Listeria In Foods Research Articles

Label-Free Immuno-Sensors for the Fast Detection of Listeria in Food.

Foodborne diseases are a major concern for both food industry and health organizations due to the economic costs and potential threats for human lives. For these reasons, specific regulations impose the research of pathogenic bacteria in food products. Nevertheless, current methods, references and alternatives, take up to several days and require many handling steps. In order to improve pathogen detection in food, we developed an immune-sensor, based on Surface Plasmon Resonance imaging (SPRi) and bacterial growth which allows the detection of a very low number of Listeria monocytogenes in food sample in one day. Adequate sensitivity is achieved by the deposition of several antibodies in a micro-array format allowing real-time detection. This label-free method thus reduces handling and time to result compared with current methods.

Label-free immuno-sensors for the fast detection of Listeria in food

Listeria monocytogenes is one of the major foodborne pathogen found in a large set of foodstuff. With the aim of decreasing listeriosis occurrence, the early detection of this pathogen in food products is of tremendous interest. So far, the reference method and all the alternative methods require a pre-enrichment step lasting 24 h to several days. Then, an aliquot of the enriched sample is collected and characterized according different methods including genetic analysis, or beads immuno-agglutination. With the aim of significantly decreasing the overall processing time, we propose an approach involving the detection of alive bacteria during their growing phase via the use of an immuno-chip SPR imaging process. Listeria specific antibodies were also produced and chemically grafted on the biochip before being selected for their ability to distinguish different Listeria strains. Finally, this approach allows the detection of a very low number of L. monocytogenes present in foodstuff.

Method Modification Study for the Thermo Scientific SureTect™ Listeria Species Assay-Matrix Extension.

The Thermo Scientific™ SureTect™ Listeria species assay is a new real-time PCR assay for the detection of all species of Listeria in food and environmental samples. The assay was originally certified as Performance Tested Methods(SM) (PTM) 071304 in 2013. This report details the method modification study undertaken to extend the performance claims of the assay for matrixes of raw ground turkey, raw ground pork, bagged lettuce, raw pork sausages, pasteurized 2% fat milk, raw cod, pasteurized brie cheese, and ice cream. The method modification study was conducted using the AOAC Research Institute (RI) PTM program to validate the SureTect PCR assay in comparison to the reference method detailed in ISO 11290-1:1996 including amendment 1:2004. All matrixes were tested by Thermo Fisher Scientific (Basingstoke, United Kingdom). In addition, three matrixes (raw cod, bagged lettuce, and pasteurized brie cheese) were analyzed independently as part of the AOAC RI-controlled independent laboratory study by the University of Guelph, Canada. Using probability of detection statistical analysis, there was no significant difference in the performance between the SureTect assay and the International Organization for Standardization reference method for any of the matrixes analyzed in this study.

Outbreaks of listeriosis associated with deli meats and cheese: an overview

Microbial pollution of foods by undesirable microorganisms is a global food safety issue. One of such undesirable microorganism is the psychrotrophic, pathogenic specie of Listeria—Listeria <em>monocytogenes </em>that survives at low temperature. The source of contamination of this microbe into foods can be many including the food processing facilities due to improper sanitation procedures. The review of the literature on this important topic shows there are increasing concerns as regards contamination from Listeria in foods leading to many cases of listeriosis disease and food recalls. Ready-to-eat products, such as delicatessen (deli) meats and soft cheeses have repeatedly been identified by foodborne disease control programs as sources of outbreaks and products that put humans at risk for listeriosis. Although, most listeriosis cases tend to be sporadic in occurrence, outbreaks do occur frequently. Due to the global phenomenon of outbreaks associated with Listeria in deli meats and cheese, it requires an urgent attention from national and international authorities through rigorous procedures for its identification, surveillance procedures that can bring more awareness to the general public. There is also a need for more reports on the cases of Listeria particularly in developing countries, the standardization of identification procedures, and an improvement on national control programs by adequate surveillance.

