Monocytogenes In Foods Research Articles

Development of a Loop‐Mediated Isothermal Amplification Assay Based on lmo0460 Sequence for Detection of Listeria monocytogenes

AbstractLoop‐mediated isothermal amplification (LAMP) based on lmo0460 sequence in genome of Listeria monocytogenes (L. monocytogenes) was designed for detection of L. monocytogenes, which is an important foodborne kind of pathogenic bacteria causing human and animal disease. The primers set for lmo0460 sequence encoding membrane‐associated lipoprotein consist of four specific primers targeting six regions on specific fragment. The LAMP assay could be optimized and the optimum condition was in 50 min at 63°C. Amplification products were visualized by 2% agarose gel electrophoresis. Sensitivity of the LAMP assay for detection of L. monocytogenes in pure cultures was 1.7 cfu per reaction. The LAMP assay was 147‐fold higher sensitive than that of the conventional polymerase chain reaction (PCR) assay (251 cfu/reaction). Fifty chicken samples were detected for L. monocytogenes in the method, and the accuracy of LAMP was shown to be 100% when compared with culture biotechnical, while the PCR assay failed to detect L. monocytogenes in one of the positive samples. It is shown that LAMP assay based on lmo0460 sequence can be a highly sensitive, efficient and easy‐to‐perform useful detection tool for L. monocytogenes and will be a potential useful and powerful tool for detection foodborne pathogens.Practical ApplicationsL. monocytogenes contamination in chicken samples has been reported. In developing countries, such as China, the LAMP assay is a suitable, useful tool for rapid detection of L. monocytogenes in food because it is more cost‐effective, simple and high sensitive than PCR.

Sensitive enumeration of Listeria monocytogenes and other Listeria species in various naturally contaminated matrices using a membrane filtration method

For the enumeration of Listeria monocytogenes (L. monocytogenes) in food, a sensitive enumeration method has been recently developed. This method is based on a membrane filtration of the food suspension followed by transfer of the filter on a selective medium to enumerate L. monocytogenes.An evaluation of this method was performed with several categories of foods naturally contaminated with L. monocytogenes. The results obtained with this technique were compared with those obtained from the modified reference EN ISO 11290-2 method for the enumeration of L. monocytogenes in food, and are found to provide more precise results. In most cases, the filtration method enabled to examine a greater quantity of food thus greatly improving the sensitivity of the enumeration. However, it was hardly applicable to some food categories because of filtration problems and background microbiota interference.

Where the problem is with Listeria monocytogenes ?

Foodborne diseases are considerate an important public health concern in many countries and each year, ingestion of foods contaminated with pathogens causes millions of episodes of diarrhea and other debilitating effects. As results, large number of hospitalizations and thousands of deaths occur, and billions of Euros and Dollars are spent every year with recalls and patients medical treatment. The emergence of some of these diseases is the result of complex interactions, such as the advances in the medical field, the expansion of the food industry, cold storage systems, as well as the change in eating habits. Since the consumption of contaminated food has been associated with the development of diseases, various studies were developed, especially in industrialized countries, in order to clarify the relationship between these pathogens and their persistence in the food processing environment, their main routes of contamination, how they can survive and multiply in foods, and their potential to cause disease. Among these microorganisms, Listeria monocytogenes is a concern for Public Health agencies. Several countries adopt a zero tolerance policy regarding the presence of L. monocytogenes in foods. However, the environment conditions of food processing industries were always partially located in secondary importance in the tracking presence of reservoirs for L. monocytogenes and other foodborne pathogens. In recent years, phenotypic and genotypic studies led to new insights into ecology, epidemiology, virulence potential and genetic evolution of various genuses of bacteria. L. monocytogenes is the causative agent of listeriosis, a severe foodborne disease that affects mainly the elderly, immunocompromised individuals, pregnant women and neonates. Listeriosis is characterised by gastroenteritis and septicaemia, leading to abortion and infections in the nervous system. Its mortality rate varies from 20 to 30%, this being common mainly when the pathogen is able to cause encephalitis and meningitis.

