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.     

Sources of Listeria monocytogenes Contamination in a Cold-Smoked Rainbow Trout Processing Plant Detected by Pulsed-Field Gel Electrophoresis Typing

Sites of Listeria monocytogenes contamination in a cold-smoked rainbow trout (Oncorhynchus mykiss) processing plant were detected by sampling the production line, environment, and fish at different production stages. Two lots were monitored. The frequency of raw fish samples containing L. monocytogenes was low. During processing, the frequency of fish contaminated with L. monocytogenes clearly rose after brining, and the most contaminated sites of the processing plant were the brining and postbrining areas. A total of 303 isolates from the raw fish, product, and the environment were characterized by pulsed-field gel electrophoresis (PFGE). PFGE yielded nine pulsotypes, which formed four clusters. The predominating L. monocytogenes pulsotypes of the final product were associated with brining and slicing, whereas contaminants of raw fish were not detected in the final product. Air-mediated contamination in the plant could not be proved. In accordance with these results, an L. monocytogenes eradication program was planned. The use of hot steam, hot air, and hot water seemed to be useful in eliminating L. monocytogenes. None of the control samples taken in the 5 months after the eradication program was implemented contained L. monocytogenes.     

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.     

Probabilistic Model for Listeria monocytogenes Growth during Distribution,Retail Storage,and Domestic Storage of Pasteurized Milk

A survey on the time-temperature conditions of pasteurized milk in Greece during transportation to retail, retail storage, and domestic storage and handling was performed. The data derived from the survey were described with appropriate probability distributions and introduced into a growth model of Listeria monocytogenes in pasteurized milk which was appropriately modified for taking into account strain variability. Based on the above components, a probabilistic model was applied to evaluate the growth of L. monocytogenes during the chill chain of pasteurized milk using a Monte Carlo simulation. The model predicted that, in 44.8% of the milk cartons released in the market, the pathogen will grow until the time of consumption. For these products the estimated mean total growth of L. monocytogenes during transportation, retail storage, and domestic storage was 0.93 log CFU, with 95th and 99th percentiles of 2.68 and 4.01 log CFU, respectively. Although based on EU regulation 2073/2005 pasteurized milk produced in Greece belongs to the category of products that do not allow the growth of L. monocytogenes due to a shelf life (defined by law) of 5 days, the above results show that this shelf life limit cannot prevent L. monocytogenes from growing under the current chill chain conditions. The predicted percentage of milk cartons—initially contaminated with 1 cell/1-liter carton—in which the pathogen exceeds the safety criterion of 100 cells/ml at the time of consumption was 0.14%. The probabilistic model was used for an importance analysis of the chill chain factors, using rank order correlation, while selected intervention and shelf life increase scenarios were evaluated. The results showed that simple interventions, such as excluding the door shelf from the domestic storage of pasteurized milk, can effectively reduce the growth of the pathogen. The door shelf was found to be the warmest position in domestic refrigerators, and it was most frequently used by the consumers for domestic storage of pasteurized milk. Furthermore, the model predicted that a combination of this intervention with a decrease of the mean temperature of domestic refrigerators by 2°C may allow an extension of pasteurized milk shelf life from 5 to 7 days without affecting the current consumer exposure to L. monocytogenes.L. monocytogenes is an important safety concern for the dairy industry. Several listeriosis outbreaks have been associated with the consumption of dairy products, including pasteurized milk (13, 22). An effective control of L. monocytogenes in pasteurized milk should be based on the selection of raw milk and the controls of the processing, packaging, distribution, and storage conditions. In general, the pathogen is effectively controlled during pasteurization. However, its presence in the finished product is possible as a result of postpasteurization contamination from sources in the plant environment. Considering that the levels of postpasteurization contamination are usually very low, the extent of L. monocytogenes growth during distribution, retail storage, and domestic storage is of major significance for the safety status of pasteurized milk at the time of consumption.The growth of L. monocytogenes during distribution and storage of pasteurized milk can be evaluated using the available predictive models. During the last decade, a large number of mathematical models for L. monocytogenes growth have been published (9, 11, 16, 19, 21, 24, 26, 31, 38), and some of them have been targeted to pasteurized milk (1, 40, 49). However, since the available data show that conditions that prevail during the chill chain vary significantly (8, 17, 23, 27, 28, 34, 44, 45, 48), the value of a deterministic application of these models as a tool in safety management of pasteurized milk would be limited.In recent years the need for taking into account the variabilities of the various factors in predictive microbiology has been increasingly recognized, leading to a more sophisticated modeling approach called probabilistic or stochastic modeling. The main characteristic of probabilistic modeling is the specific quantification of variabilities using probability distributions for the input data rather than point estimates. The importance of characterizing variability was stressed by Nauta (41), who illustrated the differences in decisions that could result if variability is ignored. Probabilistic modeling is being used with increasing frequency in the area of food safety. It has been extensively applied in quantitative microbial risk assessments (12, 18, 20), in quality and safety management systems (25, 30, 32), and recently for more specific topics, such as the evaluation of the effects of food processing (39) and the compliance of food products to safety criteria set by regulations (33).In the present study a probabilistic modeling approach was applied for evaluating the growth of Listeria monocytogenes in pasteurized milk from production to the time of consumption based on a Monte Carlo simulation. The objectives of the study were (i) to estimate the growth of the pathogen at the various stages of the chill chain, including transportation to retail, retail storage, and domestic storage, (ii) to analyze the importance of the chill chain factors, (iii) to evaluate the effects of selected intervention scenarios related to the improvement of the chill chain and handling conditions, and (iv) to evaluate the effect of a potential extension of milk shelf life on the growth of Listeria monocytogenes.     

