Prevalence and fingerprinting of Listeria monocytogenes strains isolated from raw whole milk in farm bulk tanks and in dairy plant receiving tanks

The incidence of Listeria species in raw whole milk from farm bulk tanks and from raw milk in storage at a Swedish dairy plant was studied. Listeria monocytogenes was found in 1.0% and Listeria innocua was found in 2.3% of the 294 farm bulk tank (farm tank) milk specimens. One farm tank specimen contained 60 CFU of L. monocytogenes ml(-1). L. monocytogenes was detected in 19.6% and L. innocua was detected in 8.5% of the milk specimens from the silo receiving tanks at the dairy (dairy silos). More dairy silo specimens were positive for both Listeria species during winter than during summer. Restriction enzyme analysis and pulsed-field gel electrophoresis were applied to 65 isolates of L. monocytogenes, resulting in 16 different clonal types. Two clonal types were shared by the farm tank milk and the dairy silo milk. All except one clonal type belonged to serovar 1/2a. In the dairy silo milk five clonal types were found more frequently and for a longer period than the others. No Listeria species were found in any other samples from the plant.     

Listeria species in a California coast estuarine environment

Listeria species and L. monocytogenes were found in 81 and 62%, respectively, of fresh or low-salinity waters (37 samples) in tributaries draining into Humboldt-Arcata Bay, Calif., during a winter (January-February) sampling period. The incidence of Listeria species and L. monocytogenes in sediment (46 samples) from the same sites where water was sampled was 30.4 and 17.4%, respectively. One of three bay water samples contained Listeria species (including L. monocytogenes), while of 35 samples of oysters examined, only 1 was found positive for Listeria species (L. innocua). A given species or L. monocytogenes serogroup appeared to predominate in fresh water when domesticated animals (cows, horses) were nearby, whereas greater variety with no species predominance was observed in areas with no direct animal influence.     

Listeria species in a California coast estuarine environment.

Listeria species and L. monocytogenes were found in 81 and 62%, respectively, of fresh or low-salinity waters (37 samples) in tributaries draining into Humboldt-Arcata Bay, Calif., during a winter (January-February) sampling period. The incidence of Listeria species and L. monocytogenes in sediment (46 samples) from the same sites where water was sampled was 30.4 and 17.4%, respectively. One of three bay water samples contained Listeria species (including L. monocytogenes), while of 35 samples of oysters examined, only 1 was found positive for Listeria species (L. innocua). A given species or L. monocytogenes serogroup appeared to predominate in fresh water when domesticated animals (cows, horses) were nearby, whereas greater variety with no species predominance was observed in areas with no direct animal influence.     

Estimating size and diversity of nitrifying communities in deodorizing filters using PCR and immunofluorescence

Thirty isolates of Listeria monocytogenes and 18 of L. innocua obtained from different short-ripened cheeses manufactured in Asturias (northern Spain), were compared with each other and with reference strains using serotype, phage type and pulsed-field restriction endonuclease digestion profiles analysis of the total DNA. Restriction enzymes Apa I and Sma I defined five clusters in L. monocytogenes ( m1 to m5 ) and two main clusters in L. innocua ( i1 and i2 ). Cluster i2 was further arranged into three subclusters ( i2a , i2b and i2c ) based on the different Eco 52I ( Xma III) and Crf 42I ( Sac II) patterns of its isolates. Clusters of L. innocua were clearly different whereas those of L. monocytogenes were more closely related to each other. In this latter species, serotype 4b isolates ( m4 and m5 ) constituted a more homogeneous group than serogroup 1 isolates ( m1 , m2 and m3 ). Cluster m3 contained two strains of serotype 1/2a whereas m1 and m2 harboured strains of both serotypes, 1/2a and 1/2b. Therefore, the combined use of restriction patterns and serotype may be useful to differentiate L. monocytogenes strains showing identical restriction profiles but differing in serotype. The cheese source of Listeria strains proved that isolates from cluster m1 were repeatedly detected as a contaminant in the same type of cheese. Comparison of L. monocytogenes Apa I profiles showed a genetic proximity of m4 and m5 to the recognized pathogenic strains ATCC 13932 and NCTC 11994, responsible for meningitis cases in other countries. Finally, bacteriophage typing data indicated that m4 , the sole phage typable group, had a phage type resembling that of strains causing the Auckland (New Zealand) outbreak of listeriosis in 1969. These data suggest a wide distribution of closely related types which might cause, under several circumstances, sporadic cases of listeriosis.     

