Listeria Monocytogenes In Foods Research Articles

Survey of Listeria monocytogenes in Ready-to-Eat Foods

The purpose of this study was to develop data on the risk of listeriosis to support a science-based strategy for addressing Listeria monocytogenes in foods in the United States. Eight categories of ready-to-eat foods were collected over 14 to 23 months from retail markets at Maryland and northern California FoodNet sites. The product categories included luncheon meats, deli salads, fresh soft “Hispanic-style” cheeses, bagged salads, blue-veined and soft mold-ripened cheeses, smoked seafood, and seafood salads. The presence and levels of L. monocytogenes in the samples were determined by rapid DNA-based assays in combination with culture methods. Of 31,705 samples tested, 577 were positive. The overall prevalence was 1.82%, with prevalences ranging from 0.17 to 4.7% among the product categories. L. monocytogenes levels in the positive samples varied from <0.3 MPN (most probable number) per g to 1.5 × 105 CFU/g, with 402 samples having levels of <0.3 MPN/g, 21 samples having levels of >102 CFU/g, and the rest of the samples having intermediate levels. No obvious trends with respect to seasonality were observed. Significant differences (P < 0.05) between the sampling sites were found, with higher prevalences for threes categories in northern California and for two categories in Maryland. Significantly (P < 0.001) higher prevalences were found for in-store-packaged samples than for manufacturer-packaged samples of luncheon meats, deli salads, and seafood salads, while 16 of the 21 samples with higher counts were manufacturer packaged. The data collected in this study help to fill gaps in the knowledge about the occurrence of L. monocytogenes in foods, and this new information should be useful in the assessment of the risk posed by L. monocytogenes to consumers.

Application of the Polymerase Chain Reaction for Detection of Listeria monocytogenes in Foods: A Review of Methodology

The polymerase chain reaction (PCR) as applied to the detection of Listeria monocytogenes in foods encompasses a variety of cell extraction, cell lysis, and DNA purification techniques. These techniques have been applied to foods directly and to enrichment broths with wide variations in sensitivity reported by numerous investigators. The selection of the specific gene for amplification of a selected sequence is also important in that some genes associated with pathogenicity have been found to be present in certain of the other species of Listeria. This review constitutes a detailed presentation of all such factors along with the tabulation of primer sequences, the genes from which they are derived, and the size of the amplified PCR products, in addition to descriptions of the use of nested primers and multiplex PCR.

Detection of Listeria monocytogenes in food, equivalent to EN ISO 11290-1 or ISO 10560, by a three-days polymerase chain reaction-based method

A method is presented for the detection of Listeria monocytogenes in food, which produces definitive results on the third day after the sample collection. The method is equivalent to EN ISO 11290-1 or ISO 10560 in terms of the same detection limit of 10 0 cfu per 25 or 10 g and 100% relative accuracy. The version alternative to EN ISO 11290-1 begins with a primary enrichment in half Fraser broth (24 h), secondary enrichment in Fraser broth (24 h) and post-enrichment in brain heart infusion broth (5 h). The version alternative to ISO 10560 begins with enrichment in Listeria enrichment broth (48 h) and post-enrichment in tryptone soya broth (5 h). These steps are followed by bacterial cell lysis by boiling, PCR oriented to inlB gene using a mimic internal control, and agarose gel electrophoresis. At the evaluation of the method on model food samples artificially contaminated with decimal dilutions of a L. monocytogenes culture (cheese, smoked fish, ready-to-eat meat products; 21 samples altogether), a detection limit of 10 0 cfu per 25 or 10 g was determined. Dead L. monocytogenes cells did not cause false positivity, as determined using model food samples artificially contaminated with decimal dilutions of dead L. monocytogenes cells. At the evaluation of the method on naturally contaminated food samples (same as above, 140 samples altogether) identical results (8 positives) as with the reference method were obtained.

Incidence of Listeria monocytogenes in food and environmental samples in Italy between 1990 and 1999: serotype distribution in food, environmental and clinical samples.

