Enumeration Of Listeria Monocytogenes Research Articles

Microbiological Risks of Traditional Raw Cow's Milk Cheese (Koryciński Cheeses).

Traditional and regional foods have been increasing in popularity among consumers in Poland for many years. The observed trend of searching for natural and authentic taste encourages many producers to craft products from raw milk, including Koryciński cheeses. The aim of this study was to assess the microbiological hazards resulting from the presence of pathogenic bacteria in Koryciński cheeses available in retail trade. The tests were carried out using accredited methods, including the detection of the presence of Salmonella spp., the enumeration of Listeria monocytogenes, the enumeration of coagulase-positive staphylococci, and the detection of staphylococcal enterotoxins in food when the number of coagulase-positive staphylococci in the sample exceeded the limit of 105 cfu/g. The research material consisted of 45 Koryciński cheeses. The tests conducted revealed that Salmonella spp. was not detected in any of the examined cheeses. However, coagulase-positive staphylococci were present in 68.9% of the samples. In as many as 15 tested cheeses, the level of S. aureus contamination was above 105 cfu/g; therefore, these samples were tested for the presence of staphylococcal enterotoxins. The presence of staphylococcal enterotoxins was found in one Koryciński cheese. In four cheeses, the number of L. monocytogenes exceeded the level of 102 cfu/g, the limit specified in Regulation 2073/2005 on microbiological criteria for foodstuffs. The obtained research results confirm the validity of monitoring the microbiological quality of Koryciński cheeses and the need to increase awareness of ensuring proper hygienic conditions of production, including the increased risk associated with unpasteurized milk products.

Evaluation of the Thermo Scientific SureTectTM Listeria Species PCR Assay in a Broad Range of Foods and Selected Environmental Surfaces: Pre-Collaborative and Collaborative Study, First Action 2021.06.

BackgroundThe Thermo Scientific SureTect™ Listeria species PCR assay utilizes SolarisTM reagents for performing PCR for the rapid and specific detection of Listeria species in a broad range of foods and selected environmental surfaces.ObjectiveTo demonstrate reproducibility of the Thermo Scientific SureTect Listeria species PCR assay in a collaborative study using a challenging matrix, full-fat cottage cheese (25 g), to extend the scope of the method.MethodsIn the collaborative study, the candidate method was compared to the US Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) Ch. 10 Listeria reference method. The candidate method used two PCR thermocyclers, the Applied Biosystems QuantStudio™ 5 Real-Time PCR instrument (QS5) and the Applied Biosystems 7500 Fast Real-Time PCR instrument (7500 Fast). The candidate method included its own confirmation procedure. Eighteen participants from 10 laboratories located within the United States and Europe were solicited for the collaborative study, with 12 participants submitting valid data. Statistical analysis was conducted according to the probability of detection (POD) statistical model. In addition, in order to extend the scope of the method, seven matrix studies were performed comparing the candidate method to the FDA/BAM reference method. One of these matrixes was also compared to the ISO 11290–1:2017 Microbiology of the food chain—Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp.—Part 1: Detection method reference method.ResultsIn the collaborative study, the difference in laboratory results indicates equivalence between the candidate method and reference method for the matrix evaluated and the method demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. The two PCR instruments used in the study performed equivalently. All presumptive positives were confirmed via the alternative confirmation procedure. In the pre-collaborative studies, the results showed comparable performances between the candidate method and the reference method for all matrixes tested.ConclusionBased on the data generated, the method demonstrated acceptable inter-laboratory reproducibility data and statistical analysis.HighlightsDue to the COVID-19 pandemic, some participants had to be trained remotely. Additionally, 25 g full-fat cottage cheese is known to be a challenging matrix to test. No unusual cross-contamination, or false-positive/negative data was reported, highlighting the ease of use, reproducibility, and robustness of the candidate method.

Evaluation of the Thermo ScientificTM SureTectTMListeria monocytogenes PCR Assay in a Broad Range of Foods and Selected Environmental Surfaces: Pre-Collaborative and Collaborative Study, First Action 2021.05.