Detection and characterization of the most common foodborne pathogens by using multiplex PCR procedure

Food Microbial contamination is one of the most serious problems. A large percentage of food-borne illnesses are caused by food-borne pathogens, and diarrheal agents comprise more than half of the overall prevalence of food-borne illnesses in the globe, and more commonly in developing countries. This study aimed to identify the most-common foodborne organisms from foods in Khartoum state by PCR.A total of 207 food samples (raw milk, fresh cheese, yogurt, fish, sausage, mortadella, and eggs) were collected. DNA was extracted from food samples by guanidine chloride protocol, and then species-specific primers were used to identify Escherichia coli O157: H7, Listeria monocytogenes, Salmonella spp., Vibrio cholerae, V. parahaemolyticus, and Staphylococcus aureus. Out of 207 samples, five (2.41%) were positive for L. monocytogenes, one (0.48%) was positive for S. aureus, and one (0.48%) was positive for both Vibrio cholerae and Vibrio parahaemolyticus. From 91 fresh cheese samples, 2 (2.19%) were positive for L. monocytogenes, and one (1.1%) sample was positive for two different foodborne pathogens (V. cholerae and V. parahaemolyticus). Out of 43 Cow's milk samples, three (7%) samples were positive for L. monocytogenes, and out of 4 sausage samples, one (25 %) was positive for S. aureus. Our study revealed the presence of L. monocytogenes and V. cholera in raw milk and fresh cheese samples. Their presence is considered a potential problem and needs intensive hygiene efforts and standard safety measures before, during, and after food processing operations.     