Detection of Viable but Nonculturable Cells of Listeria monocytogenes with the Use of Direct Epifluorescent Filter Technique

AbstractDirect epifluorescent filter technique was studied as a method to enumerate Listeria monocytogenes in artificially contaminated cheese. Occurrences of viable but nonculturable (VBNC) cells of L. monocytogenes prompted us to investigate the viability of this pathogen in hard cheese during refrigerated storage under different packaging conditions. This study compared the results of two enumeration methods: epifluorescent microscopic counting and plating. The microscopic enumeration was based on fluorescent staining with carboxyfluorescein diacetate (CFDA) for tracing cells that display metabolic activity. In this study, the L. monocytogenes persisted well throughout a prolonged storage of cheese, regardless of the packaging conditions. Based on a discrepancy between CFDA and plate count results, a dormant fraction of VBNC cells was distinguished, thus greater quantities of VBNC cells were estimated along with storage time. We conclude that direct epifluorescent filter technique may be an attractive approach for assessing bacterial viability, especially for pathogens with the ability to enter a dormant state.Practical ApplicationsListeria monocytogenes is a foodborne pathogen with a remarkable long‐term ability to persist in a variety of ready‐to‐eat foods. The plating method for enumeration of L. monocytogenes in food is well established, but it is time consuming and relies only on cell reproduction, so that injured and nonculturable cells have no chance to be counted. As a solution, we describe a rapid method for the enumeration of L. monocytogenes in cheese using the direct epifluorescent filter technique. This method is based on fluorescent staining of cells with a metabolic activity indicator and cell counting with the help of epifluorescence microscopy. The method presented in this study allows the rapid enumeration of both active and nonculturable cells of L. monocytogenes and could be used by predictive microbiologists for precise estimation of the safety of dairy products with respect to pathogenic bacteria.

Comparison of Culture, Conventional and Real-time PCR Methods for Listeria monocytogenes in Foods.

We compared standard culture methods as well as conventional PCR and real-time PCR for the detection of Listeria monocytogenes (L. monocytogenes) in milk, cheese, fresh-cut vegetables, and raw beef that have different levels of background microflora. No statistical differences were observed in sensitivity between the two selective media in all foods. In total, real-time PCR assay exhibited statistically excellent detection sensitivity (p<0.05) and was less time consuming and laborious as compared with standard culture methods. Conventional culture methods showed poor performance in detecting L. monocytogenes in food with high levels of background microflora, generating numerous false negative results. While the detection of L. monocytogenes in fresh cut vegetable by culture methods was hindered only by L. innocua, various background microflora, such as L. innocua, L. welshimeri, L. grayi, and Enterococcus faecalis appeared on the two selective media as presumptive positive colonies in raw beef indicating the necessity of improvement of current selective media. It appears that real-time PCR is an effective and sensitive presumptive screening tool for L. monocytogenes in various types of foods, especially foods samples with high levels of background microflora, thus complementing standard culture methodologies.

Identification of non-Listeria spp. bacterial isolates yielding a β-d-glucosidase-positive phenotype on Agar Listeria according to Ottaviani and Agosti (ALOA)

Agar Listeria according to Ottaviani and Agosti (ALOA) is the mandatory medium used for the detection and enumeration of Listeria monocytogenes in foods according to the official International Organization for Standardization (ISO) methods. On ALOA, Listeria spp. appear as bluish-green colonies due to the production of β-d-glucosidase, an enzyme that cleaves 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside, a chromogenic substrate included in the formulation of the medium. The present work reports on bacterial isolates (n=64) from ready-to-eat soft cheeses, which are able to grow on ALOA, forming bluish-green colonies and therefore phenotypically resemble Listeria spp. All isolates were also capable of growing on the selective media PALCAM and RAPID L'mono™. The isolates were characterised with biochemical tests including those specified in the ISO standards for the confirmation of Listeria spp. and identified via partial sequencing of their 16S rRNA gene. According to sequencing results the isolates represented 12 different bacterial species or species-groups belonging to seven different genera: Bacillus spp. (B. circulans, B. clausii, B. licheniformis and B. oleronius), Cellulosimicrobium spp. (C. funkei), Enterococcus spp. (E. faecalis, E. faecium/durans), Kocuria spp. (K. kristinae), Marinilactibacillus spp. (M. psychrotolerans), Rothia spp. (R. terrae) and Staphylococcus spp. (S. sciuri and S. saprophyticus subsp. saprophyticus/xylosus). Cellulosimicrobium spp. have never been previously isolated from foods. These results significantly extend the list of bacteria previously known as capable of growing on ALOA as bluish-green colonies and suggest that there may be room for further improvement in the medium's inhibitory properties towards non-Listeria spp., Gram-positive bacteria present in foods.

