytogenes

A solid agar overlay method was developed for recovery of heat-injured Listeria monocytogenes. Presolidified nonselective tryptic soy agar with 0.6% yeast extract (TSAYE, 2% agar) was overlaid on top of solidified modified Oxford agar (MOX). Heat injury of L. monocytogenes was conducted at 58 degrees C for 6 min in a jacketed flask filled with tryptic soy broth. Both noninjured and heat-treated L. monocytogenes cells were plated onto TSAYE, MOX, and TSAYE-MOX plates. No significant differences (P > 0.05) in recovery were found among the three media for noninjured bacterial cells. Recovery of heat-injured L. monocytogenes cells on TSAYE-MOX overlay plates was equivalent to that on the nonselective TSAYE medium, whereas recovery on the selective MOX medium was significantly lower (P < 0.05) compared with both TSAYE and the overlay plates. There were no significant differences (P > 0.05) among the overlay plates prepared 0, 2, 4, 6, 8, 16, and 24 h prior to plating heat-injured bacterial cells. The TSAYE-MOX overlay also allowed differentiation of L. monocytogenes from a mixture of four other types of foodborne pathogens. This solid agar overlay method for recovery of heat-injured L. monocytogenes cells is less time-consuming and less complicated than the conventional overlay-underlay technique and the double overlay modification of the thin agar layer method and may allow for greater laboratory plating efficiencies.     

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.     

Multistate outbreak of Listeria monocytogenes associated with Mexican-style cheese made from pasteurized milk among pregnant, Hispanic women

Listeriosis is a severe infection caused by Listeria monocytogenes. Since 2004, the Centers for Disease Control and Prevention has requested that listeriosis patients be interviewed using a standardized Listeria Initiative (LI) questionnaire. In January 2009, states and the Centers for Disease Control and Prevention began investigating a multistate outbreak of listeriosis among pregnant, Hispanic women. We defined a case as an illness occurring between October 2008 and March 2009 with an L. monocytogenes isolate indistinguishable from the outbreak strain by pulsed-field gel electrophoresis. We conducted a multistate case-control study using controls that were selected from L. monocytogenes illnesses in non-outbreak-related pregnant, Hispanic women that were reported to the LI during 2004 to 2008. Eight cases in five states were identified. Seven of these were pregnant, Hispanic females aged 21 to 43 years, and one was a 3-year-old Hispanic girl, who was excluded from the study. Seven (100%) cases but only 26 (60%) of 43 controls had consumed Mexican-style cheese in the month before illness (odds ratio, 5.89; 95% confidence interval, 1.07 to ∞; P = 0.04). Cultures of asadero cheese made from pasteurized milk collected at a manufacturing facility during routine sampling by the Michigan Department of Agriculture on 23 February 2009 yielded the outbreak strain, leading to a recall of cheeses produced in the plant. Recalled product was traced to stores where at least three of the women had purchased cheese. This investigation highlights the usefulness of routine product sampling for identifying contaminated foods, of pulsed-field gel electrophoresis analysis to detect multistate outbreaks, and of the LI for providing timely exposure information for case-control analyses. Recalls of contaminated cheeses likely prevented additional illnesses.     

Comparison of growth kinetics for healthy and heat-injured Listeria monocytogenes in eight enrichment broths

Detection of Listeria in food products is often limited by performance of enrichment media used to support growth of Listeria to detectable levels. In this study, growth curves were generated using healthy and heat-injured Listeria monocytogenes strain F5069 in three nonselective and five selective enrichment broths. Nonselective enrichment media included the current Food and Drug Administration Bacteriological Analytical Manual Listeria enrichment broth base (BAM), Listeria repair broth (LRB), and Trypticase soy broth. Selective enrichment media included BAM with selective agents and LRB with selective agents, BCM L. monocytogenes preenrichment broth, Fraser broth, and UVM-modified Listeria enrichment broth. The Gompertz equation was used to model the growth of L. monocytogenes. Gompertz parameters were used to calculate exponential growth rate, lag-phase duration (LPD), generation time, maximum population density (MPD), and time required for repair of injured cells. Statistical differences (P < 0.05) in broth performance were noted for LPD and MPD when healthy and injured cells were inoculated into the broths. With the exception of Fraser broth, there were no significant differences in the time required for the repair of injured cells. Results indicate that the distinction between selective and nonselective broths in their ability to grow healthy Listeria and to repair sublethally injured cells is not solely an elementary issue of presence or absence of selective agents.     

Rapid detection of Listeria monocytogenes in raw milk with loop-mediated isothermal amplification and chemosensor

Loop-mediated isothermal amplification (LAMP) allows a rapid amplification of nucleic acids under isothermal conditions. It can be combined with a rhodamine-based dual chemosensor for much more efficient, field-friendly detection of Listeria monocytogenes. In this report, LAMP was performed at 63 °C for 10 min, followed by a rapid reaction of DNA amplification and the byproduct, pyrophosphate ion, with a rhodamine-based dual chemosensor and Cu(2+) is visualized as a disappearance of red color. The detection limit of L. monocytogenes by the LAMP-chemosensor was 8 to 10 cells per reaction tube, and the total assay time including 10 min for rapid DNA extraction was approximately 30 min. Data on naturally contaminated raw milk samples indicated that the LAMP method was highly specific and sensitive, giving 100% concordance with the ISO 10560 reference method. The results showed that the LAMP-chemosensor method has the advantages of better sensitivity and speed and less dependence on equipment than the standard Polymerase Chain Reaction for specifically detecting low levels of L. monocytogenes DNA, and this can be useful in the field as a routine diagnostic tool. PRACTICAL APPLICATION: The LAMP-chemosensor method reported here provided a powerful tool for detection of L. monocytogenes in raw milk samples due to its specificity, sensitivity, and rapidity.     