We report the findings of the study of 4185 food samples and 958 environmental samples collected in Italy in the period 1990-1999 and tested for the presence of Listeria monocytogenes. The strains isolated were biochemically and serologically characterised. We found a fairly high percentage of L. monocytogenes contamination in food (12.8%), whereas the level of contamination was lower in the environment (environment and work surfaces in food processing plants) (6.1%). Serotyping showed a prevalence of a few serotypes (i.e., 1/2a, 1/2b, 1/2c and 4b), which were the same as those found in clinical samples collected during outbreaks and from sporadic cases of listeriosis reported in Italy in the period considered. The geographical distribution of the strains of L. monocytogenes isolated from food samples is very similar to that of the clinical strains.

Polymerase Chain Reaction-Based Methods for Detection of Listeria monocytogenes: Toward Real-Time Screening for Food and Environmental Samples

A review is presented of nucleic acid amplification-based methodology, specifically polymerase chain reaction (PCR)-based assays, for the detection of Listeria monocytogenes in food and environmental samples. Until recently, developmental challenges including poor sensitivity, due in part to reaction inhibition by components of the sample matrix, and the potential for false-positive reactions have limited routine application of PCR-based screening assays. Commercial assays address these challenges while offering convenient, standardized protocols, a high level of automation, and results within 2 days after the sampling date. Although sample enrichment is necessary to achieve desired detection limits, continued efforts toward template purification will facilitate the development of assays offering real-time, quantitative results. The development of ribonucleic acid (RNA) amplification-based assays may increase in importance, particularly if end-product testing is prioritized by regulatory agencies, as messenger RNA appears to serve as an accurate indicator of cell viability. Further, the increase in target copy number may improve assay sensitivity. PCR-based screening methods offer efficient, reliable results and are ideal for monitoring the presence of L. monocytogenes in foods and in the food processing environment.

On the Specificity of PCR Detection of Listeria monocytogenes in Food: a Comparison of Published Primers

A total of nine pairs of primers, seven previously published and two newly developed, have been assayed for PCR detection of Listeria monocytogenes in food. They have been tested for specificity on a total of 72 strains including reference and food isolates belonging to L. monocytogenes and other species in the genus. First of all, a polyphasic approach has been carried out in order to establish a reference strain collection. They were biochemically and genetically characterized by API-Lis and randomly amplified polymorphic DNA PCR (RAPD-PCR), respectively. Random amplification of DNA was performed with M13, T7 and T3 universal primers and a data bank was created to compile the RAPD patterns of all the analyzed strains. The UPGMA cluster analysis of RAPD profiles with primer M13 showed eight clusters at 72.3% similarity. Clusters 2 and 7 corresponded to L. monocytogenes. Clusters 1 and 6 grouped L. ivanovii strains. Clusters 3, 4, 5 and 8 corresponded to L. grayi, L. innocua, L. welshimeri and L. seeligeri, respectively. Pattern analysis revealed the existence of miss-identified reference strains which was confirmed by 16S rDNA sequence analysis. RAPD-PCR is a rapid genetic test which helped to confirm strain identity. On the basis of PCR specificity results, primers LM1-LM2 were the best combination for the detection of L. monocytogenes since they only amplified the specific fragment in strains that had been genetically and biochemically assessed as belonging to the species. Specificity of other assayed primers is discussed.

PCR Detection of Listeria monocytogenes in Milk and Milk Products and Differentiation of Suspect Isolates

