International Life Sciences Institute North America Listeria monocytogenes strain collection: development of standard Listeria monocytogenes strain sets for research and validation studies

Research and development efforts on bacterial foodborne pathogens, including the development of novel detection and subtyping methods, as well as validation studies for intervention strategies can greatly be enhanced through the availability and use of standardized strain collections. These types of strain collections are available for some foodborne pathogens, such as Salmonella and Escherichia coli. We have developed a standard Listeria monocytogenes strain collection that has not been previously available. The strain collection includes (i) a diversity set of 25 isolates chosen to represent a genetically diverse set of L. monocytogenes isolates as well as a single hemolytic Listeria innocua strain and (ii) an outbreak set, which includes 21 human and food isolates from nine major human listeriosis outbreaks that occurred between 1981 and 2002. The diversity set represents all three genetic L. monocytogenes lineages (I, n = 9; II, n = 9; and III, n = 6) as well as nine different serotypes. Molecular subtyping by EcoRI automated ribotyping and pulsed-field gel electrophoresis (PFGE) with AscI and ApaI separated the 25 isolates in the diversity set into 23 ribotypes and 25 PFGE types, confirming that this isolate set represents considerable genetic diversity. Molecular subtyping of isolates in the outbreak set confirmed that human and food isolates were identical by ribotype and PFGE, except for human and food isolates for two outbreaks, which displayed related but distinct PFGE patterns. Subtype and source data for all isolates in this strain collection are available on the Internet and are linked to the PathogenTracker database (www.pathogentracker.com), which allows the addition of new, relevant information on these isolates, including links to publications that have used isolates from this collection. We have thus developed a core L. monocytogenes strain collection, which will provide a resource for L. monocytogenes research and development efforts with centralized Internet-based data curation and integration.     

Subtyping of Listeria monocytogenes at the haplotype level by Fourier transform infrared (FT-IR) spectroscopy and multivariate statistical analysis

Listeria monocytogenes is a widespread foodborne pathogen that represents a major concern with respect to food safety. Rapid identification of this bacterium at a subspecies level is important to trace back an outbreak and improve risk-based inspection programs. A method for subtyping L. monocytogenes at the serotype and haplotype levels was developed using Fourier transform infrared (FT-IR) reflectance microscopy. Thirty strains of L. monocytogenes belonging to four different PCR serotypes (1/2a, 1/2b, 4b, and 4c) that had previously been characterized by Multilocus genotyping (MLGT) and Pulsed field gel electrophoresis (PFGE) assays were used in this study. The FT-IR based identification and classification was compared to the known MLGT and PFGE subtyping of the L. monocytogenes. Canonical variate analysis (CVA) of the spectra resulted in 96.6% correct identification of L. monocytogenes at the serotype level. Hierarchical cluster analysis (HCA) and CVA of the spectra showed 91.7% correct identification of strains at the haplotype level consistent with their MLGT groupings. FT-IR spectra of strains were also differentiated correctly in accordance with their PFGE haplotyping. Additionally, by using HCA of FT-IR spectra, each bacterium was differentiated at the strain level. Starting from a pure culture, this method enabled classification of L. monocytogenes at the serotype, haplotype, and/or strain level within 18 h, which is faster and potentially less expensive than the molecular methods and previous FT-IR methods. This is the first report of the identification of L. monocytogenes at the haplotype level using FT-IR.     

Transfer of persistent Listeria monocytogenes contamination between food-processing plants associated with a dicing machine

The possibility of the transfer of persistent Listeria monocytogenes contamination from one plant to another with a dicing machine was evaluated, and possible reasons for persistent contamination were analyzed. A dicing machine that diced cooked meat products was transferred from plant A to plant B and then to plant C. After the transfer of the dicing machine, L. monocytogenes PFGE type I, originally found in plant A, was soon also found in plants B and C. This L. monocytogenes PFGE type I caused persistent contamination of the dicing lines in plants B and C. The persistent L. monocytogenes strain and three nonpersistent L. monocytogenes strains found in the dicing line of plant C were tested for adherence to stainless steel surfaces and minimal inhibitory concentrations of a quaternary ammonium compound and sodium hypochlorite, disinfectants widely used in the dicing lines. The persistent strain showed significantly higher adherence to stainless steel surfaces than did the nonpersistent strains. The minimal inhibitory concentrations of sodium hypochlorite were similar for all strains, and the minimal inhibitory concentrations of the quaternary ammonium compound for three of the L. monocytogenes PFGE types, including the persistent PFGE type, were high. All persistent L. monocytogenes PFGE type I isolates were found in an area with high hygienic standards, with the dicing machine being the first point of contamination. These observations show that the dicing machine sustained the contamination and suggest that the dicing machine transferred the persistent L. monocytogenes PFGE type from one plant to another.     