Fate of Listeria monocytogenes in tissues of experimentally infected cattle and in hard salami

Muscle, organ, and lymphoid tissues of four Holstein cows experimentally inoculated (intravenously) with Listeria monocytogenes were examined 2, 6, or 54 days postinoculation for the presence of the organism by direct plating and cold enrichment procedures. L. monocytogenes was isolated from 66% of the tissues sampled; 38% of the isolations were attributed to the use of cold enrichment. Isolation of the organism from muscle tissue was possible only with animals inoculated 2 days before slaughter. The fate of L. monocytogenes during the manufacture and storage of fermented hard salami made from this meat also was determined. Three sausage treatments were evaluated: (i) uninoculated control sausage, (ii) "naturally" contaminated sausage (NC) made from meat of an experimentally inoculated cow, and (iii) sausage made from beef inoculated with a laboratory culture of L. monocytogenes (I). Initial Listeria levels in NC and I sausage were 10(3) CFU/g in trial 1 and 10(4) CFU/g in trial 2. Numbers of L. monocytogenes decreased by approximately 1 log10 CFU/g during fermentation and decreased further during drying and refrigerated storage. Small numbers (less than or equal to 20 CFU/g) of L. monocytogenes were present in I and NC sausage at the end of 12 weeks of refrigerated storage; recovery of these organisms generally depended on the use of an enrichment procedure. The results indicate that L. monocytogenes does not multiply during the fermentation and drying processes typical of hard salami manufacture but that survival may occur if the organism is initially present at greater than or equal to 10(3) CFU/g.     

Fate of Listeria monocytogenes in tissues of experimentally infected cattle and in hard salami.

Muscle, organ, and lymphoid tissues of four Holstein cows experimentally inoculated (intravenously) with Listeria monocytogenes were examined 2, 6, or 54 days postinoculation for the presence of the organism by direct plating and cold enrichment procedures. L. monocytogenes was isolated from 66% of the tissues sampled; 38% of the isolations were attributed to the use of cold enrichment. Isolation of the organism from muscle tissue was possible only with animals inoculated 2 days before slaughter. The fate of L. monocytogenes during the manufacture and storage of fermented hard salami made from this meat also was determined. Three sausage treatments were evaluated: (i) uninoculated control sausage, (ii) "naturally" contaminated sausage (NC) made from meat of an experimentally inoculated cow, and (iii) sausage made from beef inoculated with a laboratory culture of L. monocytogenes (I). Initial Listeria levels in NC and I sausage were 10(3) CFU/g in trial 1 and 10(4) CFU/g in trial 2. Numbers of L. monocytogenes decreased by approximately 1 log10 CFU/g during fermentation and decreased further during drying and refrigerated storage. Small numbers (less than or equal to 20 CFU/g) of L. monocytogenes were present in I and NC sausage at the end of 12 weeks of refrigerated storage; recovery of these organisms generally depended on the use of an enrichment procedure. The results indicate that L. monocytogenes does not multiply during the fermentation and drying processes typical of hard salami manufacture but that survival may occur if the organism is initially present at greater than or equal to 10(3) CFU/g.     

Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media.