Rapid detection of Listeria monocytogenes in raw milk and soft cheese by a redox potential measurement based method combined with real-time PCR.

The incidence of outbreaks of foodborne listeriosis has indicated the need for a reliable and rapid detection of the microbe in different foodstuffs. A method combining redox potential measurement and real-time polymerase chain reaction (PCR) was developed to detect Listeria monocytogenes in artificially contaminated raw milk and soft cheese. Food samples of 25 g or 25 ml were homogenised in 225 ml of Listeria Enrichment Broth (LEB) with Oxford supplement, and the redox potential measurement technique was applied. For Listeria species the measuring time was maximum 34 h. The absence of L. monocytogenes could reliably be proven by the redox potential measurement method, but Listeria innocua and Bacillus subtilis could not be differentiated from L. monocytogenes on the basis of the redox curves. The presence of L. monocytogenes had to be confirmed by real-time PCR. The combination of these two methods proved to detect < 10 cfu/g of L. monocytogenes in a cost- and time-effective manner. This method can potentially be used as an alternative to the standard nutrient method for the rapid detection of L. monocytogenes in food.

Monitoring occurrence and persistence of Listeria monocytogenes in foods and food processing environments in the Republic of Ireland.

Although rates of listeriosis are low in comparison to other foodborne pathogenic illness, listeriosis poses a significant risk to human health as the invasive form can have a mortality rate as high as 30%. Food processors, especially those who produce ready-to-eat (RTE) products, need to be vigilant against Listeria monocytogenes, the causative pathogen of listeriosis, and as such, the occurrence of L. monocytogenes in food and in the food processing environment needs to be carefully monitored. To examine the prevalence and patterns of contamination in food processing facilities in Ireland, 48 food processors submitted 8 samples every 2 months from March 2013 to March 2014 to be analyzed for L. monocytogenes. No positive samples were detected at 38% of the processing facilities tested. Isolates found at the remaining 62% of facilities were characterized by serotyping and Pulsed Field Gel Electrophoresis (PFGE). A general L. monocytogenes prevalence of 4.6% was seen in all samples analyzed with similar rates seen in food and environmental samples. Differences in prevalence were seen across different food processors, food sectors, sampling months etc. and PFGE analysis allowed for the examination of contamination patterns and for the identification of several persistent strains. Seven of the food processing facilities tested showed contamination with persistent strains and evidence of bacterial transfer from the processing environment to food (the same pulsotype found in both) was seen in four of the food processing facilities tested.

Analysing and modelling the growth behaviour of Listeria monocytogenes on RTE cooked meat products after a high pressure treatment at 400 MPa

Various predictive models are available for high pressure inactivation of Listeria monocytogenes in food, but currently available models do not consider the growth kinetics of surviving cells during the subsequent storage of products. Therefore, we characterised the growth of L. monocytogenes in sliced cooked meat products after a pressurization treatment. Two inoculum levels (107 or 104CFU/g) and two physiological states before pressurization (freeze-stressed or cold-adapted) were evaluated. Samples of cooked ham and mortadella were inoculated, high pressure processed (400MPa, 5min) and subsequently stored at 4, 8 and 12°C. The Logistic model with delay was used to estimate lag phase (λ) and maximum specific growth rate (μmax) values from the obtained growth curves. The effect of storage temperature on μmax and λ was modelled using the Ratkowsky square root model and the relative lag time (RLT) concept.Compared with cold-adapted cells the freeze-stressed cells were more pressure-resistant and showed a much longer lag phase during growth after the pressure treatment. Interestingly, for high-pressure inactivation and subsequent growth, the time to achieve a concentration of L. monocytogenes 100-fold (2-log) higher than the cell concentration prior to the pressure treatment was similar for the two studied physiological states of the inoculum. Two secondary models were necessary to describe the different growth behaviour of L. monocytogenes on ready-to-eat cooked ham (lean product) and mortadella (fatty product). This supported the need of a product-oriented approach to assess growth after high pressure processing. The performance of the developed predictive models for the growth of L. monocytogenes in high-pressure processed cooked ham and mortadella was evaluated by comparison with available data from the literature and by using the Acceptable Simulation Zone approach. Overall, 91% of the relative errors fell into the Acceptable Simulation Zone.

