Molecular characterization of Listeria monocytogenes from natural and urban environments

Characterization of 80 Listeria monocytogenes isolates from urban and natural environments differentiated 7 and 26 EcoRI ribotypes, respectively. Whereas the majority of isolates from the natural environment represented L. monocytogenes lineage II (12 of 13 isolates), urban isolates grouped evenly into lineages I and II (32 and 33 isolates, respectively) and included two lineage III isolates. Multilocus sequence typing of all natural isolates and a randomly selected subset of 30 urban isolates showed a higher overall diversity (Simpson index of discrimination [D] of 0.987 and 0.920, respectively) than did EcoRI ribotyping (D = 0.872 and 0.911, respectively). Combined analysis with ribotype and lineage data for 414 isolates from farm sources, 165 isolates from foods and food-processing environments, and 342 human clinical isolates revealed that lineage I was significantly more common among human (P < 0.0001) isolates, whereas lineage II was more common among isolates from the natural environment, farms, and foods (P < or = 0.05). Among a total of 92 ribotypes, 31 showed significant associations with specific isolate sources. One ribotype (DUP-1039C) was significantly associated with both natural environments and farms. A spatial analysis showed a marginal association between locations in the natural environment positive for L. monocytogenes and a proximity to farms. Our data indicate that (i) L. monocytogenes strains from different sources show a high level of diversity; (ii) L. monocytogenes subtypes differ significantly in their associations with different environments, even though populations overlap; and (iii) a higher proportion of isolates from environmental sources than from human clinical cases can be classified into L. monocytogenes lineage II, which supports the classification of this lineage as an environmentally adapted subgroup.     

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.     

Distribution of Listeria monocytogenes molecular subtypes among human and food isolates from New York State shows persistence of human disease--associated Listeria monocytogenes strains in retail environments

While there is considerable information available regarding Listeria monocytogenes contamination patterns in food processing plants, our understanding of L. monocytogenes contamination and transmission in retail operations is limited. We characterized 125 food, 40 environmental, and 342 human clinical L. monocytogenes isolates collected in New York State from 1997 to 2002 using automated ribotyping and hly allelic variation. All environmental isolates were obtained from retail establishments and the majority of food isolates (98 isolates) were obtained from foods that were prepared or handled at retail. Overall, food and/or environmental isolates from 50 different retail establishments were characterized. The 125 food and 40 environmental isolates were differentiated into 29 and 10 ribotypes, respectively. For 16 retail establishments, we found evidence for persistence of one or more specific L. monocytogenes strains as indicated by isolation of the same EcoRI ribotype from food or environmental samples collected in a given establishment on different days. The human isolates were differentiated into 48 ribotypes. Statistical analyses showed that two ribotypes were significantly (P < 0.0001) more common among food isolates as compared with human isolates. However, a total of 17 ribotypes found among the human clinical isolates were also found among the food and environmental isolates. We conclude that L. monocytogenes, including subtypes that have been linked to human disease, can persist in retail environments. Implementation of Listeria control procedures in retail operations, which process and handle products that permit the growth of L. monocytogenes, are thus a critical component of a farm-to-table L. monocytogenes control program.     

Characterization of Listeria monocytogenes isolates in import food products of China from 8 provinces between 2005 and 2007

A total of 48 Listeria monocytogenes isolates of different import food products from 8 provinces between 2005 and 2008 were characterized. The serotype and virulence were confirmed for each strain and molecular subtyping were analyzed by multilocus sequence typing (MLST). Twenty five strains were assigned to serotype 1/2a, and 11 isolates to serotype 1/2b, serotype 4b were found in 7 isolate, and the remaining 5 strains were grouped into serotypes 1/2c, 4a, and 4e. Molecular subtyping schemes found thirty two sequence types (STs) among these isolates and the majority of L. monocytogenes strains belonged to lineage II (56%), followed by lineage I (38%), lineage III (6%). Two molecular subtype clusters, cluster A included all isolates of lineage II, while cluster B contained the isolates of lineages I and lineages III. Two L. monocytogenes strains were not grouped in either of the two clusters. Fifty three isolates were as virulent as L. monocytogenes reference strain EGD in mouse virulence assay, while the isolates 22213 and 22265 had low pathogenicity. These results provide the first molecular insight into the L. monocytogenes strains isolated from import food products of 8 provinces in China and indicate the potential risk to cause human disease if intake by contaminated foods. MLST could be used as a routine subtyping method of L. monocytogenes isolates. In China, inspection and quarantine strategies of imported foods should be strengthened. PRACTICAL APPLICATION: There is a potential risk of listeriosis in China and routine subtyping of L. monocytogenes isolates is important. It is necessary for food hygiene management to strengthen the supervision of imported foods.     