Molecular Studies on the Ecology of Listeria monocytogenes in the Smoked Fish Processing Industry

We have applied molecular approaches, including PCR-based detection strategies and DNA fingerprinting methods, to study the ecology of Listeria monocytogenes in food processing environments. A total of 531 samples, including raw fish, fish during the cold-smoking process, finished product, and environmental samples, were collected from three smoked fish processing facilities during five visits to each facility. A total of 95 (17.9%) of the samples tested positive for L. monocytogenes using a commercial PCR system (BAX for Screening/Listeria monocytogenes), including 57 (27.7%) environmental samples (n = 206), 8 (7.8%) raw material samples (n = 102), 23 (18.1%) samples from fish in various stages of processing(n = 127), and 7 (7.3%) finished product samples (n = 96). L. monocytogenes was isolated from 85 samples (16.0%) using culture methods. Used in conjunction with a 48-h enrichment in Listeria Enrichment Broth, the PCR system had a sensitivity of 91.8% and a specificity of 96.2%. To track the origin and spread of L. monocytogenes, isolates were fingerprinted by automated ribotyping. Fifteen different ribotypes were identified among 85 isolates tested. Ribotyping data established possible contamination patterns, implicating raw materials and the processing environment as potential sources of finished product contamination. Analysis of the distribution of ribotypes revealed that each processing facility had a unique contamination pattern and that specific ribotypes persisted in the environments of two facilities over time (P ≤ 0.0006). We conclude that application of molecular approaches can provide critical information on the ecology of different L. monocytogenes strains in food processing environments. This information can be used to develop practical recommendations for improved control of this important food-borne pathogen in the food industry.     

One Group of Genetically Similar Listeria monocytogenes Strains Frequently Dominates and Persists in Several Fish Slaughter- and Smokehouses

Contamination of foods with the human pathogen Listeria monocytogenes may occur during processing, and the purpose of this study was to determine whether genetically similar strains colonize different processing plants or whether specific persistent strains are unique to each processing plant. We hypothesized that specific L. monocytogenes strains may be better adapted to specific environmental niches in the processing environment. L. monocytogenes contamination patterns were identified by the collection of 686 and 267 samples from the processing environments: raw fish and products of four fish smokehouses and four fish slaughterhouses, respectively. Samples were collected both during production and after cleaning and disinfection. Typically, these samplings were separated by 1 to 3 months. Sampling sites were targeted toward areas likely to harbor the bacterium. L. monocytogenes was isolated from 213 samples, and one strain from each positive sample was typed by RAPD (random amplified polymorphic DNA) analysis with four different primers. The 213 strains were divided into 37 RAPD types. One RAPD type was predominant; 86 of 213 strains belonged to this type. This type was found in three smokehouses and two slaughterhouses and was predominant in three of these plants. A subset of 35 strains was also analyzed by amplified fragment length polymorphism typing, which confirmed the genetic similarity of the groups. Moreover, strains of the dominant RAPD type were indistinguishable from strains isolated frequently from smoked fish products 10 years ago. One smokehouse was surveyed for a year and a half, and the dominant RAPD type persisted throughout the survey period and accounted for 94 of 118 isolates. Our study indicates that strains of L. monocytogenes that are genetically very closely related may be especially adapted to colonizing the processing equipment or especially resistant to cleaning and disinfection.     

Application of 5′-Nuclease PCR for Quantitative Detection of Listeria monocytogenes in Pure Cultures, Water, Skim Milk, and Unpasteurized Whole Milk

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Countless adaptations and applications have been described, including quantitative PCR and the latest innovation, real-time PCR. In real-time PCR, e.g., the 5′-nuclease chemistry renders the automated and direct detection and quantification of PCR products possible (P. M. Holland et al., Proc. Natl. Acad. Sci. USA 88:7276–7280, 1991). We present an assay for the quantitative detection of Listeria monocytogenes based on the 5′-nuclease PCR using a 113-bp amplicon from the listeriolysin O gene (hlyA) as the target. The assay was positive for all isolates of L. monocytogenes tested (65 isolates including the type strain) and negative for all other Listeria strains (16 isolates from five species tested) and several other bacteria (18 species tested). The application of 5′-nuclease PCR in diagnostics requires a quantitative sample preparation step. Several magnetic bead-based strategies were evaluated, since these systems are simple and relatively easy to automate. The combination of nonspecific binding of bacteria to paramagnetic beads, with subsequent DNA purification by use of the same beads, gave the most satisfactory result. The detection limit was approximately 6 to 60 CFU, quantification was linear over at least 7 log units, and the method could be completed within 3 h. In conclusion, a complete quantitative method for L. monocytogenes in water and in skimmed and raw milk was developed.     