Holko I., J. Urbanova, M. Kantikova, K. Pastorova, V. KmeE: PCR Detection of Listeria monocytogenes in Milk and Milk Products and Differentiation of Suspect Isolates. Acta Vet. Brno 71, 2002: 125-131. The main goal of our work was to solve the problems of normative diagnostic method for the detection of Listeria monocytogenes in foods by STN ISO 10560 (1999), i.e. to shorten the long time of analysis (5-10 days) and differentiate the non-haemolysed strains of Listeria monocytogenes from non-pathogenic Listeria innocua yielding the same results of biochemical and serological typing (serotype 4ab). PCR was employed to confirm the method for testing 60 suspect isolates from other laboratories of the Slovak Republic. We verified the detection of Listeria monocytogenes in milk and dairy products by nested PCR. The objective of this experiment was to analyze 100 various samples by traditional cultivation method and compare the results with those yielded by PCR. The results were in good agreement; all 18 positive and 72 negative samples were detected by both methods. However, the PCR method yields results within 2 days. This makes diagnosis in food control laboratories much more efficient. For PCR detection, two pairs of primers (PRFA 1 and 2, LIP 1 and 2) were used with affinity to prfA gene involved in the regulation of listeriolysin synthesis. The size of the PCR product was 1060 bp fragment in a first step of PCR and 273 bp fragment in nested PCR. The sensitivity and reliability of PCR was comparable with conventional methods. The PCR method solved the problem of interpretation of classical biochemical and serological typing in the only one step without the necessity of using additional examinations. Furthermore, we found that in most of the strains isolated from foods with a biochemical profile of non-haemolytic Listeria monocytogenes on blood agar serotype 4ab were not Listeria monocytogenes but a non-pathogenic Listeria innocua. This finding is very important from the point of view of food evaluation according to the STN ISO 10560 (1999). Diagnostic methods, food, Listeria innocua, non-haemolysed strains of Listeria monocytogenes, prfA gene Bacteria of the genus Listeria are widely distributed in the environment and they frequently contaminate food. Listeria monocytogenes is a pathogenic species causing listeriosis, increasing incidence of which has been reported in the last two decades. Food products most frequently associated with listeriosis are soft cheeses, particularly those made from unpasteurized milk, and ready-to-eat meat-containing food products (Farber and

Occurrence of Listeria monocytogenes in food in Chile

Out of 2145 food samples analysed 77 were found contaminated with Listeria monocytogenes in Santiago, Chile. Samples were: 603 ice-cream (3.5% contaminated), 256 soft cheese (0.8%), 155 hard cheese (0%), 229 baby milk bottles (0%), 634 processed meat products (3.6%) and 268 crustaceous shellfish (11.6%). Three different isolation media were used: for 318 samples, Modified McBride Agar (MMA), Lithium chloride Phenylethanol Moxalactam agar, and Polymyxin Acriflavine Lithium chloride Ceftazidime Aesculin Mannitol agar; for 1827 samples MMA was replaced by Listeria Selective Agar Oxford Formulation. Isolates were classified as follow: serovar 1/2a (25 isolates), serovar 4b (20), serovar 1/2b (19), serovar 3b (7), serovar 1/2c (2), untypable (4). A high variety of phagovars was detected although 52% of strains was untypable.

Rapid detection of Listeria monocytogenes in foods, by a combination of PCR and DNA probe

Listeria monocytogenes is a frequent contaminant of water and foods. Its rapid detection is needed before some foods can be prepared for marketing. In this work L. monocytogenes has been searched for in foods, by a combination of polymerase chain reaction (PCR) and a DNA probe. Both PCR and the probe were prepared for recognizing a specific region of the internalin gene, which is responsible for the production of one of the most important pathogenic factors of Listeria. The combined use of PCR and the DNA probe was used for the detection of L. monocytogenes in over 180 environmental and food samples. Several detection methods were compared in this study, namely conventional culture methods; direct PCR; PCR after an enrichment step; a DNA probe alone; a DNA probe after enrichment and another commercially available gene-probe. Finally PCR and the DNA probe were used in series on all the samples collected. When the DNA probe was associated with the PCR, specific and accurate detection of listeria in the samples could be obtained in about a working-day. The present molecular method showed some advantages in terms of rapidity and specificity in comparison to the other aforementioned tests. In addition, it resulted as being easy to handle, even for non-specialized personnel in small diagnostic microbiology laboratories.