Characterization of clinical and food Listeria monocytogenes isolates with different antibiotic resistance patterns through simulated gastrointestinal tract conditions and environmental stresses

Thirty-three Listeria monocytogenes isolates previously collected from two sources, food (n = 18) and human patients suffering from listeriosis (n = 15), with variable antibiotic susceptibility profiles (sensitive/resistant) to erythromycin, ciprofloxacin and nitrofurantoin, were studied for their ability to survive (i) the environmental stress provided by sequential conditions that simulate the digestive tract, and (ii) extreme pH values (1.5–12). The results showed a response that was only strain dependent. There were no variability in survival results based on type of stress (low or high pH), source (food or clinical), or sensitivity/resistance to antibiotics (p > 0.01). Some strains of L. monocytogenes are able to survive extreme acid and alkaline conditions, and conditions that mimic the sequential stressors found in the gastro-intestinal tract. The resistance to the antibiotics tested in this study by some L. monocytogenes strains did not confer any cross-protection to acid or alkaline stressors.

Magnetic bead based immuno-detection of Listeria monocytogenes and Listeria ivanovii from infant formula and leafy green vegetables using the Bio-Plex suspension array system

Listeriosis, a disease contracted via the consumption of foods contaminated with pathogenic Listeria species, can produce severe symptoms and high mortality in susceptible people and animals. The development of molecular methods and immuno-based techniques for detection of pathogenic Listeria in foods has been challenging due to the presence of assay inhibiting food components. In this study, we utilize a macrophage cell culture system for the isolation and enrichment of Listeria monocytogenes and Listeria ivanovii from infant formula and leafy green vegetables for subsequent identification using the Luminex xMAP technique. Macrophage monolayers were exposed to infant formula, lettuce and celery contaminated with L. monocytogenes or L. ivanovii. Magnetic microspheres conjugated to Listeria specific antibody were used to capture Listeria from infected macrophages and then analyzed using the Bio-Plex 200 analyzer. As few as 10 CFU/mL or g of L. monocytogenes was detected in all foods tested. The detection limit for L. ivanovii was 10 CFU/mL in infant formula and 100 CFU/g in leafy greens. Microsphere bound Listeria obtained from infected macrophage lysates could also be isolated on selective media for subsequent confirmatory identification. This method presumptively identifies L. monocytogenes and L. ivanovii from infant formula, lettuce and celery in less than 28 h with confirmatory identifications completed in less than 48 h.

Evaluation of the Thermo Scientific™ SureTect™ Listeria species Assay.

The Thermo Scientific™ SureTect™ Listeria species Assay is a new real-time PCR assay for the detection of all species of Listeria in food and environmental samples. This validation study was conducted using the AOAC Research Institute (RI) Performance Tested MethodsSM program to validate the SureTect Listeria species Assay in comparison to the reference method detailed in International Organization for Standardization 11290-1:1996 including amendment 1:2004 in a variety of foods plus plastic and stainless steel. The food matrixes validated were smoked salmon, processed cheese, fresh bagged spinach, cantaloupe, cooked prawns, cooked sliced turkey meat, cooked sliced ham, salami, pork frankfurters, and raw ground beef. All matrixes were tested by Thermo Fisher Scientific, Microbiology Division, Basingstoke, UK. In addition, three matrixes (pork frankfurters, fresh bagged spinach, and stainless steel surface samples) were analyzed independently as part of the AOAC-RI-controlled independent laboratory study by the University of Guelph, Canada. Using probability of detection statistical analysis, a significant difference in favour of the SureTect assay was demonstrated between the SureTect and reference method for high level spiked samples of pork frankfurters, smoked salmon, cooked prawns, stainless steel, and low-spiked samples of salami. For all other matrixes, no significant difference was seen between the two methods during the study. Inclusivity testing was conducted with 68 different isolates of Listeria species, all of which were detected by the SureTect Listeria species Assay. None of the 33 exclusivity isolates were detected by the SureTect Listeria species Assay. Ruggedness testing was conducted to evaluate the performance of the assay with specific method deviations outside of the recommended parameters open to variation, which demonstrated that the assay gave reliable performance. Accelerated stability testing was additionally conducted, validating the assay shelf life.