BackgroundThe Thermo Scientific™ SureTect™ Listeria monocytogenes PCR Assay uses Solaris reagents for performing PCR for the rapid and specific detection of Listeria monocytogenes in a broad range of foods and selected environmental surfaces.ObjectiveTo demonstrate reproducibility of the SureTect Listeria monocytogenes PCR Assay in a collaborative study using a challenging matrix, full-fat cottage cheese (25 g). To extend the scope of the method.MethodIn the collaborative study, the candidate method was compared to the United States Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) Chapter 10 Listeria reference method. The candidate method used two PCR thermocyclers, the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR instrument (QS5) and the Applied Biosystems 7500 Fast Real-Time PCR instrument (7500 Fast). Eighteen participants from 10 laboratories located within the United States and Europe were solicited for the collaborative study, with 12 participants submitting valid data. Three levels of contamination were evaluated for each matrix. Statistical analysis was conducted according to the probability of detection (POD) statistical model. In addition, to extend the scope, six matrix studies were performed comparing the candidate method to the FDA/BAM reference method. One of these matrixes was also compared to the ISO 11290–1:2017 Microbiology of the Food Chain—Horizontal Method for the Detection and Enumeration of Listeria monocytogenes and of Listeria spp.—Part 1: Detection Method Reference Method.ResultsIn the collaborative study, the difference in laboratory results indicates equivalence between the candidate method and reference method for the matrix evaluated, and the method demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. The two PCR instruments used in the study performed equivalently. All presumptive positives were confirmed via the alternative confirmation procedure. In the pre-collaborative studies, the results showed comparable performances between the candidate method and the reference method for all matrixes tested.ConclusionsBased on the data generated, the method demonstrated acceptable inter-laboratory reproducibility data and statistical analysis.HighlightsDue to the COVID-19 pandemic, some participants had to be trained remotely. Additionally, 25 g full-fat cottage cheese is known to be a challenging matrix to test. No unusual cross-contamination or false positive/negative data were reported, highlighting the ease of use, reproducibility, and robustness of the method.

Validation of N-LightTM L. monocytogenes for Detection of Listeria monocytogenes on Environmental Surfaces: AOAC Performance Tested MethodSM 122002.

The NEMIS Technologies N-LightTM L. monocytogenes assay uses chemiluminescence designed for the qualitative detection of Listeria monocytogenes from environmental surface samples. To validate the NEMIS Technologies N-Light L. monocytogenes assay as part of the AOAC Performance Tested MethodSM program for the detection of L. monocytogenes on stainless steel, plastic (polystyrene), and ceramic environmental surfaces. Using the Vitl Life Science Solutions Lu-mini luminometer, the NEMIS Technologies N-Light L. monocytogenes assay was compared to the ISO 11290-1:2017: Microbiology of the Food Chain-Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. - Part 1 using a 1" × 1" stainless steel test area in an unpaired study design. The NEMIS Technologies N-Light L. monocytogenes assay using the Vitl Life Science Solutions Lu-mini luminometer demonstrated no statistically significant differences between presumptive and confirmed results or between candidate and reference method results. Data for additional Performance Tested MethodSM studies met acceptance criteria requirements. The NEMIS Technologies N-Light L. monocytogenes assay is an effective method for the qualitative detection of L. monocytogenes from stainless steel, plastic (polystyrene), and ceramic environmental surface samples. The NEMIS method is the first chemiluminescence detection system based on a novel, patented, dioxetane compound.

Comparative Evaluation of the SMARTCHECK® Listeria monocytogenes Detection Kit for GENECHECKER® UF-300 Real-Time PCR System for Select Foods: AOAC Performance Tested MethodSM 042101.

The candidate method, SMARTCHEK® Listeria monocytogenes Detection Kit, is designed for rapid detection of L. monocytogenes in select food matrixes, for use in conjunction with the Genesystems Rapi:chipTM and GENECHECKER®UF-300 real-time PCR system. The independent laboratory evaluation consisted of an inclusivity and exclusivity study, product consistency study, robustness study, stability study, and a matrix study. The paired matrix study tested stainless-steel environmental sponges, uncured ready-to-eat deli turkey, ready-to-eat deli ham, cooked shrimp, and fresh bagged spinach. The robustness study evaluated the method by changing three parameters. The accelerated stability and product consistency study was conducted at 8, 16, 24, 32, and 40 days. The candidate method was compared to the US Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook, 8.11 Isolation and Identification of Listeria monocytogenes from Red Meat, Poultry, Ready-to-Eat Siluriformes (Fish) and Egg Products, and Environmental Samples and to the US Food and Drug Administration Bacteriological Analytical Manual Chapter 10: Detection of Listeria monocytogenes in foods and Environmental Samples, and Enumeration of Listeria monocytogenes in Foods following a paired study design. All 50 L. monocytogenes isolates were detected in all three broths tested for inclusivity, while all 37 non-L. monocytogenes isolates were correctly excluded. In the matrix study, the candidate method showed no significant differences between confirmed candidate method results and reference method results for all food matrixes evaluated. The candidate method is an effective method for the qualitative detection of L. monocytogenes in select food matrixes. The SMARTCHECK Listeria monocytogenes Detection Kit assay allows for fast, reliable detection of L. monocytogenes with results obtained in approximately 35 min after 18-48 h pre-enrichment, depending on the matrix.