InstantLabs Listeria monocytogenes food safety kit. Performance tested method 051304.

The InstantLabs Listeria monocytogenes Food Safety Kit was validated against the International Organization for Standardization (ISO) reference method 11290-1 for the detection of Listeria monocytogenes and other Listeria species. The matrixes (stainless steel, sealed concrete, ice cream, whole milk, cheddar cheese, raw shrimp, hot dogs, deli turkey, and lettuce) were inoculated with approximately 1 CFU/test portion of L. monocytogenes to generate fractional positives (5-15) in 20 inoculated samples. Enrichments were also fractionally inoculated with L. grayii for side-by-side testing of the Listeria Species Food Safety Kit. Stainless steel and sealed concrete samples were validated using 4 x 4" and 1 x 1 " test areas, respectively, and enriched in Buffered Listeria Enrichment Broth (BLEB) at 35 +/- 1degreesC for 22-28 h. All food samples were tested at 25 g and enriched in BLEB at 35 +/- 1 degreesC for 24-28 h. All samples were confirmed using the ISO reference method, regardless of initial screen result. The InstantLabs test method performed as well as or better than the reference method for the detection of L. monocytogenes on stainless steel and sealed concrete and in ice cream, whole milk, cheddar cheese, raw shrimp, hot dogs, deli turkey, and lettuce. Inclusivity and exclusivity testing revealed no false negatives and no false positives among the 50 L. monocytogenes serovars and 30 non-L. monocytogenes species examined. The method was shown to be robust when the enrichment times, volumes for DNA extraction, and heat block times were varied.

InstantLabs Listeria species food safety kit. Performance tested methods 041304.

The InstantLabs Listeria Species Food SafetyKitwas validated against the International Organization for Standardization (ISO) reference method 11290-1 for the detection of Listeria monocytogenes and other Listeria species. The matrixes (stainless steel, sealed concrete, cheddar cheese, raw shrimp, and hot dogs) were inoculated with approximately 1 CFU/test portion of various Listeria species to generate fractional positives (5-15) in 20 inoculated samples. Enrichments were also fractionally inoculated with L. monocytogenes for side-by-side testing of the InstantLabs Listeria monocytogenes Food Safety Kit. Stainless steel and sealed concrete samples were validated using 4 x 4" and 1 x 1" test areas, respectively, and enriched in Buffered Listeria Enrichment Broth (BLEB) at 35 +/- 1 degrees C for 22-28 h. All food samples were tested at 25 g or 25 mL and enriched in BLEB at 35 +/- 1 degrees C for 24-28 h. All samples were confirmed using the ISO reference method, regardless of initial screen result. The InstantLabs test method performed as well as or better than the reference method for the detection of Listeria species on stainless steel, sealed concrete, cheddar cheese, raw shrimp, and hot dogs. Inclusivity and exclusivity testing revealed no false negatives and no false positives among the 80 Listeria species and 30 non-Listeria species examined. The method was shown to be robust when variations were introduced to the enrichment time, the volume for DNA extraction, and the heat block time (data not shown).