Identification of sixListeriaspecies by real-time PCR assay

The Listeria genus comprises 10 recognized species. Listeria monocytogenes causes listeriosis in humans and other animals primarily via contaminated food or animal feed. Listeria ivanovii causes listeriosis in animals and on rare occasions in humans. The identification of nonpathogenic species of Listeria in foods indicates that conditions exist that support the growth of pathogenic strains and is used to facilitate the implementation of control and prevention measures. This study shows the development and evaluation of a 5'exonuclease real-time PCR assay for the rapid identification of Listeria seeligeri, Listeria welshimeri, L. monocytogenes, L. ivanovii, Listeria grayi and Listeria innocua. The assay consists of two triplexes that were evaluated using 53 cultures of Gram-positive bacteria, including 49 Listeria spp. from human, animal, food or food-processing environments. The assay was rapid, specific and reproducible and could identify each of the six species from a mixture of strains. The developed assay proved to be a powerful means of rapidly identifying Listeria species and could be usefully implemented in busy specialist reference laboratories. The identification of species of Listeria from foods is important to monitor pathogenic strains and facilitates the implementation of control measures. This study shows the development and evaluation of a 5'exonuclease real-time PCR assay for the rapid identification of L. seeligeri, L. welshimeri, L. monocytogenes and L. ivanovii, L. grayi, L. innocua. The developed assay proved to be specific, rapid and reproducible and therefore could be implemented in busy specialist reference laboratories.

Detection of Listeria in milk using non-targeted metabolic profiling of Listeria monocytogenes: A proof-of-concept application

Listeria is represented by ten known species, comprising pathogenic and non-pathogenic variants. Listeria monocytogenes is the type species and is primarily pathogenic to humans and causes serious illness. As a result, most countries have a zero tolerance towards the presence of Listeria in foods. Therefore, in order to ensure food safety, robust techniques for detection are required. This paper describes a proof-of-concept application of a metabolomic technique for the rapid detection of Listeria, applied to nutrient media and a complex food sample (milk) inoculated with a pathogenic Listeria strain (L. monocytogenes). It was found that a profile of intracellular and extracellular metabolites associated with L. monocytogenes could be obtained using gas chromatography coupled to orthogonal acceleration time-of-flight mass spectrometry (GC-oaToFMS). Chemometric analysis showed that it is possible to differentiate between the uninoculated samples and samples inoculated with Listeria based on L. monocytogenes metabolic activity. This research demonstrates that metabolomics has the potential for rapidly identifying food contaminated with Listeria and could provide a means for enhancing monitoring programmes and ensuring food safety.

Use of bacteriophage cell wall-binding proteins for rapid diagnostics of Listeria.

Diagnostic protocols for food-borne bacterial pathogens such as Listeria need to be sensitive, specific, rapid, and inexpensive. Conventional culture methods are hampered by lengthy enrichment and incubation steps. Bacteriophage-derived high-affinity binding molecules (cell wall-binding domains, CBDs) specific for Listeria cells have recently been introduced as tools for detection and differentiation of this pathogen in foods. When coupled with magnetic separation, these proteins offer advantages in sensitivity and speed compared to the standard diagnostic methods. Furthermore, fusion of CBDs to differently colored fluorescent reporter proteins enables differentiation of Listeria strains in mixed cultures. This chapter provides protocols for detection of Listeria in food by CBD-based magnetic separation and subsequent multiplexed identification of strains of different serotypes with reporter-CBD fusion proteins.