Development of a multilocus variable-number of tandem repeat typing method for Listeria monocytogenes serotype 4b strains

Listeria monocytogenes serotype 4b strains have been identified as the causative agent in many human listeriosis epidemics as well as in a considerable number of sporadic cases. Due to the genetic homogeneity of serotype 4b isolates, development of rapid subtyping methods with high discriminatory power for serotype 4b isolates is required to allow for improved outbreak detection and source tracking. In this study, multilocus variable-number tandem repeat analysis (MLVA) was developed and used to characterize 60 serotype 4b isolates from various sources. All isolates were also characterized by automated EcoRI ribotyping, single enzyme pulsed-field gel electrophoresis (PFGE) with ApaI, and a multilocus sequence typing (MLST) scheme targeting six virulence and virulence-associated genes. Discriminatory power of MLVA (as determined by Simpson Index of Discrimination) was higher than the discriminatory power of any of the other three methods. MLVA markers targeted were found to be stable and did not change when three isolates were passaged daily for 70 days. Cluster analyses of MLVA, PFGE and MLST consistently grouped the same isolates into three major clusters, each of which includes one of the three major L. monocytogenes epidemic clones (i.e., ECI, ECIa and ECII). We conclude that the MLVA method described here (i) provides for more discriminatory subtyping of L. monocytogenes serotype 4b strains than the other three methods, (ii) identifies three major groups within the serotype 4b, which are consistent with the groups identified by other subtyping methods, and (iii) is easy to interpret. Use of MLVA may thus be recommended for subtyping of serotype 4b isolates, including as a secondary more discriminatory subtyping method that could be used after initial isolate characterization by PFGE or ribotyping.     

Antimicrobial resistance of Listeria monocytogenes isolated from various cabbage farms and packing sheds in Texas

Twenty-one isolates of Listeria monocytogenes from cabbage, environmental, and water samples were evaluated for antimicrobial resistance by the disk diffusion method. Ninety-five percent (20 of 21) of the isolates tested were resistant to two or more antimicrobial agents. This finding is significant, since multiresistant strains of Listeria spp. are not commonly found in nature. Eighty-five percent (17 of 20) of the multiresistant strains were resistant to penicillin, and the remaining multiresistant isolates were somewhat sensitive to penicillin. A multiresistant strain showing intermediate sensitivity to penicillin was resistant to gentamicin. One isolate was susceptible to all antimicrobial agents except penicillin. Penicillin- and gentamicin-resistant L. monocytogenes have not previously been reported from human, food, or environmental samples. This study provides evidence of the emergence of multiresistant L. monocytogenes strains, pointing to an increase in the potential threat to human health posed by this pathogen.     

Listeria monocytogenes in Gorgonzola: subtypes, diversity and persistence over time

L. monocytogenes represents a primary concern in the production of Gorgonzola, a Protected Designation of Origin (PDO) Italian blue-veined cheese produced only in the Piedmont and Lombardy regions. L. monocytogenes isolates (N=95) obtained from Gorgonzola rinds, paste, and production/ripening environments were serotyped and then genotyped using Pulsed Field Gel Electrophoresis (PFGE). The goal of this study was to investigate the variability of L. monocytogenes PFGE-types across different PDO Gorgonzola manufacturers (N=22). The majority of the strains (88%) were serotyped as 1/2a. PFGE identified 2 major pulse-types grouping 62 strains, detected from different plants and years, suggesting the presence of persistent and niche-adapted L. monocytogenes. In 9 plants, environmental strains shared the same pulse-types with strains from rinds or paste, suggesting a possible transmission pathway. Encouragingly, L. monocytogenes was retrieved from only 1 paste, indicating that production processes were under control in 21 plants. In the remaining plant, un-effective pasteurization or cross-contamination during production processes could be the cause of the contamination. Consequently, it is imperative that producers operate under the total respect of the Good Manufacturing Practices and following the principles of the Hazard Analysis Critical Control Point plans, in order to contain contamination throughout the whole processing.     