Microbiological quality of fresh vegetable salads from the Novi Sad market

Increased consumption, big production units and very efficient delivery service networks of fresh vegetables and salads caused a higher number of foodborne illnesses in the last two decades all over the world. In this study 15 fresh vegetable salads from different marketplaces in Novi Sad were tested by reference microbiological methods (detection of Salmonella spp., enumeration of Listeria monocytogenes, mesophilic aerobic bacteria, yeasts and molds, sporogenic mesophilic aerobic bacteria, sporogenic sulfite-reducing bacteria, Bacillus cereus, Enterobacteriaceae, Escherichia coli and coagulase positive staphylococci). Salmonella spp. was found in one sample which, according to the safety criteria for such a product (if treated as ready-to-eat), was considered to be microbiologically unsafe. The number of Listeria monocytogenes in all tested samples was <100 cfu/g, which is a satisfactory result according to the safety criteria. E. coli was detected in only one sample, and the number of Enterobacteriaceae was in the range from 4.4 to 6.9 log CFU/g. The isolated Enterobacteriaceae strains were identified as Enterobacter cloacae, Klebsiella pneumoniae ssp. pneumoniae and Citrobacter spp.

Identifikasi Listeria monocytogenes pada Susu Kambing di Kabupaten Purworejo Jawa Tengah

Listeria monocytogenes is a pathogenic Listeria species, especially for high-risk groups and it that can be transmitted through contaminated food. Goat milk produced by traditional milking process and storage has a high risk of contamination. The aim of this study was to identify the presence of L. monocytogenes in fresh goat milk in Puworejo regency, Central Java. This study used 60 samples of raw goat milk that were obtained from seven farms by disease detection sampling method. All of the used method in this research refer to Indonesian National Standard (SNI) ISO 11290-1: 2012 about Microbiology of food and feed for detection and enumeration of Listeria monocytogenes. A total of 60 samples of raw goat milk that used in this study were not contaminatedwith L. monocytogenes. Based on the results of this study, it can be concluded that all the samples of raw goat milk were free from L. monocytogenes and have fulfilled the Indonesian National Standard (SNI) ISO No. 7388: 2009 about Limit of Microbial Contamination in Food.

Listeria monocytogenes in ready-to-eat foods in Italy: Prevalence of contamination at retail and characterisation of strains from meat products and cheese

In the framework of a European Union (EU) Coordinated Monitoring Programme, different types of ready-to eat (RTE) products, including soft and semi-soft cheese (n = 398) and cooked meat products (n = 403), were collected at retail in Italy and tested for detection and enumeration of Listeria monocytogenes. An Integrative Survey of 2696 samples, including soft and semi-soft cheese (n = 894) and cooked meat products (n = 1802) was carried out to have statistically representative results at the national level. Considering the results obtained both from the EU and the national Integrative Survey, prevalence of contamination of meat products was 1.66% (95% CL: 1.02–2.73%) at the arrival of the samples at the laboratory and 1.92% (95% CL: 1.31%–2.82%) at the end of shelf-life. Spalla cotta was the most frequently contaminated meat product. Prevalence of contamination in cheese was 2.13% (95% CL: 1.37%–3.3%) at the arrival at the laboratory and 1.01% (95% CL: 0.41%–2.55%) at the end of shelf-life. To get information about differences between cheese rinds and pastes, these two parts were separately analysed in the Integrative Survey samples. L. monocytogenes was detected in 4.02% (95% CL: 2.60%–6.19%) of cheese rinds (n = 473), whereas only the 0.34% (95% CL: 0.12%–0.98%) of cheese pastes (n = 894) were contaminated. This difference was statistically significant (χ2 = 10.026, P < 0.05). Gorgonzola and Taleggio were the most frequently contaminated cheeses. Non-compliance with EU official criteria (100 CFU/g) was reported in 0.55% of meat products at arrival at the laboratory, in 0.46% at the end of shelf-life, and in 1.9% of cheese rinds. PFGE showed that cheese rinds could have been the origin of the few pastes' contamination, highlighting genetic similarity between isolates found in these two cheese components. Genetic subtyping also showed the presence of different pulsotypes, usually belonging to different clusters, in isolates from different food types. The presence of some pulsotypes predominantly prevalent over the others, in products from the same manufacturer, could suggest environmental strains as the main source of contamination.