Cell-mediated killing of Listeria monocytogenes by leucocin C producing Escherichia coli

Listeria monocytogenes is a foodborne pathogen causing listeriosis. Listeria in foods can be inhibited with bacteriocins or bacteriocin producing cultures. The aim of this study was to enhance the killing of L. monocytogenes by binding bacteriocin producing Escherichia coli cells to Listeria cells. Antilisterial E. coli was obtained by transferring leucocin C production from Leuconostoc carnosum 4010. For binding of E. coli cells to Listeria cells, the Listeria phage endolysin PlyP35 cell wall binding domain (CBD) was displayed on E. coli cell surface as FliC::CBD chimeric protein in flagella. CBD insertion in flagella was confirmed by Western analysis and enterokinase cleavage. By mixing isolated flagella with L. monocytogenes WSLC 1019 cells, the FliC::CBD flagella was shown to bind to Listeria cells. However, the wild type flagella also attached to Listeria cells masking putative additional binding mediated by the CBD. Yet, the cell-mediated leucocin C killing resulted in two-log reduction of Listeria, whereas the corresponding amount of leucocin C in spent culture medium could only inhibit growth without bacteriocidal effect. Cells binding Listeria and secreting antilisterial peptides may have applications in protection against listeriosis as they kill Listeria better than free antilisterial peptides.

Widespread Listeriosis Outbreak Attributable to Pasteurized Cheese, Which Led to Extensive Cross-Contamination Affecting Cheese Retailers, Quebec, Canada, 2008

A major Listeria monocytogenes outbreak occurred in the province of Quebec, Canada, in 2008, involving a strain of L. monocytogenes (LM P93) characterized by pulsed-field gel electrophoresis (PFGE) and associated with the consumption of pasteurized milk cheese. This report describes the results of the ensuing investigation. All individuals affected with LM P93 across the province were interviewed with a standardized questionnaire. Microbiological and environmental investigations were conducted by the Quebec's Food Inspection Branch of Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec among retailers and cheese plants involved in the outbreak. Between 8 June and 31 December 2008, 38 confirmed cases of LM P93 were reported to public health authorities, including 16 maternal-neonatal cases (14 pregnant women, and two babies born to asymptomatic mothers). The traceback of many brands of cheese that tested positive for LM P93 collected from retailers identified two cheese plants contaminated by L. monocytogenes strains on 3 and 4 September. PFGE profiles became available for both plants on 8 September, and confirmed that a single plant was associated with the outbreak. Products from these two plants were distributed to more than 300 retailers in the province, leading to extensive cross-contamination of retail stock. L. monocytogenes is ubiquitous, and contamination can occur subsequent to heat treatment, which usually precedes cheese production. Contaminated soft-textured cheese is particularly prone to bacterial growth. Ongoing regulatory and industry efforts are needed to decrease the presence of Listeria in foods, including pasteurized products. Retailers should be instructed about the risk of cross-contamination, even with soft pasteurized cheese and apply methods to avoid it.

Comparing rapid methods for detecting Listeria in seafood and environmental samples using the most probably number (MPN) technique

The standard Bacteriological Analytical Manual (BAM) protocol for detecting Listeria in food and on environmental surfaces takes about 96 h. Some studies indicate that rapid methods, which produce results within 48 h, may be as sensitive and accurate as the culture protocol. As they only give presence/absence results, it can be difficult to compare the accuracy of results generated. We used the Most Probable Number (MPN) technique to evaluate the performance and detection limits of six rapid kits for detecting Listeria in seafood and on an environmental surface compared with the standard protocol. Three seafood products and an environmental surface were inoculated with similar known cell concentrations of Listeria and analyzed according to the manufacturers' instructions. The MPN was estimated using the MPN-BAM spreadsheet. For the seafood products no differences were observed among the rapid kits and efficiency was similar to the BAM method. On the environmental surface the BAM protocol had a higher recovery rate (sensitivity) than any of the rapid kits tested. Clearview™, Reveal®, TECRA® and VIDAS® LDUO detected the cells but only at high concentrations (>10(2) CFU/10 cm(2)). Two kits (VIP™ and Petrifilm™) failed to detect 10(4) CFU/10 cm(2). The MPN method was a useful tool for comparing the results generated by these presence/absence test kits. There remains a need to develop a rapid and sensitive method for detecting Listeria in environmental samples that performs as well as the BAM protocol, since none of the rapid tests used in this study achieved a satisfactory result.