Microbial flora of in-use, display eye shadow testers and bacterial challenges of unused eye shadows.

We surveyed 15 different brands of eye shadow on display for customer use in different retail stores for microbial contamination. This was the first reported microbial surveillance of in-use eye shadow display testers in retail establishments. Cultures were obtained at each retail store. Sterile dacron swabs were rolled and rubbed over the entire used surface of each shadow, and each inoculum was streaked onto the surfaces of blood agar plates. Of the 1,345 individual samples obtained, 67% were contaminated with one or more species of microorganisms representing the genera Staphylococcus, Micrococcus, Corynebacterium, Acinetobacter, Bacillus, and Moraxella. We also purchased two different brands of water-miscible eye shadows in replicate unit containers. Each brand was challenged separately with a few hundred to several thousand colony-forming units of Staphylococcus aureus, Pseudomonas aeruginosa, or Acinetobacter calcoaceticus. Both brands permitted growth of P. aeruginosa but not growth of S. aureus. A. calcoaceticus was inhibited after inoculation into one brand. With the other brand, the inoculum of Acinetobacter multiplied in one of the two different lots tested. This experimental challenge procedure can serve as a useful model system for studying the behavior of microbes in eye shadows and similar matrices.     

Isolation and characterization of Listeria monocytogenes from tropical seafood of Kerala, India

Listeria monocytogenes, which is an intracellular pathogen, causes various illnesses in human as well as in animals. The pathogenicity of this organism depends upon the presence of different virulence genes. A total of 324 tropical seafood and fishery environmental samples were screened for L. monocytogenes. The incidence of the human pathogenic species L. monocytogenes was 1.2 % of the samples. Listeria spp. was detected in 32.3, 27.1, and 5 % of fresh, frozen, and dry fish samples, respectively. Listeria innocua was found to be the most prevalent species of Listeria in the tropical seafood and environmental samples of Kerala. Listeria monocytogenes and L. innocua isolates were confirmed by multiplex PCR. L. monocytogenes isolates from the four positive samples showed phosphatidylinositol-specific phospholipase C reaction on Chromocult® Listeria selective agar. Molecular characterization of L. monocytogenes isolates for virulence genes revealed the presence of β-hemolysin (hly), plcA, actA, metalloprotease (mpl), iap and prfA genes in all the isolates recovered from the positive samples.     

Occurrence ofListeria monocytogenes in bovine milk in Hyderabad, Pakistan

In the present study 200 milk samples (50 each from cow, buffalo, market and mastitic milk) were collected from Hyderabad. Among the isolated presumptiveListeria spp., all the isolates were identified asListeria monocytogenes and showed a prevalence of 6, 8, 10 and 12%, respectively. Through serotyping analysis all isolates were confirmed asListeria genus, since they agglutinated with polyvalent ‘O’ serum; 12 of the 18L. monocytogenes isolates were ascribed to serotype 1, and 6 to serotype 4. Furthermore, the pathogenicity was investigated in rabbit and mice through Anton and inoculation tests, respectively. The organism produces keratoconjunctivitis in rabbit while it causes death and presence of necrotic foci in liver, spleen and kidneys of mice.Listeria monocytogenes was found sensitive to ampicillin, chloramphenicol, ciprofloxacin, gentamycin and kanamycin.     