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.

QUANTIFICATION OF LISTERIA MONOCYTOGENES IN MILK BY MPN-PCR AND MPN-CULTURE METHODS

The aim of this study was to compare the MPN-PCR (Most Probable Number- Polymerase Chain Reaction) and MPN-Culture methods in enumerating of Listeria monocytogenes in milk. In order to compare the accuracy of these methods, 103 cell/ml Listeria monocytogenes and different background bacteria which may be present in raw milk, were inoculated in sterilized milk. After preparing serial dilutions, three replicates per dilution were inoculated in tubes containing listeria enrichment broth. After 48 hours of incubation, for MPN-Culture three inoculated replicates were subcultured on Oxford agar and suspected colonies were confirmed by performing by biochemical tests. For MPN-PCR assay, the DNA extraction was performed from the three inoculated replicates which were already used for MPN-Culture and PCR assay was performed using primers specific for Listeria monocytogenes. The experiment was repeated three times and the average of enumerated bacteria was calculated by each method separately. Statistical analysis using one sample Wilcoxon signed rank test showed that enumeration by MPN-PCR method was more accurate than enumeration by MPN-Culture method. The result of this study showed that MPN-PCR method in comparision with MPN-Culture even in the presence of different background microorganisms is more rapid and reliable. It is concluded that MPN-PCR method facilitates the enumeration of Listeria monocytogenes without excessive work and could be considered as an alternative to MPN-Culture technique.

Manual for Reporting on Zoonoses, Zoonotic Agents and Antimicrobial Resistance in the framework of Directive 2003/99/EC and on some other pathogenic microbiological agents for information derived from the year 2012

Manual for Reporting on Zoonoses, Zoonotic Agents and Antimicrobial Resistance in the framework of Directive 2003/99/EC and on some other pathogenic microbiological agents for information derived from the year 2012

Presence of Listeria spp. in fish samples, fish products and sea products

Listeria monocytogenes is a bacterium that is pathogenic for man and for most animal species. Listeriosis is a generalized infection that starts after ingestion of the causative agent L. monocytogenes (Finlay, 2001). Food that is not properly thermically processed, long storage of such food, food that is produced in inadequate hygienic food plants, as well as cooked-cooled ready to eat food (RTE) is the ideal medium for listeria multiplication. High risk food originated from fish, fish products and sea products comprised of: molluscs (fresh of frozen shellfish, crustaceans shelled or not), fresh fish (ready to eat without cooking), fish products packed with brine (NaCl<6%), (salted, marinated, fermented, cold smoked and fish in brine), thermically treated fish and crustacean products (pasteurization, cooking, hot smoking, including pre-cooking and panning). In this research, fish samples, fish products and sea products from Serbian markets were examinated such as: fresh fish (cooled), frozen food (fish and sea products - cuttlefish, squid, octopus, shellfish, crustaceans and shrimps), panned products; smoked fish, salted fish, thermally treated fish and fish products, semi-canned fish and canned fish. Microbiological testing has been performed according to internationally prescribed standards ISO 11290-1 (1996) "Microbiology of food and animal feed stuffs - Horizontal method for the detection and enumeration of Listeria monocytogenes". There were 470 samples from fish products and sea products. Presence of Listeria spp was registered in 58 samples (12.34%). Listeria monocytogenes was found in 1.92% samples (9 isolates), which makes 15.52% of the total Listeria that were isolated from the tested food. Other isolated Listeria belong to the following species: L. innocua (8.51%), L.welshimeri (1.28%), L.welshimeri/innocua (0.21%), L. grayi (0.21%) and L. seeligeri (0.21%). Exceptional viability of Listeria monocytogenes in the food tested was documented, also (freezing temperature - 18oC, five months).