Genetic variation of Listeria monocytogenes isolates from domestic and imported foods in Japan

Phylogenetic analyses were carried out on a total of 118 Listeria monocytogenes isolates from foods or food processing environments, and 7 isolates from listeriosis patients in Japan to evaluate the genetic variation in the pathogen in this country. Isolates of serotypes 1/2a, 1/2b and 4b were mainly examined to assess the risk of exposure of humans to L. monocytogenes from foods in Japan. The nucleotide sequences of the part of the iap gene that contains the region encoding the threonine-asparagine repeat units were determined in order to construct phylogenetic trees of the isolates investigated. A phylogram showed high genetic diversity among lineage 2 isolates, while the lineage 1 isolates showed clonal characteristics. The results of the genetic analyses suggested the presence of rare putative lineage 3 isolates and epidemic clone I (ECI) isolates in foods in Japan. The results showed that ECI was also isolated from listeriosis patients. The genetic variation in L. monocytogenes in Japan reported here suggests the necessity of monitoring the pathogen in foods and environments in addition to surveillance of listeriosis patients.     

Low prevalence of Listeria monocytogenes in human stool

Listeria monocytogenes is a foodborne pathogen that is found widely in the environment and in a variety of ready-to-eat foods, yet human invasive infection is relatively rare (five cases per million people annually in the United States). Despite wide exposure to this organism, little is known about the prevalence of L. monocytogenes in human stool, and it is not known whether human fecal dispersal contributes to human foodborne transmission. We cultured 827 stool specimens (well formed and loose-watery) from individuals from four large metropolitan areas of New York state for L. monocytogenes and found only 1 (0.12%) positive specimen. L. monocytogenes was also isolated from the blood of the person with the single positive specimen, and the two isolates were indistinguishable by molecular subtyping (both were ribotype DUP-1042B). This provides further evidence that human L. monocytogenes fecal carriage among persons with and without diarrheal disease is remarkably low. Unlike the case for other foodborne pathogens (e.g., Salmonella), human shedders probably do not contribute significantly to L. monocytogenes contamination of foods. However, we observed a single individual with invasive listeriosis that shed the pathogen in feces, indicating the potential for fecal dispersal of L. monocytogenes from persons with listeriosis.     

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.     

Attributing risk to Listeria monocytogenes subgroups: dose response in relation to genetic lineages

The objective of this study was to evaluate the hypothesis that the dose-response relationship for Listeria monocytogenes in humans varies with genotypic lineage or subtype. The linkages between molecular subtyping data and enumeration data for L. monocytogenes subtypes in foods consumed by the at-risk population were examined to test this hypothesis. We applied a conditional probability model to conduct a subtype-specific dose-response analysis, with the focus on invasive listeriosis. L. monocytogenes differed not only in the molecular subtype and lineage but also in the contamination level when isolates of the pathogen occurred in retail samples of ready-to-eat foods. Using the exponential model parameter r-value as a measure (essentially the probability of a single cell causing illness), we found that the virulence varied among L. monocytogenes lineages by several orders of magnitude. Under the assumptions made, for L. monocytogenes lineages I and II the consumption of a single cell would result in listeriosis with log average probabilities of -7.88 (equivalent to once in 10(7.78) times) and -10.3, respectively, as compared with -9.72 for L. monocytogenes independent of subtype. A greater difference in r-values was found for selected ribotypes. The uncertainty about the r-value estimates was small compared with the large differences in the virulence parameters themselves. Thus, for L. monocytogenes both subtype and the number of cells consumed matter, highlighting the usefulness of considering both exposure concentration and subtype prevalence in dose-response analysis. As advances are made in molecular subtyping and quantitative tools for dose-response analysis, further studies integrating genomic data into quantitative risk assessments will enable better attribution of disease risk to L. monocytogenes subtypes.     