单核细胞增生李斯特菌的毒力基因检测及PFGE分型的研究

目的了解福建省食品中单核细胞增生李斯特菌携带hly、plcA、plcB和prfA毒力基因的情况及脉冲场凝胶电泳(PFGE)的分型情况。方法将hly、plcA、plcB和prfA基因作为靶序列选取4对引物,通过聚合酶链反应(PCR)检测61株单核细胞增生李斯特菌和2株可疑单核细胞增生李斯特菌的毒力基因,用PulseNet单核细胞增生李斯特菌标准方法进行7株单核细胞增生李斯特菌的PFGE分子分型。结果61株单核细胞增生李斯特菌毒力基因为hly 、plcA 、plcB 和prfA ,2株可疑单核细胞增生李斯特菌的毒力基因分别为hly-、plcA 、plcB-、prfA-和hly-、plcA-、plcB-、prfA-。7株单核细胞增生李斯特菌的PFGE分为5个型。结论实验结果表明福建省食品中分离到的单核细胞增生李斯特菌均含有hly、plcA、plcB和prfA基因,属于致病株。对2株可疑单核细胞增生李斯特菌进行了进一步的鉴定,排除了单核细胞增生李斯特菌,该毒力基因检测方法可用于可疑单核细胞增生李斯特菌的进一步鉴别。7株菌中有两对2株PFGE型别一致,一致的菌株来自不同年份不同销售地点的同一品牌,应用PFGE方法可以进一步调查该厂冻鸡肉中单核细胞增生李斯特菌的传播途径和污染源。     

Listeria monocytogenes--threat to a safe food supply: a review

Listeria monocytogenes can cause circling disease, encephalitis, meningitis, septicemia, and mastitis in dairy cattle. Shedding of the pathogen from the udder or contamination from the environment can lead to presence of L. monocytogenes in raw milk. Surveys indicate the pathogen is in about 4% of US raw milks. Although HTST pasteurization commonly inactivates L. monocytogenes, evidence suggests that under unusual circumstances minimal survival is possible. The pathogen grows well in liquid dairy products at 4 to 35 degrees C and achieves higher populations in chocolate than in unflavored milks. When present in cheese milk, growth of L. monocytogenes may be retarded but not stopped by lactic starter cultures. The pathogen is concentrated in the curd with only a small fraction of cells in milk appearing in whey. Once in curd, the behavior of the pathogen ranges from growth (feta cheese making) to death of most but not all cells (cottage cheese making). During ripening of cheese, the numbers of L. monocytogenes decrease gradually (as in Cheddar or Colby cheese), decrease precipitously early during ripening, and then stabilize (as in blue cheese) or increase markedly (as in Camembert cheese). Consumption of foods containing L. monocytogenes can lead to listeriosis in susceptible humans (adults with a compromised immune system), pregnant women, and infants). In large outbreaks of human listeriosis, mortality rates of ca. 30% are common.     

High-resolution genotyping of Listeria monocytogenes by fluorescent amplified fragment length polymorphism analysis compared to pulsed-field gel electrophoresis, random amplified polymorphic DNA analysis, ribotyping, and PCR-restriction fragment length polymorphism analysis

The purpose of this study was to evaluate fluorescent amplified fragment length polymorphism (AFLP) analysis for the inter- and intraspecies differentiation of a collection of 96 strains of Listeria monocytogenes and 10 non-L. monocytogenes strains representing six other Listeria species of different origin. The AFLP technique was compared with three other molecular typing methods--ribotyping, random amplified polymorphic DNA analysis (RAPD), and pulsed-field gel electrophoresis (PFGE)--in terms of discriminatory ability. PCR-restriction fragment length polymorphism was included for virulence gene allele characterization. The 96 L. monocytogenes strains were divided into two major clusters by AFLP fingerprinting at a similarity level of 82% in concordance with the results of PFGE, RAPD, and ribotyping. One main cluster consisted of all of the 24 L. monocytogenes hly allele 1 strains, while another main cluster consisted of all of the 72 L. monocytogenes hly allele 2 strains. This indicates the existence of two distinct phylogenetic divisions. Isolates of the remaining Listeria species were not included in the clusters. AFLP, PFGE, and RAPD typing were highly discriminatory methods, with discrimination (D) indices of 0.974, 0.969, and 0.954, respectively, whereas ribotyping had a lower D index of 0.874. AFLP, PFGE, and RAPD typing showed some level of agreement in terms of strain grouping and differentiation. However, all three methods subdivided types of strains grouped by the other methods. Isolates with identical DNA profiles were distributed across the spectrum of origin. It was not possible to associate certain types with specific food sectors or clinical cases, which is indicative of the spread of L. monocytogenes clones across species. Overall, AFLP fingerprinting was suitable for the high-resolution genotyping of L. monocytogenes and had an equally high or higher differentiation power compared to PFGE or RAPD typing.     