Proposed technical specifications for a survey on Listeria monocytogenes in selected categories of ready‐to‐eat food at retail in the EU

Proposed technical specifications for a survey on Listeria monocytogenes in selected categories of ready‐to‐eat food at retail in the EU

Enumeration of Listeria monocytogenes at Low Contamination Levels in Several Food Matrices Using a Membrane Filtration Method

Enumeration of Listeria monocytogenes at Low Contamination Levels in Several Food Matrices Using a Membrane Filtration Method

Listeria monocytogenes in RTE foods marketed in Italy: Prevalence and automated EcoRI ribotyping of the isolates

The aims of the present study were: (a) to investigate the prevalence and the enumeration of Listeria monocytogenes in 200 samples of ready to eat (RTE) foods of animal and vegetal origin collected from different outlets and processing plants in Sardinia; (b) to characterize the isolates by phenotypical and molecular methods; (c) to analyze a subset of 42 L. monocytogenes by automated EcoRI ribotyping in order to predict the strain's potential virulence for humans. The strains were isolated from: smoked fish products, cooked marinated products, meat products and pre-packaged mixed vegetable salads. Of the samples tested, 22% were positive for Listeria spp. The prevalence of L. monocytogenes was 9.5%, while the level of L. monocytogenes in the positive samples was <10 cfu/g in 94.7% of cases. EcoRI ribotyping differentiated the isolates into 16 distinct ribotypes (similarity>93%), belonging to 17 different DuPont Identification Library Codes (DUP-IDs) clones. The Simpson's numerical index of discrimination was 0.911. Cluster analysis pointed out a high similarity among strains isolated from meat, fish, and vegetables of different origin. These results confirmed the existence of a widespread population of L. monocytogenes, characterized by highly related strains existing in different geographical areas. 65% of these strains belonged to lineage II (serotypes 1/2a and 1/2c), subtypes known to be associated with sporadic human listeriosis outbreaks. The remaining 35% of the isolates (serotypes 1/2b, 3b and 4b) were allocated to lineage I and belong to distinct clonal groups (DUP-ID 1038 and 1042), which again have been associated with several outbreaks of human listeriosis. Neither atypical profiles nor lineage III strains were found. EcoRI ribotyping was confirmed as a rapid and reliable method for L. monocytogenes typing, providing useful data for epidemiologic and clonality surveys of L. monocytogenes strains isolated from RTE foods.

Impact of−2°C Superchilling before Refrigerated Storage (4 and 8°C) on the Microbiological and Sensory Qualities of Cold-Smoked Salmon

Detection and enumeration of Listeria monocytogenes and total spoilage bacteria in 40 batches of cold-smoked salmon (one batch = 42 products from the same day of manufacture) straight from the factory were carried out. If L. monocytogenes was detected in at least one of the nine samples analyzed on receipt at the laboratory, 9 products of the same batch were stored for 10 days at 4°C, which was followed by 18 days at 8°C (control), 12 products were superchilled for 14 days at −2°C, and 12 other products were superchilled for 28 days at −2°C and then stored under the same conditions as the control was stored. L. monocytogenes was detected in 7% of the 40 batches analyzed immediately after receipt at the laboratory. L. monocytogenes prevalence was similar (approximately 25%) throughout the storage at 4 and 8°C, both in control and super-chilled products at −2°C for 14 days. After superchilling for 28 days at −2°C, L. monocytogenes was found in 9% of products, and in 39% at the end of the storage above 0°C. Moreover, the L. monocytogenes count was higher after 3 and 4 weeks of storage at 4 and 8°C in products superchilled 28 days at −2°C than in control products or in products superchilled for 14 days. Serotype 1/2a-3a and nine genetic groups were identified and found throughout the storage scenario. At the end of shelf life, sensory characteristics of products superchilled for 28 days at −2°C were slightly modified. A decrease in firmness associated with increased tearing of salmon slices was observed as well as a slight amine odor.