Validation of ISO method 11290 Part 1 — Detection of Listeria monocytogenes in foods

The European and International Standard method for the detection of Listeria monocytogenes, described in EN ISO 11290 Part 1: 1997 (International Organisation for Standardisation, Geneva) was validated by order of the European Commission (Standards, Measurement and Testing Fourth Framework Programme Project SMT4-CT96-2098). Nineteen laboratories in 14 countries in Europe participated in a collaborative trial to determine the performance characteristics of the method, which are intended for publication in the corresponding standard. An additional objective of this project was to devise a new series of parameters to indicate the ‘precision’ of microbiological qualitative methods. The method was challenged with three food types, namely fresh cheese, minced beef and dried egg powder and a reference material. Inoculation levels ranged from 5 to 100 cfu/25 g. Each participant examined five replicates of each food type at three inoculum levels and five reference materials. Both PALCAM and Oxford media were assessed. All test materials were subjected to stringent homogeneity and stability testing before being used in the collaborative trial. The results demonstrated that the method prescribed in EN ISO 11290-1 had an overall sensitivity of 85.6% and a specificity of 97.4%. L. monocytogenes was detected in most cases after primary enrichment, although secondary enrichment often yielded further positives. However, a significant number of false-negative results were obtained with all food types when large numbers of L. innocua were present in the test materials. L. innocua tended to dominate L. monocytogenes during the selective enrichment stages and thus masked small numbers of colonies of L. monocytogenes on the isolation media. There was no evidence from this collaborative study to demonstrate a significant difference in performance between Oxford and PALCAM media. Due to the problem of false-negative results with this method as highlighted in this trial, recommendations have been made to ISO to launch a revision of the standard to improve the detection of low numbers of L. monocytogenes in foods. New statistical methods devised to advance the measurement of the performance of qualitative microbiological methods are also described.

Microbiological criteria for Listeria monocytogenes in foods under special consideration of risk assessment approaches

This paper shortly summarizes data related to risk assessment of Listeria monocytogenes. From available data on risk assessment, it is concluded that the levels of L. monocytogenes consumed is an important factor affecting the incidence of listeriosis. Foods that do not support the growth of L. monocytogenes are unlikely to be a source of listeriosis, whereas foods that support the growth to high levels, should be the target of risk management efforts. Based on current epidemiological information from several countries, a concentration of L. monocytogenes not exceeding 100/g of food at the time of consumption is of low risk to the consumers. In order not to exceed these levels at the point of consumption, lower levels may need to be applied at the port of entry, for those foods in which growth can occur within the shelf life. In order to establish such levels, knowledge of the shelf life and behaviour of L. monocytogenes in the food during prevailing storage and distribution conditions is needed.

Rapid Polymerase Chain Reaction/DNA Probe Membrane-Based Assay for the Detection of Listeria and Listeria monocytogenes in Food

We describe the development of polymerase chain reaction (PCR)/DNA probe membrane-based colorimetric assays for the detection and identification of Listeria and L. monocytogenes. PCR primers designed from the 16S to 23S rRNA intergenic spacer region amplified products that were reverse hybridized to membrane-bound oligonucleotide probes specific for Listeria and L. monocytogenes with a detection limit of 1 to 10 CFU/25 ml in inoculated raw and pasteurized milk samples. These qualitative assays have the potential to be integrated into testing laboratories for monitoring the microbiological quality of foods.

A new method for rapid detection of Listeria monocytogenes in food

Listeria monocytogenes represents serious danger for human health. Thus detection of this pathogen in food, which represents its main means of entry into the organism, is a topic of special importance. The original classic methods for the determination of Listeria monocytogenes are in general laborious and time-consuming procedures. In order to address this issue we developed a new rapid method for specific detection of Listeria monocytogenes in food samples. The method consists of three steps: i) enrichment of food microflora (16 h), ii) selective isolation of Listeria sp. exploiting immunomagnetic separation (2–3 h) followed by iii) precise identification of Listeria sp. and Listeria monocytogenes using duplex PCR. PCR primers specific to part of 16S rRNA were used in order to identify the members of Listeria genus. The specific identification of Listeria monocytogenes was accomplished exploiting a pair of primers specific to gene encoding invasion-associated protein – iap (4–5 h). Amplification products, 1003 bp and 593 bp respectively, were separated by electrophoresis and visualized by UV detection. The optimized IMS-PCR method was used to test the presence of Listeria sp. and Listeria monocytogenes in food samples (ground meat, low-fat milk and cheese [olomoucké tvarůžky]). A comparison of the efficiency of the bacteria enrichment step by IMS and centrifugation was also performed. The analysis time including enrichment is less than 24 h. The detection limit for Listeria monocytogenes was found between 101–102 cfu per 25 g of food sample.