Lessons from an outbreak of listeriosis related to vacuum-packed gravad and cold-smoked fish

The first lesson learned from this outbreak was that vacuum-packed rainbow trout is not only an excellent medium for the growth of Listeria monocytogenes, but may also cause human listeriosis. Another lesson is that one single fish processing plant may spread multiple clonal types of L. monocytogenes by selling contaminated products to consumers. Thus, when investigating fish-borne outbreaks of listeriosis one should identify and type several isolates of L. monocytogenes from each food and environmental sample, since multiple clonal types might be present. The outbreak described in this paper involved at least eight human cases, three clonal types of L. monocytogenes, and lasted for 11 months. During the outbreak investigation, L. monocytogenes was also isolated from another brand of rainbow trout found in the refrigerator of one of the patients. These latter isolates belonged to a clonal type not associated with the outbreak. However, this clonal type is of considerable interest since it has been associated with foodborne outbreaks of listeriosis in several countries, and is also the second most common clonal type among human clinical isolates of L. monocytogenes in Sweden. Besides the described outbreak, it is likely that vacuum-packed, cold-smoked and gravad rainbow trout have been involved in additional cases of foodborne listeriosis in Sweden.     

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.     

Molecular Subtyping and Tracking of Listeria monocytogenes in Latin-Style Fresh-Cheese Processing Plants

Latin-style fresh cheeses, which have been linked to at least 2 human listeriosis outbreaks in the United States, are considered to be high-risk foods for Listeria monocytogenes contamination. We evaluated L. monocytogenes contamination patterns in 3 Latin-style fresh-cheese processing plants to gain a better understanding of L. monocytogenes contamination sources in the manufacture of these cheeses. Over a 6-mo period, 246 environmental samples were collected and analyzed for L. monocytogenes using both the Food and Drug Administration (FDA) method and the Biosynth L. monocytogenes detection system (LMDS). Finished cheese samples from the same plants (n = 111) were also analyzed by the FDA method, which was modified to include L. monocytogenes plating medium (LMPM) and the L. monocytogenes confirmatory plating medium (LMCM) used in the LMDS method. Listeria monocytogenes was detected in 6.3% of cheese and 11.0% of environmental samples. Crates, drains, and floor samples showed the highest contamination rates, with 55.6, 30.0, and 20.6% L. monocytogenes positive samples, respectively. Finished products and food contact surfaces were positive in only one plant. The FDA method showed a higher sensitivity than the LMDS method for detection of L. monocytogenes from environmental samples. The addition of LMPM and LMCM media did not further enhance the performance of the FDA method for L. monocytogenes detection from finished products. Molecular subtyping (PCR-based allelic analysis of the virulence genes actA and hly and automated ribotyping) was used to track contamination patterns. Ribotype DUP-1044A, which had previously been linked to a 1998 multistate human listeriosis outbreak in the United States, was the most commonly identified subtype (20/36 isolates) and was isolated from 2 plants. This ribotype was persistent and widespread in one factory, where it was also responsible for the contamination of finished products. We hypothesize that this ribotype may represent a clonal group with a specific ability to persist in food processing environments. While previous listeriosis outbreaks were linked to Latin-style fresh cheeses made from unpasteurized milk, the presence of this organism in pasteurized cheese products illustrates that persistent environmental contamination also represents an important source of finished product contamination.     

Prevalence, distribution, and diversity of Listeria monocytogenes in retail environments, focusing on small establishments and establishments with a history of failed inspections

Despite growing concerns about cross-contamination of ready-to-eat foods with Listeria monocytogenes, our knowledge about the ecology and transmission of L. monocytogenes in retail establishments has remained limited. We conducted a cross-sectional study to characterize the prevalence, distribution, and subtype diversity of L. monocytogenes in 120 New York State retail deli establishments that were hypothesized to present an increased risk for environmental L. monocytogenes contamination (i.e., small establishments and establishments with a history of failed New York State Agriculture and Markets inspections). Analysis of these data along with previously reported data for 121 predominantly larger retail establishments in New York State identified establishment size, geographic location, and inspection history as significant predictors of L. monocytogenes presence and prevalence. The odds of an establishment being L. monocytogenes positive were approximately twice as high for large establishments, establishments located in New York City, or establishments with poor inspection history (as compared with establishments without these attributes), even though correlation between location and inspection history complicated interpretation of results. Within an establishment, L. monocytogenes was significantly more prevalent on nonfood contact surfaces than on food contact surfaces; prevalence was particularly high for floors and in floor drains, sinks, the dairy case, and milk crates. L. monocytogenes subtype diversity differed between sites, with lineage I isolates significantly associated with nonfood contact surfaces and lineage II isolates significantly associated with food contact surfaces. Isolates belonging to the same ribotype were often found dispersed across multiple sites within an operation.     