Isolation rates for Listeria monocytogenes and the other Listeria spp. typically improve when samples are enriched in more than one primary enrichment medium. This study evaluated the abilities of two primary enrichment media, University of Vermont-modified Listeria enrichment broth (UVM) and Listeria repair broth (LRB), to recover different ribotypes of Listeria spp. from raw meat and poultry samples. Forty-five paired 25-g retail samples of ground beef, pork sausage, ground turkey, and chicken (160 samples) underwent primary enrichment in UVM and LRB (30 degrees C for 24 h) followed by secondary enrichment in Fraser broth (35 degrees C for 24 and 40 h) and plating on modified Oxford agar. After 24 h of incubation of 35 degrees C, 608 Listeria colonies from selected positive samples were biochemically confirmed as L. monocytogenes (245 isolates), L innocua (276 isolates), and L. welshimeri (89 isolates) and then ribotyped with the automated Riboprinter microbial characterization system (E. I. du Pont de Nemours & Co., Inc.). Thirty-six different Listeria strains comprising 16 L. monocytogenes (including four known clinical ribotypes), 12 L. innocua, and 8 L. welshimeri ribotypes were identified from selected positive samples (15 samples of each product type; two UVM and two LRB isolates per sample). Twenty-six of 36(13 L. monocytogenes) ribotypes were detected with both UVM and LRB, whereas 3 of 36 (1 L. monocytogenes) and 7 of 36 (3 L. monocytogenes) Listeria ribotypes were observed with only UVM or LRB, respectively. Ground beef, pork sausage, ground turkey, and chicken yielded 22 (8 L. monocytogenes), 21 (12 L. monocytogenes), 20 (9 L. monocytogenes), and 19 (11 L. monocytogenes) different Listeria ribotypes, respectively, with some Listeria ribotypes confined to a particular product. More importantly, major differences in both the number and distribution of Listeria ribotypes, including previously recognized clinical and nonclinical ribotypes of L. monocytogenes, were observed when 10 UVM and 10 LRB isolates from five samples of each product were ribotyped. When a third set of six samples per product type was examined from which two Listeria isolates were obtained by using only one of the two primary enrichment media, UVM and LRB failed to detect L. monocytogenes (both clinical and nonclinical ribotypes) in two and four samples, respectively. These findings stress the importance of using more than one primary enrichment medium and picking a sufficient number of colonies per sample when attempting to isolate specific L. monocytogenes strains during investigations of food-borne listeriosis.     

Prevalence of Listeria sp. in droppings from urban rooks (Corvus frugilegus)

Droppings from 112 urban rooks ( Corvus frugilegus ) were cultured for the presence of Listeria sp. Overall, 46% of rooks sampled harboured one or more Listeria species. Of all birds examined, 33%, 24% and 8%, respectively, were infected with Listeria monocytogenes, Listeria innocua and Listeria seeligeri. Differentiation of L. monocytogenes and L. seeligeri carried out by several phenotypic typing methods proved the diversity of strains and the major role of rooks which widely contribute to spreading this bacteria in our environment. The results also suggest that the ability to recover specific Listeria strains from the same sample is at least partially dependent on the methodology. These findings reinforce the need for strain-specific typing of multiple L. monocytogenes isolates from the same sample.     

The risk of Listeria monocytogenes infection in beef cattle operations

Aim:  To determine the prevalence of Listeria monocytogenes and associated risk factors among beef operations (cow-calf and feedlot) in central and southern California.
Methods and Results:  A repeated cross-sectional study where faecal and environmental samples were collected from 50 operations three times a year at different seasons was carried out. Samples were tested for presence of L. monocytogenes using a combination of enrichment and polymerase chain reaction tests. Data on putative risk factors were also collected. Listeria monocytogenes was detected in faecal samples from cows, calves and other animals on calf-cow operations at proportions of 3·1%, 3·75% and 2·5%, respectively. The organism was detected in 5·3% of cut-grass, 5·3% of soil, 14·3% of irrigation ditches, 3·1% of the ponds and 6·5% of water troughs samples. Listeria monocytogenes was less common in faecal (0·3%) and soil (0·75%) samples collected from feedlots.
Conclusions:  Listeria monocytogenes was present at a higher proportion among cow-calf operations than feedlots. There was no significant seasonal variation in the occurrence of this pathogen within the two types of operations.
Significance and Impact of the Study:  If risk mitigation strategies were implemented to reduce the public health risk these should focus in cow-calf operations.     

Evaluation of BBL CHROMagar Listeria agar for the isolation and identification of Listeria monocytogenes from food and environmental samples

The performance of BBL CHROMagar Listeria chromogenic agar for the detection of Listeria monocytogenes was evaluated for its ability to isolate and identify L. monocytogenes from food and environmental samples. The medium was compared to non-chromogenic selective agars commonly used for Listeria isolation: Oxford, Modified Oxford, and PALCAM. BBL CHROMagar Listeria had a sensitivity of 99% and 100% for the detection of L. monocytogenes from 200 natural and artificially inoculated food samples, respectively, with a colony confirmation rate of 100%. The sensitivity of non-chromogenic selective media for the detection of L. monocytogenes from these same samples was 97-99% with colony confirmation rates of 65-67.5%. From 93 environmental samples, BBL CHROMagar Listeria agar results correlated 100% with a Listeria spp. visual immunoassay (TECRA) performed on these same samples and the USDA-FSIS standard culture method for the isolation of L. monocytogenes. From environmental samples, the L. monocytogenes confirmation rate was 100% for BBL CHROMagar Listeria as compared to 50% for conventional agars tested. On BBL CHROMagar Listeria, L. monocytogenes forms a translucent white precipitation zone (halo) surrounding blue-pigmented colonies of 2-3 mm in diameter, with an entire border. BBL CHROMagar Listeria offers a high degree of specificity for the confirmation of suspect L. monocytogenes colonies, whereas non-chromogenic selective agars evaluated were not differential for L. monocytogenes from other Listeria species.     