Incidence and control of Listeria monocytogenes in foods in Denmark

The Danish regulatory policy on Listeria monocytogenes in foods is based on the principles of HACCP and was developed using a health risk assessment approach. The Danish policy focuses examinations and criteria for L. monocytogenes in ready-to-eat foods and is based on a combination of inspection and product-testing. Based on current epidemiological information from several countries, a concentration of L. monocytogenes not exceeding 100 cfu/g of food at the time of consumption, seems to be of low risk to the consumers. In Denmark, ready-to-eat foods have been placed into six categories where absence of L. monocytogenes in 25 g is required in foods heat treated in the final package and in heat-treated as well as preserved, non heat-treated foods which can support growth within the shelf life. This level is necessary in foods capable of supporting growth, in order not to exceed 100 L. monocytogenes per g at the point of consumption. In heat-treated and preserved foods, which are not supportive of growth within the shelf-life and for raw, ready to eat foods, a level below 10 L. monocytogenes per g is regarded acceptable. A level between 10 and 100 L. monocytogenes per g is not satisfactory and a level above 100/g is not acceptable. Data on the qualitative and quantitative occurrence of L. monocytogenes in foods in Denmark are presented and discussed. In 1997 and 1998, greater than 15,000 samples from different categories of food were examined (semi-quantitatively) for the presence of L. monocytogenes. A significant difference could be seen in the number of samples containing more than 100 L. monocytogenes per g, between different categories of foods (1997, P=0.001; 1998, P=0.016). In 1997, preserved meat products and preserved fish products and to a lesser extent vegetables and meat or vegetable mayonnaise were more likely to contain high numbers (i.e. above 100 cfu/g) of L. monocytogenes than other food categories. In 1998, preserved meat products, but also heat-treated meat products, vegetables and meat or vegetable mayonnaise had the highest frequency of samples with >100 L. monocytogenes per g. In a survey performed in 1994 and 1995, 1.3% of ready-to-eat food samples (heat-treated meat products, preserved meat and fish products) were found to be contaminated with L. monocytogenes at a level above 100 cfu/g. The samples included in this survey were primarily products produced by authorized companies and were comprised mainly of vacuum packed products or products packed in modified atmosphere and with long shelf lives, typically above several weeks. The corresponding percentages of positive samples primarily processed in the retail outlets (heat-treated meat products, preserved meat and fish products) in 1997 and 1998 were 0.3% and 0.6%, respectively. The results suggest that ready-to-eat meat and fish products with extended shelf-lives produced by authorized companies are more likely to contain high numbers (>100 cfu/g) of L. monocytogenes than products processed in the retail sector which often have a shorter shelf life.

Simple method to observe the adaptive response of Listeria monocytogenes in food.

A simple, novel method for determining stress-adaptive response of Listeria monocytogenes in food systems is presented. The method involves plating samples on Listeria-selective agar (LSA) acidified to pH 5.25 with incubation at 36 degrees C for 60 h to detect acid adaptation and plating on LSA with 70 gl-1 NaCl and incubation at 7 degrees C for 7 d to detect cold-osmotic adaptation. Adapted cells produced larger colonies (> 1 mm) under these conditions than unadapted cells. Scot A (97%) and Brie-1 (100%) cells incubated in milk at pH 5 for 3 h manifested the acid-adapted colony type compared with 6% and 21% of viable cells in the unstressed control population. After a 5-d adaptation period at 4 degrees C in milk with 80 gl-1 salt, 29% of Scot A and 91% of Brie-1 viable cells exhibited the adapted colony type compared with < 1% of the unstressed control population. Stress-adapted L. monocytogenes were isolated from soft cheese held for 42 d at 10 C.