Occurrence and ribotypes of Listeria monocytogenes in Gorgonzola cheeses

In this study 1656 Gorgonzola cheeses, collected from October 2003 to April 2004 in the same industrial plant located in Lombardia (Italy), were analysed in order to evaluate their level of contamination with Listeria monocytogenes after packaging, as well as at the end of the shelf life. A subset of Gorgonzola isolates was submitted to automated EcoRI ribotyping to evaluate their DUP-IDs (DuPont identification library code) and their level of genetic diversity. The isolate ribotyping profiles were included in an on-line database named PathogenTracker database. The strain DUP-IDs and the similarities between Gorgonzola isolates and PathogenTracker human sporadic strains allowed to evaluate the potential virulence of Gorgonzola-associated strains. The L. monocytogenes detection rates observed in the cheese samples monitored after packaging and at the end of the shelf life were 2.1% and 4.8%, respectively. Seventy percent of the strains genotyped were classified in the same ribotype, labelled as 204 S5, indicating that L. monocytogenes population associated to Gorgonzola cheese shows a low level of genetic diversity. Ninety percent of the strains were classified in DUP-IDs belonging to the II pathogenicity lineage identified for L. monocytogenes. That lineage includes serotype 1/2a, 1/2c and 3c strains, associated to the 35% of the human sporadic isolates described in the literature as causing listeriosis. Moreover, 16.7% of Gorgonzola isolates showed a similarity >or=99% with PathogenTracker human sporadic strains. The results of this study showed that the incidence of L. monocytogenes in Gorgonzola cheeses commercialised by the plant tested was lower than that observed in other Italian blue-veined cheeses by other authors. However, it increased during cheese storage and it become double at the end of the cheese shelf life, ranging from 30 to 60 days after packaging.     

Listeria monocytogenes virulence and pathogenicity,a food safety perspective

Several virulence factors of Listeria monocytogenes have been identified and extensively characterized at the molecular and cell biologic levels, including the hemolysin (listeriolysin O), two distinct phospholipases, a protein (ActA), several internalins, and others. Their study has yielded an impressive amount of information on the mechanisms employed by this facultative intracellular pathogen to interact with mammalian host cells, escape the host cell's killing mechanisms, and spread from one infected cell to others. In addition, several molecular subtyping tools have been developed to facilitate the detection of different strain types and lineages of the pathogen, including those implicated in common-source outbreaks of the disease. Despite these spectacular gains in knowledge, the virulence of L. monocytogenes as a foodborne pathogen remains poorly understood. The available pathogenesis and subtyping data generally fail to provide adequate insight about the virulence of field isolates and the likelihood that a given strain will cause illness. Possible mechanisms for the apparent prevalence of three serotypes (1/2a, 1/2b, and 4b) in human foodborne illness remain unidentified. The propensity of certain strain lineages (epidemic clones) to be implicated in common-source outbreaks and the prevalence of serotype 4b among epidemic-associated stains also remain poorly understood. This review first discusses current progress in understanding the general features of virulence and pathogenesis of L. monocytogenes. Emphasis is then placed on areas of special relevance to the organism's involvement in human foodborne illness, including (i) the relative prevalence of different serotypes and serotype-specific features and genetic markers; (ii) the ability of the organism to respond to environmental stresses of relevance to the food industry (cold, salt, iron depletion, and acid); (iii) the specific features of the major known epidemic-associated lineages; and (iv) the possible reservoirs of the organism in animals and the environment and the pronounced impact of environmental contamination in the food processing facilities. Finally, a discussion is provided on the perceived areas of special need for future research of relevance to food safety, including (i) theoretical modeling studies of niche complexity and contamination in the food processing facilities; (ii) strain databases for comprehensive molecular typing; and (iii) contributions from genomic and proteomic tools, including DNA microarrays for genotyping and expression signatures. Virulence-related genomic and proteomic signatures are expected to emerge from analysis of the genomes at the global level, with the support of adequate epidemiologic data and access to relevant strains.     