Utilization of Oligopeptides by Listeria monocytogenes Scott A

For effective utilization of peptides, Listeria monocytogenes possesses two different peptide transport systems. The first one is the previously described proton motive force (PMF)-driven di- and tripeptide transport system (A. Verheul, A. Hagting, M.-R. Amezaga, I. R. Booth, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 61:226–233, 1995). The present results reveal that L. monocytogenes possesses an oligopeptide transport system, presumably requiring ATP rather than the PMF as the driving force for translocation. Experiments to determine growth in a defined medium containing peptides of various lengths suggested that the oligopeptide permease transports peptides of up to 8 amino acid residues. Peptidase activities towards several oligopeptides were demonstrated in cell extract from L. monocytogenes, which indicates that upon internalization, the oligopeptides are hydrolyzed to serve as sources of amino acids for growth. The peptide transporters of the nonproteolytic L. monocytogenes might play an important role in foods that harbor indigenous proteinases and/or proteolytic microorganisms, since Pseudomonas fragi as well as Bacillus cereus was found to enhance the growth of L. monocytogenes to a large extent in a medium in which the milk protein casein was the sole source of nitrogen. In addition, growth stimulation was elicited in this medium when casein was hydrolyzed by using purified protease from Bacillus licheniformis. The possible contribution of the oligopeptide transport system in the establishment of high numbers of L. monocytogenes cells in fermented milk products is discussed.     

Distribution and Characteristics of Listeria monocytogenes Isolates from Surface Waters of the South Nation River Watershed, Ontario, Canada

Listeria monocytogenes is a facultative intracellular pathogen thought to be widely distributed in the environment. We investigated the prevalence and characteristics of L. monocytogenes isolates from surface waters derived from catchments within the South Nation River watershed (Ontario, Canada). This watershed is dominated by urban and rural development, livestock and crop production, and wildlife habitats. From June to November 2005, a total of 314 surface water samples were collected biweekly from 22 discrete sampling sites characterized by various upstream land uses. Presumptive Listeria spp. were isolated using a selective enrichment and isolation procedure, and 75 L. monocytogenes isolates were identified based on colony morphology, hemolytic activity, and amplification of three pathogenicity genes: iap, inlA, and hlyA. Thirty-two of 314 (10%) surface water samples were positive for the presence of L. monocytogenes, but detection ranged between 0 and 27% depending on the sampling date. Isolates belonging to serovar group 1/2a, 3a (50%) and group 4b, 4d, 4e (32%) were dominant. L. monocytogenes populations were resolved into 13 EcoRI ribotypes and 21 ApaI and 21 AscI pulsotypes. These had Simpson indexes of discrimination of up to 0.885. Lineage I-related isolates were dominant (61%) during the summer, whereas lineage II isolates were dominant (77%) in the fall. Isolates were, on average, resistant to 6.1 ± 2.1 antibiotics out of 17 tested. Half of the L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, and some isolates were found to be moderately to highly virulent by in vitro Caco-2 plaque formation assay (up to 28% of entry). There was a statistically significant link between the occurrence of L. monocytogenes and proximity to an upstream dairy farm and degree of cropped land. Our data indicate that L. monocytogenes is widespread in the studied catchments, where it could represent a public health issue related to agricultural land use.     

Reservoirs of Listeria Species in Three Environmental Ecosystems

Soil and water are suggested to represent pivotal niches for the transmission of Listeria monocytogenes to plant material, animals, and the food chain. In the present study, 467 soil and 68 water samples were collected in 12 distinct geological and ecological sites in Austria from 2007 to 2009. Listeria was present in 30% and 26% of the investigated soil and water samples, respectively. Generally, the most dominant species in soil and water samples were Listeria seeligeri, L. innocua, and L. ivanovii. The human- and animal-pathogenic L. monocytogenes was isolated exclusively from 6% soil samples in regions A (mountainous region) and B (meadow). Distinct ecological preferences were observed for L. seeligeri and L. ivanovii, which were more often isolated from wildlife reserve region C (Lake Neusiedl) and from sites in proximity to wild and domestic ruminants (region A). The higher L. monocytogenes detection and antibiotic resistance rates in regions A and B could be explained by the proximity to agricultural land and urban environment. L. monocytogenes multilocus sequence typing corroborated this evidence since sequence type 37 (ST37), ST91, ST101, and ST517 were repeatedly isolated from regions A and B over several months. A higher L. monocytogenes detection and strain variability was observed during flooding of the river Schwarza (region A) and Danube (region B) in September 2007, indicating dispersion via watercourses.     