Modelling of growth, growth/no-growth interface and nonthermal inactivation areas of Listeria in foods

Growth, growth boundary and inactivation models have been extensively developed in predictive microbiology and are commonly applied in food research nowadays. Few studies though report the development of models which encompass all three areas together. A tiered modelling approach, based on the Gamma hypothesis, is proposed here to predict the behaviour of Listeria. Datasets of Listeria spp. behaviour in laboratory media, meat, dairy, seafood products and vegetables were collected from literature, unpublished sources and from the databases ComBase and Sym'Previus. The explanatory factors were temperature, pH, water activity, lactic and sorbic acids. For the growth part, 697 growth kinetic datasets were fitted. The estimated growth rates and 2021 additional growth primary datasets were used to fit the secondary growth models. In a second step, the fitted model was used to predict the growth/no-growth boundary. For the inactivation modelling phase, 535 inactivation curves were used. Gamma models with and without interactions between the explanatory factors were used for the growth and boundary models. The correct prediction percentage (predicted growth when growth is observed+predicted inactivation when inactivation is observed) varied from 62% to 81% for the models without interactions, and from 85% to 87% for the models with interactions. The median error for the predicted population size was less than 0.34 log(10)(CFU/mL) for all models. The kinetics of inactivation were fitted with modified Weibull primary models and the estimated bacterial resistance was then modelled as a function of the explanatory factors. The error for the predicted microbial population size was less than 0.71 log(10)(CFU/mL) with a median value of less than 0.21 for all foods. The model enables the quantification of the increase or decrease in the bacterial population for a given formulation or storage condition. It might also be used to optimise a food formulation or storage condition in the case of a targeted increase or decrease of the bacterial population.

Human heat-shock protein 60 receptor-coated paramagnetic beads show improved capture of Listeria monocytogenes in the presence of other Listeria in food

To investigate the suitability of human Hsp60, a receptor for Listeria adhesion protein (LAP), on paramagnetic beads (PMB) to capture Listeria monocytogenes from food in the presence of other Listeria to facilitate rapid and specific detection of this pathogen. Commercially available streptavidin-coated PMBs were linked with biotinylated Hsp60 (PMB-Hsp60), and the bacterial capture efficiency from pure culture and meat samples was determined. Capture rate was also compared with the monoclonal antibody (MAb)-C11E9-coated beads (PMB-C11E9) and the commercial Dynabeads anti-Listeria. Captured cells were detected and quantified by plating on selective medium, quantitative real-time PCR (qPCR) and a light-scattering sensor. Overall, all ligand-coated beads had similar capture efficiency (varied from 1·8 to 9·2%) for L. monocytogenes under the conditions employed, and the minimum cell number required to achieve such capture was 10³ CFU ml⁻¹. PMB-Hsp60 had significantly greater capture efficiency for pathogenic Listeria (P < 0·0001) than the nonpathogenic Listeria. In contrast, PMB-C11E9 and Dynabeads anti-Listeria had similar capture efficiency for both. The efficacy of all PMBs to capture L. monocytogenes in the presence of Listeria innocua from food matrices was compared. Although Dynabeads anti-Listeria had the overall best capture efficiency, PMB-Hsp60 was able to selectively capture L. monocytogenes even in the presence of 10-100-fold more L. innocua cells from enriched meat samples. Data show that the human cell receptor, Hsp60, is suitable for the capture of pathogenic Listeria on PMB in the presence of other Listeria in food. As pathogen interaction with host cells is highly specific, host cell receptors could be used as alternate capture molecules on PMB to aid in specific detection of pathogens.