Rapid Detection of Listeria monocytogenes by Real-Time PCR in Processed Meat and Dairy Products

The objectives of this study were to evaluate the detection of Listeria monocytogenes in different ready-to-eat foods using real-time PCR (RT-PCR). Various concentrations (10(0) to 10(5) CFU/ml) of L. monocytogenes ATCC 19115 were inoculated into ham, sausage, ground meat, processed milk, cheese, and infant formula. L. monocytogenes ATCC 19115 in the samples was then enumerated on Oxford agar, and DNA was extracted from the samples before and after incubation at 36°C for 4 h. A set of primers and hybridization probe designed in this study was then used to detect the pathogen. The standard curve was then prepared by plotting cycle threshold values for each dilution versus L. monocytogenes cell counts (log CFU). The specificity of the set of primers and hybridization probe was appropriate. A 4-h incubation at 36°C before DNA extraction produced optimum standard curves in comparison to the results for a 0-h incubation. Thus, a 4-h incubation at 36°C was applied for monitoring L. monocytogenes in collected food samples. To monitor L. monocytogenes in foods, 533 samples (ham, 129; sausage, 226; ground meat, 72; processed cheese, 54; processed milk, 42; and infant formula, 10) were collected from retail markets and from the step before pasteurization in plants. Of all 533 samples, 4 samples (0.8%) showed positive signals in RT-PCR. Two samples from hams (1.6%) and two samples from sausages (0.9%) were determined to be positive for L. monocytogenes at < 100 CFU/g. The results indicate that the RT-PCR detection method with the set of primers and hybridization probe designed in this study should be useful in monitoring for L. monocytogenes in processed meat and milk products.

Purification and characterization of the bacteriocin produced by Lactobacillus sakei MBSa1 isolated from Brazilian salami

The study aimed at determining the biochemical characteristics of the bacteriocin produced by Lactobacillus sakei MBSa1, isolated from salami, correlating the results with the genetic features of the producer strain. Identification of strain MBSa1 was performed by 16S rDNA sequencing. The bacteriocin was tested for spectrum of activity, heat and pH stability, mechanism of action, molecular mass and amino acid sequence when purified by cation-exchange and reversed-phase HPLC. Genomic DNA was tested for bacteriocin genes commonly present in Lact. sakei. Bacteriocin MBSa1 was heat-stable, unaffected by pH 2·0 to 6·0 and active against all tested Listeria monocytogenes strains. Maximal production of bacteriocin MBSa1 (1600 AU ml(-1)) in MRS broth occurred after 20 h at 25°C. The molecular mass of produced bacteriocin was 4303·3 Da, and the molecule contained the SIIGGMISGWAASGLAG sequence, also present in sakacin A. The strain contained the sakacin A and curvacin A genes but was negative for other tested sakacin genes (sakacins T-α, T-β, X, P, G and Q). In the studied conditions, Lact. sakei MBSa1 produced sakacin A, a class II bacteriocin, with anti-Listeria activity. The study covers the purification and characterization of the bacteriocin produced by a lactic acid bacteria isolated from salami (Lact. sakei MBSa1), linking genetic and expression information. Its heat-resistance, pH stability in acid conditions (pH 2·0-6·0) and activity against L. monocytogenes food isolates bring up a potential technological application to improve food safety.

Implementation of classical, molecular biological and immunoenzymatic methods in isolation and detection of Listeria monocytogenes in food

Food borne pathogens and spoilage bacteria are influencing the safety and quality of food and animal feed and can result in serious adverse effects to human and animal health as well as to the food quality. Consequently, microbiological quality control in the food industry has become the priority of the food producers and it aims towards minimizing the risks related to food pathogens and spoilage bacteria. One of the most important pathogens in the food industry is Listeria monocytogenes. Listeriosis has a significant public health and economic impact because of its high hospitalization and mortality rate. Most people infected with Listeria are hospitalized and mortality is approximately 30 %. Therefore it is necessary to undertake efficient control measures, especially concerning the necessity of a rapid and accurate detection of this pathogen in the food industry as well as in retail food samples. Recently used conventional methods are often time consuming and require intensive work. However, obtained results can often be false considering the presence of viable but not cultivable microorganisms. Advances in biotechnology and bioinformatics have resulted in the development of novel testing technologies that enable tracking, more reliable and faster detection of food pathogens. Furthermore, molecular-biology methods, although still not applied routinely in everyday practice, are the promising alternative which can replace current reference methods in this area.