Diversity of Listeria monocytogenes isolates from cold-smoked salmon produced in different smokehouses as assessed by Random Amplified Polymorphic DNA analyses

One hundred and forty-eight Listeria monocytogenes isolates originating from vacuum packed cold-smoked salmon produced in 10 different Danish smokehouses were compared by Random Amplified Polymorphic DNA (RAPD) profiling. A total of 16 different reproducible RAPD profiles were obtained using a standardised RAPD analysis by four primers separately. The grouping of the 148 strains was exactly the same for the four primers used. For a sub-set of 20 strains typed by Pulsed Field Gel Electrophoresis (PFGE), only one strain was allocated into a different group as compared to the grouping by RAPD typing. Different RAPD types dominated in products from different smokehouses. Some identical RAPD types were isolated in several smokehouses. In each of four smokehouses, one particular RAPD type could be repeatedly isolated from products. Each smokehouse/product carried its own specific RAPD type and this may indicate a possible persistence of closely related strains of L. monocytogenes in smokehouses.     

Bioprotective Leuconostoc strains against Listeria monocytogenes in fresh fruits and vegetables

Ten Leuconostoc mesenteroides and one Ln. citreum strains isolated from fresh fruit and vegetables were tested for their antagonistic capacity against Listeria monocytogenes. Genetic differences among strains were analyzed by Random Amplified Polymorphic DNA (RAPD). All the isolates clustered together and differed from the type strain Ln. mesenteroides ATCC 8293 as well as from Ln. fallax and Ln. citreum. Organic acids, hydrogen peroxide and bacteriocins were detected as main inhibition mechanisms. Characterization of culture supernatants from the bacteriocinogenic strains, CM135 and CM160 revealed a high resistance of antibacterial activity to temperature and pH, and a bactericidal mode of action against L. monocytogenes. Produced bacteriocins belonged to the Class IIa and sequencing of genes showed complete homology with mesentericin Y105. A study of the effect of the relative dose of pathogen and LAB on control of L. monocytogenes in wounds of Golden Delicious apples and Iceberg lettuce leaf cuts was performed. A comparison of the dose of bioprotective strain needed for a ten fold reduction of the viable pathogen concentration (ED(90)) revealed that strain CM160 was the most effective against L. monocytogenes. ED(90) values varied from 1.3.10(4) to 5.0.10(5) cfu.g(-1) or wound, at ranges of pathogen levels from 1.0.10(3) to 5.0.10(4) cfu.g(-1) of lettuce or wound of apple. The efficiency of the strains was also calculated as the ratio of the ED(90) value to the pathogen dose inoculated. The lowest ratio was found for strain CM160 at 5 to 50 cells of LAB per cell of pathogen. The strain offers potential application for prevention of the presence of L. monocytogenes in fresh fruit and vegetables.     

Molecular epidemiology and disinfectant susceptibility of Listeria monocytogenes from meat processing plants and human infections

We have investigated the molecular epidemiology of Listeria monocytogenes from the meat processing industry producing cold cuts and from cases of human listeriosis by discriminative pulsed-field gel electrophoresis (PFGE). A subset of the isolates was also investigated for susceptibility to a disinfectant based on quaternary ammonium compounds (QAC) frequently used in the meat processing industry. The purpose of this investigation was to obtain knowledge of sources, routes of contamination and genetic types of L. monocytogenes present along the production line in the meat processing industry, and to compare meat industry isolates and human isolates. Of the 222 isolates from four meat-processing plants, 200 were from two plants responsible for nearly 50% of the production of cold cuts in the Norwegian market. The strain collection included historical routinely sampled isolates (1989-2002) and isolates systematically sampled through a one year period (November 2001 to November 2002) from fresh meat and production environments in three plants. No isolates were obtained in samples from employees (throat, faeces). Human strains included all available reported isolates from Norwegian patients in selected time periods. The L. monocytogenes PFGE data showed a large genetic heterogeneity, with isolates separated into two genetic lineages and further subdivided into 56 different PFGE profiles. Certain profiles were observed on both sides of production (before and after heat treatment) indicating contamination of end products by fresh meat or fresh meat environments. While fresh meat isolates almost exclusively grouped within lineage I, isolates from end products showed a more balanced distribution between lineages I and II. Ten profiles were common among isolates from human and meat industry. Typing of human isolates identified a previously unrecognised outbreak. Generally, a higher QAC resistance incidence was observed among isolates from the meat processing industry than among human isolates although large plant to plant differences were indicated. No correlation between resistance and PFGE profile or resistance and persistence was observed.     