A rapid method for the identification and partial serotyping of Listeria monocytogenes in food by PCR and restriction enzyme analysis

Two highly specific primers for Listeria monocytogenes were used to yield from foods such as milk, soft cheese and meat, PCR products that were cleaved with the restriction enzyme HindIII. The fragments generated allowed a distinction between two groups of L. monocytogenes serovars: serovars 1/2a and 1/2c cluster in one group and serovars 1/2b, 3b and 4b in the other subgroup. Since this procedure can be completed in 24 h, an epidemiological association between human disease and suspected sources can be rapidly confirmed at the subgroup level in the laboratory.

Genetic typing of human and food isolates of Listeria monocytogenes from episodes of listeriosis.

Ten clinical and food Listeria monocytogenes strains isolated during the epidemiological investigations of episodes of listeriosis (one outbreak and two sporadic cases) that occurred in northern Italy during 1993-1995 have been examined by DNA macrorestriction pattern analysis obtained by PFGE and RAPD typing, in order to confirm the food vehicle of infections. The same DNA profiles within the isolates from the three episodes were obtained by both techniques. The Apal and Smal PFGE profiles and RAPD patterns with primer OPM-01 confirmed the close relationship between strains from two distinct episodes. However, RAPD analysis with primer UBC-127 distinguished between these L. monocytogenes isolates.

Listeriolysin O-Based Latex Agglutination Test for the Rapid Detection of Listeria monocytogenes in Foods

A latex agglutination-based test for the rapid detection of Listeria monocytogenes in foods was developed. An antilisteriolysin O (LLO) monoclonal antibody (HID5E12D7; IgG2b) covalently bound to polystyrene amidine-modified latex beads was used in a slide agglutination assay. The latex reagent detected 0.1 ng/ml of LLO in phosphate-buffered saline plus bovine serum albumin. It reacted with culture supernatants of L. monocytogenes but not with other Listeria species or Streptococcus groups A through G. The listeriolysin O latex agglutination assay (LLOLAT) was applied to 24-h and 48-h USDA primary enrichment cultures of 208 food samples obtained from refrigerators of listeriosis patients enrolled in a study to determine the role of foods in sporadic listeriosis. Of 19 samples positive by cultural techniques, 17 were positive by the LLOLAT. Cultures with low (<0.3 CFU/g) levels of L. monocytogenes were positive in the LLOLAT. No cross-reactivity occurred when using a heterogeneous monoclonal antibody. The LLOLAT is a sensitive, specific and rapid test and may be useful for screening foods for L. monocytogenes.

Prevalence of Listeria monocytogenes in foods: Incidence in dairy products

Listeria monocytogenes is ubiquitous and is recognized as an important foodborne pathogen. Epidemiological evidence and outbreaks have pointed to dairy products as alleged vehicles for cases of listeriosis. Incidents involving dairy products resulted in a number of surveillance programmes, the discovery of this pathogen in contaminated dairy products, and numerous product recalls. Results of raw milk sampling surveys indicate only 3–4% of the raw milk supply can be expected to contain Listeria species. These surveys have also shown that the levels in most samples are low (less than 10 cfu/ml). As pasteurization effectively destroys the Listeria organism, the presence of Listeria in finished product must be the result of post-pasteurization contamination from environmental sources in the plant. There are a number of mitigating factors in both product characteristics and processing which have an impact on the organism's ability to survive and multiply. Results of finished product sampling surveys, such as those conducted for frozen products (ie, ice cream), show low numbers of the organism (less than 15 cfu/g) in contaminated samples. In fact, no clinical evidence exists which associates frozen dairy products with listeriosis outbreaks. A number of preventive measures specifically targeted to those areas of likely exposure have been developed and implemented in the frozen dessert industry.

US position on Listeria monocytogenes in foods

Public health and regulatory agencies in the USA have established a zero tolerance for Listeria monocytogenes in cooked, ready-to-eat food. This policy is science-based: the organism causes human illness, can grow at refrigeration temperatures and the infectious dose is unknown. The US response to listeriosis has included emphasis on Good Manufacturing Practices and Hazard Analysis Critical Control Point systems for processing, recommendations for sanitation and other measures for retail handling, and consumer education targeted to populations at highest risk for listeriosis.