The incidence of Listeria monocytogenes in raw milk from farm bulk tanks in north-east Scotland

Over 180 farm bulk milk tanks were tested for the presence of Listeria monocytogenes on three separate occasions which included periods when cows were grazing and confined inside on a silage diet. The incidence of L. monocytogenes contamination was low, ranging from 3.8% in the summer samples to 1.0% in October. The level of contamination was estimated to be lower than one L. monocytogenes bacterium per ml in positive samples, as most required cold enrichment of 10-20 ml volumes before recovery. The distribution was sporadic; only one farm gave positive isolations on all three sampling occasions, one other on two, and all others were from different farms. No correlation between the presence of L. monocytogenes and hygiene standards or the feeding of silage was found.     

The incidence of Listeria monocytogenes in raw milk from farm bulk tanks in North-East Scotland

Over 180 farm bulk milk tanks were tested for the presence of Listeria monocytogenes on three separate occasions which included periods when cows were grazing and confined inside on a silage diet. The incidence of L. monocytogenes contamination was low, ranging from 3.8% in the summer samples to 1.0% in October. The level of contamination was estimated to be lower than one L. monocytogenes bacterium per ml in positive samples, as most required cold enrichment of 10–20 ml volumes before recovery. The distribution was sporadic; only one farm gave positive isolations on all three sampling occasions, one other on two, and all others were from different farms. No correlation between the presence of L. monocytogenes and hygiene standards or the feeding of silage was found.     

Characteristics and frequency of detection of fecal Listeria monocytogenes shed by livestock, wildlife, and humans

Listeria monocytogenes is a facultative intracellular pathogen that can be carried asymptomatically in various animals and can be shed in feces. We investigated the prevalence and characteristics of L. monocytogenes isolated from livestock, wildlife, and human potential sources of contamination in 2 areas in Ontario, Canada. From February 2003 to November 2005, a total of 268 fecal samples were collected from different animals. Listeria monocytogenes was isolated using selective enrichment, isolation, and confirmation procedures, and 15 samples (6%) yielded to the isolation of 84 confirmed strains. Listeria monocytogenes was isolated from livestock (beef and dairy), wildlife (deer, moose, otter, and raccoon), and human (biosolids and septic) fecal sources. Thirty-two isolates were from serovar 1/2a, 34 from serovar 1/2b, 1 from serovar 3a, and 17 from serovar 4b. Listeria monocytogenes populations were resolved into 13 EcoRI ribotypes, and 18 ApaI and 18 AscI pulsotypes, with Simpson indexes of discrimination of 0.878 and 0.907, respectively. A majority (59%) of L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, which was supported by higher entry into Caco-2 cells (9.3%) than isolates producing truncated and secreted internalin A (1.3% of entry). Listeria monocytogenes fecal isolates were on average resistant to 6.4 +/- 2.5 antibiotics out of 17 tested, and potentially virulent isolates exhibited an enhanced resistance to kanamycin, gentamicin, streptomycin, and rifampicin. Livestock, wildlife, and human L. monocytogenes fecal communities exhibited overlapping but distinct populations, and some genotypes and phenotypes were similar to those previously described for surface water isolates in the same area.     