Variable adhesion of Listeria monocytogenes isolates from food-processing facilities and clinical cases to inert surfaces

One hundred one strains of Listeria monocytogenes isolated from seafood and cheese industry samples and from patients with listeriosis were assessed using a microtiter plate method for adhesion to polystyrene and stainless steel surfaces. The adhesion rate for these strains ranged from 3.10 to 35.29% with an inoculum of 8 x 10(8) cells per well. A strong correlation was found between adhesion to polystyrene and stainless steel microtiter plates, indicating that the intrinsic ability of L. monocytogenes to adhere to inert surfaces is stronger than the influence of the surface's physicochemical properties. The clinical strains were less adherent to inert surfaces than were the industrial strains. By integrating other factors such as location of the industrial strains, contamination type of the clinical strains, serotype, and pulsotype into the analysis, some weak but significant differences were noted. For the industrial isolates, the number of cells attached to both surfaces differed significantly depending on whether they were isolated from food or food-processing environments in the seafood and cheese industry. For clinical isolates, sporadic strains exhibited greater adhesion to polystyrene than did epidemic strains. Strains belonging to the pulsed-field gel electrophoretype clusters A and M (lineages II and I, respectively) were less able to adhere to polystyrene and stainless steel than were strains in the more common clusters.     

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.     

Comparison of multiplex PCR with conventional biochemical methods for the identification of Listeria spp. isolates from food and clinical samples in Queensland, Australia

Listeria monocytogenes is an important foodborne pathogen with high mortality. L. monocytogenes and five other Listeria species can frequently be found in the same sample. To identify Listeria isolates found in foods to the species level, two multiplex PCRs were designed. The PCR and conventional biochemical methods were compared for the identification of 456 Listeria isolates collected from routine food quality monitoring schemes between June 2004 and February 2006 and for 62 L. monocytogenes isolates from patients between 1999 and 2005. The results showed that the PCR and biochemical methods had 100% agreement in Listeria identification. The distribution of Listeria species from foods was as follows: L. monocytogenes, 50.4%; L. innocua, 33.8%; L. welshimeri, 14.9%; L. seeligeri, 0.7%; L. grayi, 0.2%; and L. ivanovii, 0.0%. Additional analyses were performed to identify the major serotypes (1/2a, 1/2b, 1/2c, and 4b) and the three lineages of L. monocytogenes isolates from foods and patients, with 1/2a (69.6%) and 1/2b (21.7%) dominating the food isolates and 1/2b (54.8%) and 4b (30.7%) dominating the patient isolates. The lineage results showed that isolates of 1/2a and 1/2c belonged to lineage II and that isolates of 1/2b and 4b belonged to lineage I. The multiplex PCRs for Listeria identification that have been established provide an accurate and rapid method for food quality control. This study has provided the basic knowledge of distribution of Listeria species and L. monocytogenes serotypes in Queensland, Australia, which is useful for epidemiological investigations of listeriosis.     

Growth and persistence of Listeria monocytogenes isolates on the plant model Arabidopsis thaliana

While the majority of human listeriosis cases appear to be linked to consumption of processed ready-to-eat foods (e.g., deli meats), a few listeriosis outbreaks have been linked to consumption of contaminated vegetables. In this study, we assessed four isolates representing the major Listeria monocytogenes lineages for their abilities to attach to and grow on Arabidopsis thaliana, a well-characterized plant model. When plants were dipped for 5min into 3ml of water containing 8.8logCFU of L. monocytogenes and rinsed repeatedly, L. monocytogenes was recovered from the leaves at densities from 1.52 to 2.17logCFU/cm(2). Ten days after exposure, bacterial numbers had increased over initial numbers by 2.60-2.95logCFU/cm(2). Using L. monocytogenes expressing GFP, bacteria were visualized in the intercellular spaces of A. thaliana leaves, suggesting internalization through stomata. These data indicate that L. monocytogenes can rapidly attach to and multiply on plant surfaces and colonize intercellular spaces in A. thaliana leaves where it may be protected from sanitation treatments. When A. thaliana seeds were exposed to L. monocytogenes, between 4.23 and 4.57logCFU/cm(2) were recovered from leaves 7 days post-germination, suggesting that contaminated seeds can produce contaminated plants. Overall, our study demonstrates that prevention of L. monocytogenes contamination of plants throughout growing stages is critical, consistent with recommendations for other produce-transmitted foodborne pathogens.     

Outbreaks and sporadic cases of Salmonella serovar panama studied by DNA fingerprinting and antimicrobial resistance.