Traditional methods for isolation of Listeria monocytogenes.

Conventional methods for the detection of Listeria monocytogenes in foods and environmental samples relies on selective pre-enrichment, enrichment, and plating. This is followed by confirmation of suspected colonies by testing a limited number of biochemical markers.

Evaluation of the Thermo Scientific™ SureTect™ Listeria monocytogenes Assay

The Thermo Scientific SureTect Listeria monocytogenes Assay is a new real-time PCR assay for the detection of Listeria monocytogenes in food and environmental samples. This assay was validated using the AOAC Research Institute (AOAC-RI) Performance Tested Methods program in comparison to the reference method detailed in International Organization for Standardization 11290-1:1996, including Amendment 1:2004 with the following foods and food contact surfaces: smoked salmon, processed cheese, fresh bagged spinach, fresh cantaloupe, cooked prawns (chilled product), cooked sliced turkey meat (chilled product), ice cream, pork frankfurters, salami, ground raw beef meat (12% fat), plastic, and stainless steel. All matrixes were tested by Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. In addition, three matrixes (pork frankfurters, bagged lettuce, and stainless steel) were analyzed independently as part of the AOAC-RI controlled laboratory study by the University of Guelph, Canada. Using probability of detection (POD) statistical analysis, a significant difference was demonstrated between the candidate and reference methods for salami, cooked sliced turkey and ice cream in favor of the SureTect assay. For all other matrixes, no significant difference by POD was seen between the two methods during the study. Inclusivity and exclusivity testing was also conducted with 53 and 30 isolates, respectively, which demonstrated that the SureTect assay was able to detect all serotypes of L. monocytogenes. None of the exclusivity isolates analyzed were detected by the SureTect assay. Ruggedness testing was conducted to evaluate the performance of the assay with specific method deviations outside the recommended parameters open to variation, i.e., enrichment time and temperature and lysis temperature, which demonstrated that the assay gave reliable performance. Accelerated stability testing was also conducted, validating the assay shelf life.

Prevalence of Listeria monocytogenes in ready-to-eat food of animal origin

In this study, the presence of Listeria monocytogenes in ready- to- eat meat, milk and fish products has been investigated. In addition, the presence of L. monocytogenes on food - contact surfaces, as a potential source of food contamination, has been investigated as well. Samples were analyzed by fluorescent immunoenzyme assay on miniVidas® device and by standard microbiological SRPS EN ISO 11290-1: 2010 and SRPS EN ISO 11290-2: 2010 methods. Out of 881 food samples tested, 12,25% were Listeria spp. positive, out of which 8,4% were positive for L. monocytogenes. L. monocytogenes was most commonly found in smoked salmon, which confirmed fact that smoked fish is a high risk food for the L. monocytogenes growth and survival. Out of 512 samples from the food - contact surfaces, L. monocytogenes was found in 8,78% of swab samples. This paper highlightes the importance of implementing appropriate prevention and control measures, verification procedures, and monitoring and maintenance programs that will help to prevent L. monocytogenes food contamination.

Electroforesis en Gel de Campo Pulsado (PFGE) para la diferenciación molecular de Listeria monocytogenes

The reporting of L. monocytogenes in food in Colombia is not a mandatory; however, foods considered high-risk are monitored, and the organism is only reported clinically as Gram-positive when it causes meningitis. L. monocytogenes is a foodborne, intracellular, pathogen which causes listeriosis, a disease lethal to humans and animals. Outbreaks of this disease worldwide can bring about human and economic losses. Only a few studies in Colombia have been able to identify and molecularly serotype isolates allowing only the theoretical distribution of serotypes by lineage. This review explains the characteristics of the pathogen, its importance in public health and in the food industry, and provides an overview of PFGE-CHEF; identifying the standard work protocol and the appropriate restriction enzymes to cut DNA. We found that the enzyme combination, XbaI-AscI, followed by ApaI offers the best results to differentiate isolates, by grouping them by lineages, and displaying intra-serotype variations. Additionally, we found that in several Latin American countries the results are analyzed using PulseNet; this ensures the comparison of PFGE patterns in equivalent conditions.