Screening of glutamate decarboxylase activity and bile salt resistance of human asymptomatic carriage, clinical, food, and environmental isolates of Listeria monocytogenes

Following consumption, stomach acidity is the first major barrier encountered by the food-borne pathogen Listeria monocytogenes. Analysis of low pH sensitivity and glutamate decarboxylase (GAD) acid resistance system of 14 isolates of L. monocytogenes carried asymptomatically by humans showed that levels of GAD activity were subjected to strain variation. Similar variations were observed for strains responsible for 18 cases of listeriosis, whereas in comparison, 13 strains isolated from food and food-processing plant environments showed lower GAD activity. Following survival of the stomach barrier, L. monocytogenes also has to resist bile salts encountered in the small intestines. Analysis revealed that all strains tested were able to grow in the presence of bile salts with concentrations as high as those encountered in the small intestines and had previously identified bile salt hydrolase (BSH) activity. Strain variation was observed but there was no relationship between the origin of the strains and the ability to degrade bile salts.     

Effects of physicochemical surface characteristics of Listeria monocytogenes strains on attachment to glass

Seven strains of Listeria monocytogenes frequently involved in foodborne disease (epidemic strains) and 14 sporadic strains were examined to compare the attachment and subsequent biofilm growth on glass slides at 37 degrees C. Epidemic strains at 3 h incubation had significantly higher attachment values than sporadic strains (P<0.001), but subsequent biofilm growth over 24 h was not dependent on initial attachment. To better understand this phenomenon, the surface hydrophobicity and charge, as well as the extracellular carbohydrate content of the 21 L. monocytogenes strains were studied to determine if these surface characteristics had an effect on bacterial attachment to glass. Hydrophobicity was measured by the bacterial adherence to hydrocarbon (BATH) and polystyrene adherence methods. Hydrophobicity values obtained with the BATH method were linearly correlated with those from the polystyrene adherence method (r=0.64, P<0.001), but no correlation was found between hydrophobicity and bacterial attachment to glass. Hydrophobicity and surface charge measured as electrophoretic mobility (EM) were correlated (r=0.77, P<0.001); however, there was no correlation between the degree of attachment and surface charge. Colorimetric measurements of the total extracellular carbohydrates revealed that attached cells produced significantly (P<0.05) higher levels than planktonic cells after a 3 h time period. Analysis of co-variance (Nested ANCOVA) furthermore demonstrated that total carbohydrates produced by planktonic cells had a significant positive effect on 24 h biofilm growth (P=0.006). This is the first report to indicate that the ability of a L. monocytogenes strain to produce high levels of extracellular carbohydrates may increase its ability to form a biofilm. Genetic studies targeting carbohydrate synthesis pathways of L. monocytogenes will be required to fully understand the importance of this observation.     

A cross-sectional study on the prevalence of Listeria monocytogenes and Salmonella in New York dairy herds

As part of our long-term objective of assessing risk for Listeria monocytogenes and Salmonella spp. in dairy herds, we carried out a cross-sectional study to determine the prevalence of the two organisms. The study population consisted of a sample of dairy herds enrolled in the Quality Milk Promotion Services at Cornell during the period of April 1998 to March 1999. The sample was stratified by geographical region to assure representation. Four hundred and four dairy farms were enrolled in the study. In-line milk filters were collected from each farm for bacteriological examination of L. monocytogenes and Salmonella spp. Four hypothesized risk factors were evaluated for their association with the likelihood of the presence of each of the two organisms using logistic regression analysis. Listeria monocytogenes was isolated from 51 (12.6%) of the milk filters. We found region-specific differences in the rate of farms with positive milk filters for this pathogen. Salmonella spp. were isolated from 6 (1.5%) milk filters. One isolate was confirmed as Salmonella enterica Serotype Typhimurium DT 104. There was no significant association between any of the hypothetical risk factors and the likelihood of Salmonella spp. isolation. Our study demonstrated that both L. monocytogenes and Salmonella spp. were prevalent in milk filters in New York dairy herds and that Salmonella was isolated at a significantly lower rate then L. monocytogenes.     