Prevalence of Campylobacter spp., Escherichia coli, and Salmonella Serovars in Retail Chicken, Turkey, Pork, and Beef from the Greater Washington, D.C., Area

A total of 825 samples of retail raw meats (chicken, turkey, pork, and beef) were examined for the presence of Escherichia coli and Salmonella serovars, and 719 of these samples were also tested for Campylobacter spp. The samples were randomly obtained from 59 stores of four supermarket chains during 107 sampling visits in the Greater Washington, D.C., area from June 1999 to July 2000. The majority (70.7%) of chicken samples (n = 184) were contaminated with Campylobacter, and a large percentage of the stores visited (91%) had Campylobacter-contaminated chickens. Approximately 14% of the 172 turkey samples yielded Campylobacter, whereas fewer pork (1.7%) and beef (0.5%) samples were positive for this pathogen. A total of 722 Campylobacter isolates were obtained from 159 meat samples; 53.6% of these isolates were Campylobacter jejuni, 41.3% were Campylobacter coli, and 5.1% were other species. Of the 212 chicken samples, 82 (38.7%) yielded E. coli, while 19.0% of the beef samples, 16.3% of the pork samples, and 11.9% of the turkey samples were positive for E. coli. However, only 25 (3.0%) of the retail meat samples tested were positive for Salmonella. Significant differences in the bacterial contamination rates were observed for the four supermarket chains. This study revealed that retail raw meats are often contaminated with food-borne pathogens; however, there are marked differences in the prevalence of such pathogens in different meats. Raw retail meats are potential vehicles for transmitting food-borne diseases, and our findings stress the need for increased implementation of hazard analysis of critical control point (HACCP) and consumer food safety education efforts.     

Comparison of the Prevalences and Diversities of Listeria Species and Listeria monocytogenes in an Urban and a Rural Agricultural Watershed

Foods and related processing environments are commonly contaminated with the pathogenic Listeria monocytogenes. To investigate potential environmental reservoirs of Listeria spp. and L. monocytogenes, surface water and point source pollution samples from an urban and a rural municipal water supply watershed in Nova Scotia, Canada, were examined over 18 months. Presumptive Listeria spp. were cultured from 72 and 35% of rural and urban water samples, respectively, with 24% of the positive samples containing two or three different Listeria spp. The L. innocua (56%) and L. welshimeri (43%) groups were predominant in the rural and urban watersheds, respectively. Analysis by the TaqMan assay showed a significantly (P < 0.05) higher prevalence of L. monocytogenes of 62% versus 17% by the culture-based method. Both methods revealed higher prevalences in the rural watershed and during the fall and winter seasons. Elevated Escherichia coli (≥100 CFU/100 ml) levels were not associated with the pathogen regardless of the detection method. Isolation of Listeria spp. were associated with 70 times higher odds of isolating L. monocytogenes (odds ratio = 70; P < 0.001). Serogroup IIa was predominant (67.7%) among the 285 L. monocytogenes isolates, followed by IVb (16.1%), IIb (15.8%), and IIc (0.4%). L. monocytogenes was detected in cow feces and raw sewage but not in septic tank samples. Pulsotyping of representative water (n = 54) and local human (n = 19) isolates suggested genetic similarities among some environmental and human L. monocytogenes isolates. In conclusion, temperate surface waters contain a diverse Listeria species population and could be a potential reservoir for L. monocytogenes, especially in rural agricultural watersheds.     