Dynamic modeling of Listeria monocytogenes growth in pasteurized vanilla cream after postprocessing contamination

A product-specific model was developed and validated under dynamic temperature conditions for predicting the growth of Listeria monocytogenes in pasteurized vanilla cream, a traditional milk-based product. Model performance was also compared with Growth Predictor and Sym'Previus predictive microbiology software packages. Commercially prepared vanilla cream samples were artificially inoculated with a five-strain cocktail of L. monocytogenes, with an initial concentration of 102 CFU g(-1), and stored at 3, 5, 10, and 15 degrees C for 36 days. The growth kinetic parameters at each temperature were determined by the primary model of Baranyi and Roberts. The maximum specific growth rate (mu(max)) was further modeled as a function of temperature by means of a square root-type model. The performance of the model in predicting the growth of the pathogen under dynamic temperature conditions was based on two different temperature scenarios with periodic changes from 4 to 15 degrees C. Growth prediction for dynamic temperature profiles was based on the square root model and the differential equations of the Baranyi and Roberts model, which were numerically integrated with respect to time. Model performance was based on the bias factor (B(f)), the accuracy factor (A(f)), the goodness-of-fit index (GoF), and the percent relative errors between observed and predicted growth. The product-specific model developed in the present study accurately predicted the growth of L. monocytogenes under dynamic temperature conditions. The average values for the performance indices were 1.038, 1.068, and 0.397 for B(f), A(f), and GoF, respectively for both temperature scenarios assayed. Predictions from Growth Predictor and Sym'Previus overestimated pathogen growth. The average values of B(f), A(f), and GoF were 1.173, 1.174, and 1.162, and 1.267, 1.281, and 1.756 from Growth Predictor and Sym'Previus, respectively.     

Recovery of heat-injured Listeria innocua

Listeria innocua was subjected to thermal inactivation and the extent of heat-injured cells was quantified. Cultures were heated in liquid medium for different times, using temperatures in the range of 52.5 to 65.0 degrees C, and plated on Tryptic Soy Agar with 0.6% yeast extract (TSAYE) used as non-selective medium and on TSAYE plus 5% NaCl (TSAYE+NaCl) and Palcam agar with selective supplement (Palcam agar) as selective media. The difference observed in counts in non-selective and in selective media gave an indication of cell injury during the heat treatment. D- and z- values were calculated for all conditions considered. For each temperature, D-values obtained using non-selective recovery procedures were higher than the ones obtained using the two selective media. When comparing the selective media, it can be concluded that Palcam agar allowed recovery and growth of thermally injured cells and so it was less inhibitor than TSAYE+NaCl. Another important result was the influence of temperature on the degree of cellular injury. As temperature increases, the degree of heat-injured cells also increases, and consequently concern has to be taken with the temperature and the counting medium used in food processing studies. The results of this work clearly demonstrated that selective media used for Listeria monocytogenes enumeration/detection might not be suitable for the recovery of heat-injured cells, which can dangerously underestimate the presence of this foodborne pathogen.     

食品中单核细胞增多李斯特菌的快速检测

单核细胞增多李斯特菌是一种能引起人畜共患病和食源性疾病的致病菌。本文简述了单增李斯特菌的特性和毒力因子,介绍了从分子生物学和免疫学发展起来的核酸探针杂交、PCR、ELISA、免疫传感器等快速检测方法。这些快速检测方法与传统检测方法相比不仅缩短了检测时间,提高了检测效率,还提高了检测方法的灵敏度和特异性,对于单增李斯特菌的检测具有良好的应用前景。      

Combination of growth conditions and InlB-specific dot-immunoassay for rapid detection of Listeria monocytogenes in raw milk

The gram-positive bacterium Listeria monocytogenes is an important foodborne pathogen contaminating dairy products. Closely related to L. monocytogenes saprophytic Listeria spp. are also frequent contaminators of food and, particularly, dairy products. To distinguish L. monocytogenes from nonpathogenic Listeria spp. and other bacteria, a dot-immunoassay was developed. The immunoassay is based on the polyclonal antibody to the secreted form of the surface virulence-associated L. monocytogenes-specific InlB protein. To increase InlB production, bacteria were grown on the brain-heart infusion agar supplemented with 0.2% activated charcoal (BHIC agar). Direct plating of artificially contaminated raw milk samples on the BHIC agar followed by the dot-immunoassay allowed a rapid identification of L. monocytogenes in concentrations as little as 10 cfu/mL. Using the developed approach, preliminary results were obtained within 14 h, and the final results were obtained after 26 h. The dot-immunoassay was tested on L. monocytogenes strains belonging to different clonal complexes and phylogenetic lineages, Listeria spp., and other bacterial species. Results showed the exceptional specificity of the developed dot-immunoassay for the rapid identification of L. monocytogenes.