Listeria monocytogenes in poultry and poultry products: epidemiological investigations in seven Danish abattoirs

Listeria monocytogenes was isolated from 11/236 (4·7%) caecal samples from parent flocks, providing broilers to the abattoirs investigated. Caecal samples from 2078 broilers representing 90 randomly selected broiler flocks were negative for L. monocytogenes. A total of 3080 samples from seven abattoirs including poultry processing line samples, and final products were also examined for L. monocytogenes. Listeria monocytogenes was isolated in 0·3% to 18·7% of the samples collected in the different abattoirs. Epidemiological typing of 247 L. monocytogenes isolates, including serotyping, phage typing, pulsed-field gel electrophoresis and ribotyping revealed 62 different clones. Based upon typability and discriminatory power, DNA typing methods used were found equally suitable as epidemiological markers. Serotyping and phage typing were not found useful as epidemiological markers for poultry isolates of L. monocytogenes since only 120/247 (48·6%) isolates were typable by phage typing and 230/247 (93·1%) L. monocytogenes belonged to serotype 01 while 6/247 (2·4%) belonged to 04. The discovery of a few dominating clones in each abattoir might indicate an endemic occurrence of L. monocytogenes. It is concluded that L. monocytogenes in the broiler production is primarily localized to the abattoirs. The incidence of L. monocytogenes may be reduced by improving the hygiene.     

Effects of pH on distribution of Listeria ribotypes in corn, hay, and grass silage.

Listeria app, isolated from 13 of 129 (10%) corn silage samples, 21 of 76 (28%) hay silage samples, and 3 of 5 (60%) grass silage samples during a previous Vermont survey were subjected to automated ribotype (RT) analysis. The 13 positive corn silage samples contained 3 Listeria monocytogenes isolated (three RTs, including one known clinical RT) and 10 L. innocua isolates (four RTs). Similarly, 2 L. monocytogenes isolates (two RTs) and 19 L. innocua isolates (three RTs) were identified in the 21 positive hay silage samples. The three positive grass silage samples contained two L. innocua isolates (two RTs) and one isolate of L. welshimeri. One hundred seven of 129 (83%) high-quality (pH < 4.0) corn silage samples accounted for 8 of 13 Listeria isolates from corn silage, including isolates belonging to one L. monocytogenes clinical RT. In contrast, low-quality hay silage (70 of 76 [92%] samples having a pH of > or = 4.0) harbored 20 of 21 isolates, including isolates belonging to two nonclinical L. monocytogenes RTs. Poor-quality silage is readily discernible by appearance; however, these findings raise new concerns regarding the safety of high-quality (pH < 4.0) corn silage, which can contain Listeria spp., including L. monocytogenes strains belonging to RTs of clinical importance in cases of food-borne listeriosis.     

Comparative study of two plating media (PALCAM and Oxford) for detection of Listeria species in a range of meat products following a variety of enrichment procedures

Fifty-two different varieties of sausage, salami and pâté were examined using Oxford (Oxoid) and PALCAM (Merck) listeria-selective agars. Seven (13%) samples were positive for Listeria monocytogenes and 14 (27%) samples were positive for other Listeria species, while 31 (59%) samples were negative for Listeria species. The effectiveness of PALCAM and Oxford medium for the isolation of L. monocytogenes from the meat samples was compared. PALCAM medium was consistently more effective in suppressing other micro-organisms thus enhancing the possibility of detecting Listeria species present in low numbers. The isolation of Listeria species for identification was also easier using PALCAM medium.     

Elucidation of Listeria monocytogenes contamination routes in cold-smoked salmon processing plants detected by DNA-based typing methods

The contamination routes of Listeria monocytogenes in cold-smoked salmon processing plants were investigated by analyzing 3,585 samples from products (produced in 1995, 1996, 1998, and 1999) and processing environments (samples obtained in 1998 and 1999) of two Danish smokehouses. The level of product contamination in plant I varied from 31 to 85%, and no L. monocytogenes was found on raw fish (30 fish were sampled). In plant II, the levels of both raw fish and product contamination varied from 0 to 25% (16 of 185 raw fish samples and 59 of 1,000 product samples were positive for L. monocytogenes). A total of 429 strains of L. monocytogenes were subsequently compared by random amplified polymorphic DNA (RAPD) profiling, and 55 different RAPD types were found. The RAPD types detected on the products were identical to types found on the processing equipment and in the processing environment, suggesting that contamination of the final product (cold-smoked salmon) in both plants (but primarily in plant I) was due to contamination during processing rather than to contamination from raw fish. However, the possibility that raw fish was an important source of contamination of the processing equipment and environment could not be excluded. Contamination of the product occurred in specific areas (the brining and slicing areas). In plant I, the same RAPD type (RAPD type 12) was found over a 4-year period, indicating that an established in-house flora persisted and was not eliminated by routine hygienic procedures. In plant II, where the prevalence of L. monocytogenes was much lower, no RAPD type persisted over long periods of time, and several different L. monocytogenes RAPD types were isolated. This indicates that persistent strains may be avoided by rigorous cleaning and sanitation; however, due to the ubiquitous nature of the organism, sporadic contamination occurred. A subset of strains was also typed by using pulsed-field gel electrophoresis and amplified fragment length polymorphism profiling, and these methods confirmed the type division obtained by RAPD profiling.     