Coselection of Cadmium and Benzalkonium Chloride Resistance in Conjugative Transfers from Nonpathogenic Listeria spp. to Other Listeriae

Resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) may be an important contributor to the ability of Listeria spp. to persist in the processing plant environment. Although a plasmid-borne disinfectant resistance cassette (bcrABC) has been identified in Listeria monocytogenes, horizontal transfer of these genes has not been characterized. Nonpathogenic Listeria spp. such as L. innocua and L. welshimeri are more common than L. monocytogenes in food processing environments and may contribute to the dissemination of disinfectant resistance genes in listeriae, including L. monocytogenes. In this study, we investigated conjugative transfer of resistance to BC and to cadmium from nonpathogenic Listeria spp. to other nonpathogenic listeriae, as well as to L. monocytogenes. BC-resistant L. welshimeri and L. innocua harboring bcrABC, along with the cadmium resistance determinant cadA2, were able to transfer resistance to other nonpathogenic listeriae as well as to L. monocytogenes of diverse serotypes, including strains from the 2011 cantaloupe outbreak. Transfer among nonpathogenic Listeria spp. was noticeably higher at 25°C than at 37°C, whereas acquisition of resistance by L. monocytogenes was equally efficient at 25 and 37°C. When the nonpathogenic donors were resistant to both BC and cadmium, acquisition of cadmium resistance was an effective surrogate for transfer of resistance to BC, suggesting coselection between these resistance attributes. The results suggest that nonpathogenic Listeria spp. may behave as reservoirs for disinfectant and heavy metal resistance genes for other listeriae, including the pathogenic species L. monocytogenes.     

Molecular Ecology of Listeria monocytogenes: Evidence for a Reservoir in Milking Equipment on a Dairy Farm

A longitudinal study aimed to detect Listeria monocytogenes on a New York State dairy farm was conducted between February 2004 and July 2007. Fecal samples were collected every 6 months from all lactating cows. Approximately 20 environmental samples were obtained every 3 months. Bulk tank milk samples and in-line milk filter samples were obtained weekly. Samples from milking equipment and the milking parlor environment were obtained in May 2007. Fifty-one of 715 fecal samples (7.1%) and 22 of 303 environmental samples (7.3%) were positive for L. monocytogenes. A total of 73 of 108 in-line milk filter samples (67.6%) and 34 of 172 bulk tank milk samples (19.7%) were positive for L. monocytogenes. Listeria monocytogenes was isolated from 6 of 40 (15%) sampling sites in the milking parlor and milking equipment. In-line milk filter samples had a greater proportion of L. monocytogenes than did bulk tank milk samples (P < 0.05) and samples from other sources (P < 0.05). The proportion of L. monocytogenes-positive samples was greater among bulk tank milk samples than among fecal or environmental samples (P < 0.05). Analysis of 60 isolates by pulsed-field gel electrophoresis (PFGE) yielded 23 PFGE types after digestion with AscI and ApaI endonucleases. Three PFGE types of L. monocytogenes were repeatedly found in longitudinally collected samples from bulk tank milk and in-line milk filters.Listeria monocytogenes can cause listeriosis in humans. This illness, despite being underreported, is an important public health concern in the United States (23) and worldwide. According to provisional incidence data provided by the Centers for Disease Control and Prevention (CDC), 762 cases of listeriosis were reported in the United States in 2007. In previous years (2003 to 2006), the number of reported annual listeriosis cases in the United States ranged between 696 and 896 cases per year (5).Exposure to food-borne L. monocytogenes may cause fever, muscle aches, and gastroenteritis (30), but does not usually cause septicemic illness in healthy nonpregnant individuals (7, 30). Elderly and immunocompromised people, however, are susceptible to listeriosis (22, 10), and they may develop more-severe symptoms (10). Listeriosis in pregnant women may cause abortion (22, 30) or neonatal death (22).Dairy products have been identified as the source of several human listeriosis outbreaks (4, 7, 10, 22). Listeria is ubiquitous on dairy farms (26), and it has been isolated from cows'' feces, feed (3, 26), and milk (21, 35). In ruminants, L. monocytogenes infections may be asymptomatic or clinical. Clinical cases typically present with encephalitis and uterine infections, often resulting in abortion (26, 39). Both clinically infected and healthy animals have been reported to excrete L. monocytogenes in their feces (20), which could eventually cause contamination of the bulk tank milk or milk-processing premises (39).On-farm epidemiologic research provides science-based information to improve farming and management practices. The Regional Dairy Quality Management Alliance (RDQMA) launched a combined United States Department of Agriculture (USDA)-RDQMA pilot project in January 2004 to scientifically validate intervention strategies in support of recommended best management practices among northeast dairy farms. The primary goal of the project was to track dynamics of infectious microorganisms on well-characterized dairy farms. Target species included Salmonella spp. (6, 36, 37), Mycobacterium avium subsp. paratuberculosis (13, 24), and L. monocytogenes.The objectives of this study were to describe the presence of L. monocytogenes on a dairy farm over time and to perform molecular subtyping by pulsed-field gel electrophoresis (PFGE) on L. monocytogenes isolates obtained from bulk tank milk, milk filters, milking equipment, feces, and the environmental samples to identify diversity among L. monocytogenes strains, persistence, and potential sources of bulk tank milk contamination.     