Serological and molecular ecology of Listeria monocytogenes isolates collected from 13 French pork meat salting-curing plants and their products

The purpose of this study was dual: 1. to evaluate the serotype distribution of 1028 Listeria monocytogenes isolates collected in 13 French salting factories and their products and 2. to identify sources of L. monocytogenes contamination in these factories and trace the routes of spread by PFGE (Pulsed-Field Gel Electrophoresis) typing. Serotypes 1/2a, 1/2b, 1/2c, 4b and 4e occurred. Pulsotype diversity was high among strains collected in plants and products. Furthermore, strains showing similar pulsotypes occurred on the same surfaces after an interval of at least two weeks and in unrelated factories. Forty five strains were genetically closely related to a 4b serotype L. monocytogenes strain isolated from a human clinical case of listeriosis. Our results highlighted the fact that L. monocytogenes is introduced into meat processing plants through raw meat. To overcome such contamination, suppliers of raw material should adhere to specific microbiological control measures. In addition, more attention should be focused on the appropriateness and compliance with procedures of cleaning and disinfection.     

Molecular discrimination of new isolates of Bifidobacterium animalis subsp. lactis from reference strains and commercial probiotic strains

Fifteen new isolates from pig faeces were identified as Bifidobacterium animalis subsp. lactis by polymerase chain reaction (PCR) using primers specific for this subspecies. Ten of the isolates could be differentiated at strain level by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). These new strains were different from the type strain and other reference strains of this subspecies, and from all commercial dairy strains with probiotic functionality. Thus, possibly some of the isolates are potential candidates for new probiotic Bifidobacterium strains that can be unambiguously identified by RAPD-PCR and PFGE, which could be of interest for the food industry. In contrast, reference strains and commercial dairy strains revealed great genetic homogeneity, showing almost identical DNA fingerprints using RAPD-PCR and PFGE. Some reference strains and all commercial probiotic strains that had been originally designated as B. animalis or B. lactis have to be assigned to B. animalis subsp. lactis to be correctly labelled.     

Processing plant persistent strains of Listeria monocytogenes appear to have a lower virulence potential than clinical strains in selected virulence models

Listeria monocytogenes is an important foodborne bacterial pathogen that can colonize food processing equipment. One group of genetically similar L. monocytogenes strains (RAPD type 9) was recently shown to reside in several independent fish processing plants. Persistent strains are likely to contaminate food products, and it is important to determine their virulence potential to evaluate risk to consumers. We compared the behaviour of food processing persistent and clinical L. monocytogenes strains in four virulence models: Adhesion, invasion and intracellular growth was studied in an epithelial cell line, Caco-2; time to death in a nematode model, Caenorhabditis elegans and in a fruit fly model, Drosophila melanogaster and fecal shedding in a guinea pig model. All strains adhered to and grew in Caco-2 cells in similar levels. When exposed to 10(6) CFU/ml, two strains representing the persistent RAPD type 9 invaded Caco-2 cells in lower numbers (10(2)-10(3) CFU/ml) as compared to the four other strains (10(4)-10(6) CFU/ml), including food and human clinical strains. In the D. melanogaster model, the two RAPD type 9 strains were among the slowest to kill. Similarly, the time to reach 50% killed C. elegans worms was longer (110 h) for the RAPD type 9 strains than for the other four strains (80 h). The Scott A strain and one RAPD type 9 strain were suspended in whipping cream before being fed to guinea pigs and the persistent RAPD type 9 strain was isolated from feces in a lower level (approximately 10(2) CFU/g) than the Scott A strain (approximately 10(5) CFU/g) (P<0.05). The addition of NaCl has been shown to cause autoaggregation and increases adhesion of L. monocytogenes to plastic. However, growth in the presence of NaCl did not alter the behaviour of the tested L. monocytogenes strains in the virulence models. Overall, the two strains representing a very common fish processing plant persistent group (RAPD type 9) appear to have a lower virulence potential in all four virulence models than Scott A and a strain isolated from a clinical case of listeriosis.