Pregnancy-associated listeriosis

Infection by Listeria monocytogenes in pregnant women may result in fetal loss or invasive disease in the newborn. We examined listeriosis cases reported through the U.S. Listeria Initiative during 2004-2007. Cases were classified as pregnancy-associated if illness occurred in a pregnant woman or an infant aged <28 days. Of 758 reported Listeria cases, 128 (16.9%) were pregnancy-associated. Maternal infection resulted in four neonatal deaths and 26 (20.3%) fetal losses. Invasive illnesses in newborns (n=85) were meningitis (32.9%) and sepsis (36.5%). Pregnant women with Listeria were more likely to report Hispanic ethnicity (52.8% vs. 25.6%, respectively; OR 3.3 95% CI 2.2-4.8) than mothers giving live birth in the USA during 2005 and were more likely to report consumption of Mexican-style cheese (OR 2.6, 95% CI 1.6-4.2) than were non-pregnant patients with Listeria infection. Pregnant woman comprised a considerable proportion of reported listeriosis cases. Further declines in pregnancy-associated listeriosis will require education about avoiding high-risk foods, and continued regulatory and industry efforts to decrease Listeria in foods.

Comparative Validation Study to Demonstrate the Equivalence of a Minor Modification to AOAC Method 996.09 Visual Immunoprecipitate (VIP®) for E. coli O157:H7 Method to the Reference Culture Method

The Visual Immunoprecipitate (VIP) for the Detection of Listeria in Foods and Environmental Surfaces, AOAC Official Method 997.03, has been modified to change the color of the test and control lines of the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Two food matrixes and one environmental surface were analyzed. In total, there were valid results from 100 samples and controls. Results showed that the modified VIP Gold for Listeria spp. is equivalent to the reference culture methods for the detection of Listeria.

Comparative Validation Study to Demonstrate the Equivalence of a Minor Modification to AOAC Method 997.03 Visual Immunoprecipitate (VIP®) for Listeria to the Reference Culture Method

Abstract The Visual Immunoprecipitate (VIP®) for the Detection of Listeria in Foods and Environmental Surfaces, AOAC Official Method 997.03, has been modified to change the color of the test and control lines of the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Two food matrixes and one environmental surface were analyzed. In total, there were valid results from 100 samples and controls. Results showed that the modified VIP Gold® for Listeria spp. is equivalent to the reference culture methods for the detection of Listeria.

Comparative Validation Study to Demonstrate the Equivalence of a Minor Modification to AOAC Method 997.03 [Visual Immunoprecipitate (VIP®) for Listeria] to the Reference Culture Methods

The Visual Immunoprecipitate (VIP) for the Detection of Listeria in Foods and Environmental Surfaces, AOAC Official Method 997.03, has been modified to use a simplified housing for the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture methods. Two food matrixes and one environmental surface were analyzed. In total, valid results were obtained from 145 samples and controls. Results showed that the modified VIP for Listeria spp. is equivalent to the reference culture methods for the detection of Listeria.

Validation Study to Demonstrate the Equivalence of a Minor Modification of Assurance Enzyme Immunoassay Method, Official Method 996.14, for Detection of Listeria in Foods and Environmental Surfaces to the Reference Culture Methods

The Assurance Enzyme Immunoassay (EIA) for the Detection of Listeria in Foods and Environmental Surfaces, AOAC Official Method 996.14, has been modified to combine the separate antibody and conjugate addition steps into one. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture methods. Two food matrixes and one environmental surface were analyzed. In total, there were valid results from 145 samples and controls. Results showed that the Assurance EIA for Listeria spp. is equivalent to the reference culture methods for the detection of Listeria.