Molecular Epidemiological Survey of Listeria monocytogenes in Seafoods and Seafood-Processing Plants

To evaluate the role of seafoods in the epidemiology of human listeriosis and the role of the processing environment as a source of Listeria monocytogenes in seafood products, 305 L. monocytogenes isolates were characterized by multilocus enzyme electrophoresis using 21 genetic loci and restriction enzyme analysis of total DNA. Forty-four isolates were recovered from patients in Norway; 93 were isolated from seafoods, seafood-processing environments, and seawater from 55 different producers; and the remaining 168 isolates originated from six seafood-processing plants and one transport terminal examined in detail for L. monocytogenes. The patient isolates fell into 11 electrophoretic types, with four of them being responsible for 77% of the listeriosis cases in 1992 to 1996. Isolates from Norwegian seafoods and processing environments showed great genetic diversity, indicating that seafoods and seafood-processing environments do not offer a niche for specific L. monocytogenes strains. On the other hand, isolates from individual processing plants were genetically more homogenous, showing that plants are likely to be colonized with specific subclones of L. monocytogenes. The isolation of identical subclones of L. monocytogenes from both human patients and seafoods, including ready-to-eat products, suggests that such products may have been possible sources for listeriosis cases in Norway.     

Anthropogenic impact on the presence of L. monocytogenes in soil, fruits, and vegetables

The aim of this study was to determine the prevalence of Listeria sp. and Listeria monocytogenes in soil samples with reference to type of fertilizers (natural and artificial) and distance from places intensively exploited by men, as well as to determine the relationship between the presence of L. monocytogenes in the soil and in fruits and vegetables. The examined 1,000 soil samples originated from 15 different areas, whilst 140 samples of fruits and 210 samples of vegetables were collected from those areas. L. monocytogenes was isolated only from 5.5 % of all soil samples coming exclusively from meadows intensively grazed by cattle (27.8 %) and areas near food processing plants (25 %) and wild animal forests (24 %). Listeria sp. and L. monocytogenes were not present on artificially fertilized areas and wastelands. L. monocytogenes was detected in 10 % of samples of strawberry, 15 % of potato samples, and 5 % of parsley samples. Our data indicate that Listeria spp. and particularly L. monocytogenes were found in the soil from (1) arable lands fertilized with manure, (2) pasture (the land fertilized with feces of domestic animals), and (3) forests (again, the land fertilized with feces of animals, not domestic but wild). The bacteria were not detected in the soil samples collected at (1) artificially fertilized arable lands and (2) wastelands (the lands that were not fertilized with manure or animal feces). Moreover, a correlation was determined in the presence of L. monocytogenes between soil samples and samples of the examined fruits and vegetables.