Chromogenic media for the detection and/or enumeration of Listeria monocytogenes - results of trials performed by a working group of the International Organization for Standardization - ISO/TC 34/SC 9

The solid selective media PALCAM and Oxford agar originally described in the ISO (International Organization for Standardization) Standard 11290 part 1 and part 2 Microbiology of food and animal feeding stuffs - Horizontal method for the detection and enumeration of Listeria suffer from the disadvan-tage that L monocytogenes cannot be differentiated from non-pathogenic liste-rias.On the basis of the results of trials, organised by an ISO/TC 34/SC 9 working group with blood-based and chromogenic media, ISO members agreed that the ALOA medium and Rapid'L.mono agar performed significantly better than the other media tested.Moreover, the ready-to-use ALOA plates performed similarly to the medium prepared from the individual components. Finally Substitution of the expensive component phosphatidylinositol (Pl), Substrate for phospholipase C activity of L monocytogenes, by lecithin had no noticeable effect on the elective features of the ALOA medium, provided the lecithin contained a sufficiently high concentration of Pl.

Experimental Evaluation of Different Precision Criteria Applicable to Microbiological Counting Methods

The dispersion of microbiological counting measurements, when repeating the analysis on the same material both within a laboratory (repeatability) and between laboratories (reproducibility) can be characterized by the organization of interlaboratory studies, where several sets of identical test materials are sent to several laboratories. Using the example of data generated by an interlaboratory study on enumeration of Listeria monocytogenes in foods by the standardized reference method (colony-count technique), 2 types of robust estimators of reproducibility standard deviations, based on the median, were examined, in comparison with the classical estimators, based on the mean. Experimental evaluation indicated that the 3 approaches gave consistent results for most of the combinations. The usual log10 transformation of the enumeration results was also questioned before these calculations were conducted.

Detection and quantification of the iap gene of Listeria monocytogenes and Listeria innocua by a new real-time quantitative PCR assay

A real-time quantitative polymerase chain reaction (PCR) assay for direct detection and enumeration of Listeria monocytogenes and Listeria innocua was developed and applied to artificially contaminated milk samples. The iap gene present in both species was used as a target for amplification of a 175-bp ( L. monocytogenes) and a 309-bp ( L. innocua) fragment. To ensure that L. monocytogenes and L. innocua are specifically detectable, tests were carried out using 42 L. monocytogenes strains and 33 L. innocua strains belonging to different serovars. Specificity was also confirmed using 22 bacterial strains not belonging to the genus Listeria, including closely related bacteria. In addition to specificity, the reported assay is characterized by a wide dynamic range of quantification and a high sensitivity, as we could detect as few as six copies of the iap gene per PCR using purified DNA as template. When applied to direct detection and quantification of L. monocytogenes in milk, the more rapid real-time quantitative PCR assay was as sensitive as the traditional plate count method, but real-time quantitative PCR-derived iap gene copy numbers were one to two logs higher than colony-forming units obtained by the plate count method.

Enhanced detection and enumeration of Listeria monocytogenes from foodstuffs and food-processing environments

Listeria monocytogenes blood agar (LMBA) was compared to Listeria selective agar based on lithium chloride and ceftazidime (LA) and to the Oxford and Palcam media recommended by ISO and IDF for the detection and enumeration of L. monocytogenes from foodstuffs and food-processing environments. LMBA is based on trypticase soy agar with the following additions: sheep blood (5%) and as selective agents lithium chloride (10 g/l), polymyxin B sulphate (10 mg/l) and ceftazidime (20 mg/l), whereas the selectivity of LA is based on lithium chloride (15 g/l) and ceftazidime (15 g/l). The media were compared in the detection of L. monocytogenes after enrichment from naturally contaminated foodstuffs ( n=423) and from food-processing environments ( n=93), and in the enumeration of the species from naturally contaminated foodstuffs ( n=287). LMBA was superior both to the standard media and to LA in detection after enrichment and also in enumeration, except in the case of fresh broiler cut samples. The overall sensitivities of the Palcam, Oxford, LA and LMBA media were 68%, 67%, 74% and 96% in detection after enrichment and 64%, 73%, 76% and 80% in the enumeration of the species from ready to eat foods. The superiority of LMBA is based on distinguishing L. monocytogenes from other Listeria species by detection of β-hemolysis, whereas the other media gave false-negative results because of the overgrowth of Listeria spp. other than L. monocytogenes, especially in detection after enrichment. A more selective medium than LMBA would have been required for the enumeration of the species from samples with high levels of competitive bacteria other than Listeria spp. The results indicate the need for a more specific isolation medium for L. monocytogenes in addition to those recommended by ISO and IDF for both detection and enumeration. LMBA offers an alternative to be used in combination with either Palcam or Oxford as well as with LA.