Construction of Listeria monocytogenes Mutants with In‐Frame Deletions in the Phosphotransferase Transport System (PTS) and Analysis of Their Growth under Stress Conditions

Listeria monocytogenes is a foodborne pathogen that is difficult to eliminate due to its ability to survive under different stress conditions such as low pH and high salt. To better control this pathogen in food, it is important to understand its survival mechanisms under these stress conditions. LMOf2365_0442, 0443, and 0444 encode for phosphotransferase transport system (PTS) permease (fructose-specific IIABC components) that is responsible for sugar transport. LMOf2365_0445 encodes for glycosyl hydrolase. These genes were induced by high pressure and inhibited under salt treatments; therefore, we hypothesized that genes encoding these PTS proteins may be involved in general stress responses. To study the function of these genes, deletion mutants of the PTS genes (LMOf2365_0442, LMOf2365_0443, and LMOf2365_0444) and the downstream gene LMOf2365_0445 were created in L. monocytogenes strain F2365. These deletion mutants were tested under different stress conditions. The growth of ∆LMOf2365_0445 was increased under nisin (125 μg/mL) treatments compared to the wild-type (P < 0.01). The growth of ∆LMOf2365_0442 in salt (brain-heart infusion medium with 5% NaCl) was significantly increased (P < 0.01), and ∆LMOf2365_0442 showed increased growth under acidic conditions (pH 5.0) compared to the wild-type (P < 0.01). The results from phenotypic arrays demonstrated that some of these mutants showed slightly slower growth under different carbon sources and basic conditions. The results indicate that deletion mutants ∆LMOf2365_0442 and ∆LMOf2365_0445 were more resistant to multiple stress conditions compared to the wild-type, suggesting that they may contribute to the general stress response in L. monocytogenes. An understanding of the growth of these mutants under multiple stress conditions may assist in the development of intervention strategies to control L. monocytogenes in food.

Quantifying Listeria monocytogenes prevalence and concentration in minced pork meat and estimating performance of three culture media from presence/absence microbiological testing using a deterministic and stochastic approach

Listeria monocytogenes poses a serious threat to public health, and the majority of cases of human listeriosis are associated with contaminated food. Reliable microbiological testing is needed for effective pathogen control by food industry and competent authorities. The aims of this work were to estimate the prevalence and concentration of L. monocytogenes in minced pork meat by the application of a Bayesian modeling approach, and also to determine the performance of three culture media commonly used for detecting L. monocytogenes in foods from a deterministic and stochastic perspective. Samples (n = 100) collected from local markets were tested for L. monocytogenes using in parallel the PALCAM, ALOA and RAPID'L.mono selective media according to ISO 11290-1:1996 and 11290-2:1998 methods. Presence of the pathogen was confirmed by conducting biochemical and molecular tests. Independent experiments (n = 10) for model validation purposes were performed. Performance attributes were calculated from the presence–absence microbiological test results by combining the results obtained from the culture media and confirmative tests. Dirichlet distribution, the multivariate expression of a Beta distribution, was used to analyze the performance data from a stochastic perspective. No L. monocytogenes was enumerated by direct-plating (<10 CFU/g), though the pathogen was detected in 22% of the samples. L. monocytogenes concentration was estimated at 14–17 CFU/kg. Validation showed good agreement between observed and predicted prevalence (error = −2.17%). The results showed that all media were best at ruling in L. monocytogenes presence than ruling it out. Sensitivity and specificity varied depending on the culture-dependent method. None of the culture media was perfect in detecting L. monocytogenes in minced pork meat alone. The use of at least two culture media in parallel enhanced the efficiency of L. monocytogenes detection. Bayesian modeling may reduce the time needed to draw conclusions regarding L. monocytogenes presence and the uncertainty of the results obtained. Furthermore, the problem of observing zero counts may be overcome by applying Bayesian analysis, making the determination of a test performance feasible.