Evaluation of enhanced haemolysis agar for detection of Listeria spp. and L. monocytogenes from production lines of fresh to cold-smoked fish.

Enhanced haemolysis agar (EHA) was compared to the two conventional Listeria isolation agars Oxford and PALCAM for its ability to detect Listeria spp. from production lines of fresh to cold-smoked fish. The ability of EHA for distinguishing L. monocytogenes colonies from other Listeria spp. was also evaluated.A total of 243 fish and environmental samples were analysed. Overall, 42 samples were found to contain Listeria spp. Only 34 samples were positive simultaneously by the three plating media. Two samples considered to be negative by the two conventional agars were found to be positive after isolation on EHA. All three selective agars were shown to be less effective in recovering Listeria spp. after primary enrichment in half-Fraser broth, compared to secondary enrichment in Fraser broth after 24 and 48 h.From 79 Listeria but presumptive negative L. monocytogenes colonies, EHA identified correctly 76 Listeria spp. and presented three false-negative results_three colonies further identified as L. monocytogenes but showing no noticeable haemolysis on EHA. Twenty-three of the thirty-three L. monocytogenes presumptive positive colonies, were confirmed positive and ten were identified as L. seeligeri.Despite its ability of distinguishing L. monocytogenes from the other Listeria spp., unless it is produced as a commercial medium, EHA cannot be an alternative to time-consuming classical identification because the preparation of this medium is both time and labour intensive.     

Detection and prevalence of Listeria monocytogenes in the agricultural ecosystem

The sensitivity of four different enrichment procedures to detect Listeria monocytogenes in the presence of high levels of Streptococcus faecalis was investigated. Defined mixed cultures of Strep. faecalis and L. monocytogenes gave better results with one-stage enrichment techniques. For manure samples, however, two-stage enrichment techniques gave the best performance. The so-called cold enrichment techniques were found to be unsatisfactory for samples from natural environments. The following materials were examined for the presence of L. monocytogenes: fresh pig faeces (16% positive), fresh cattle faeces (20% positive), stored liquid manure (0% positive), manured soil samples (0% positive) and ground water samples (5% positive). After 3 weeks of storage L. monocytogenes could be detected in only one of the initially nine positive fresh faeces samples. Two months after inoculation of stored liquid pig manure, stored liquid cattle manure and soil with L. monocytogenes, this bacterium could not be traced in any of these materials. Radishes (Raphanus sativus) and carrots (Daucus carota), sown in soil inoculated with L. monocytogenes, were gathered after 3 months and examined for the presence of L. monocytogenes. Three of six radish samples were found to be positive. Remarkably, however, all carrot samples (six) were free of L. monocytogenes.     

Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay.

A new detection system, the magnetic immuno-polymerase chain reaction (PCR) assay (MIPA) has been developed to detect Listeria monocytogenes in food. This method separates Listeria cells from PCR-inhibitory factors present in enrichment broths containing food samples by using magnetic beads coated with specific monoclonal antibodies (MAbs). The separated bacteria were lysed, and the supernatant containing the bacterial DNA was subjected to the PCR. Detection of L. monocytogenes in three naturally contaminated cheese samples with two different MAbs and PCR primers specific for the gene encoding the delayed-hypersensitivity factor showed that with MAb 55 all three samples were positive whereas with MAb A two samples were positive. A further improvement of the method was obtained by using a PCR step based on the listeriolysin O gene. A MIPA employing MAb 55 and the listeriolysin O gene primer set detected L. monocytogenes after 24 h of culture in Listeria Enrichment Broth samples from Port Salut artificially contaminated with 40 CFU/25 g. We could detect 1 CFU of L. monocytogenes per g of cheese after a second enrichment for 24 h in Fraser broth. The analysis time including both enrichments is approximately 55 h.