In the Principality of Asturias (PA), Spain. three Salmonella serovar Panama outbreaks were registered in August 1998. In order to achieve an accurate identification of the strains implicated in the outbreaks and to study the molecular epidemiology of this serovar in the PA, the isolates collected over 1990-1999 were examined by DNA fingerprinting and antimicrobial resistance analysis. The origin of the isolates was: human (65, of which 20 were associated with the three outbreaks), octopus (2), beef (2), eggs (7), poultry faeces (2), sea water (5), sewage (2) and unknown (1). Sixteen lineages were defined by ribotyping, enterobacterial repetitive intergenic consensus sequences analysis, and randomly amplified polymorphic DNA segment analysis. One lineage was endemic in the PA and was also represented by isolates from other Spanish regions. The organisms of this lineage can be differentiated (by resistance-, plasmid- and integron-profiles) into 19 types. The three outbreaks were caused by organisms falling into a single type (nalidixic acid-resistant, plasmid- and integron-free) belonging to the endemic lineage, which was associated with poultry as the reservoir. Isolates showing drug-resistance (71%) fell into six lineages and 23 types. Ten multidrug-resistant types carried class I integrons with three types of variable regions containing resistance gene cassettes.     

Ribotype analysis of strain distribution in Listeria monocytogenes

Changes in the temporal and spatial patterns of strain distribution for the foodborne pathogen Listeria monocytogenes were studied by ribotyping using the Qualicon Riboprinter system. Ribotype patterns were obtained by using the restriction enzymes EcoRI and PvuII for 72 isolates of L. monocytogenes recovered from smoked salmon samples over a period of 3 years. Each pattern was classified both by comparison to a pattern library and by comparison among the 72 isolate patterns. Eleven EcoRI-based ribogroups and 16 PvuII groups were identified. Eight of the 11 EcoRI ribogroups were found in isolates obtained over a period of >12 months, and 75% of the EcoRI ribogroups that were found in more than one food sample were distributed nationally. Within the set of isolates, there were 26 instances where more than one isolate was obtained from a single food sample. In 35% of these instances, the co-isolates produced different ribotype patterns, indicating that multiple strains of L. monocytogenes commonly coexist in the same environment. Overall, these data indicate that the population of L. monocytogenes consists of a number of widely dispersed strains with little geographic or temporal stratification.     

Distribution of epidemic clonal genetic markers among Listeria monocytogenes 4b isolates

Recent genome sequencing of isolates of Listeria monocytogenes serotype 4b implicated in some major outbreaks of foodborne listeriosis has revealed unique genetic markers in these isolates. The isolates were grouped into two distinct epidemic clones, ECI and ECII. In the present study, selected ECI- and ECII-specific genetic markers were detected in 16 and 15 of 89 L. monocytogenes 4b isolates, respectively. The ECI markers were found in 6 of 34 clinical isolates, 9 of 50 food isolates, and 1 of 5 environmental isolates, and the ECII markers were detected in 7 of 34 clinical isolates, 7 of 50 food isolates, and 1 of 5 environmental isolates. Hence, of the isolates with the epidemic clonal genetic markers, 38% (13 of 34) were of clinical origin, 32% (16 of 50) were of food origin, and 40% (2 of 5) were of environmental origin. The predominance of the epidemic clonal markers among the clinical and environmental isolates supports the hypothesis that these markers are correlated with the pathogenic potential of strains and with their environmental persistence. Several isolates had only one epidemic clonal marker, either the ECI-specific marker 133 or the ECII-specific marker 4bSF18. Pulsed-field gel electrophoresis analysis revealed higher genomic diversity among the strains with ECII-like characteristics than among those strains carrying the ECI-specific genetic markers.     

Tracking of Listeria monocytogenes in smoked fish processing plants

Four smoked fish processing plants were used as a model system to characterize Listeria monocytogenes contamination patterns in ready-to-eat food production environments. Each of the four plants was sampled monthly for approximately 1 year. At each sampling, four to six raw fish and four to six finished product samples were collected from corresponding lots. In addition, 12 to 14 environmental sponge samples were collected several hours after the start of production at sites selected as being likely contamination sources. A total of 234 raw fish, 233 finished products, and 553 environmental samples were tested. Presumptive Listeria spp. were isolated from 16.7% of the raw fish samples, 9.0% of the finished product samples, and 27.3% of the environmental samples. L. monocytogenes was isolated from 3.8% of the raw fish samples (0 to 10%, depending on the plant), 1.3% of the finished product samples (0 to 3.3%), and 12.8% of the environmental samples (0 to 29.8%). Among the environmental samples, L. monocytogenes was found in 23.7% of the samples taken from drains, 4.8% of the samples taken from food contact surfaces, 10.4% of the samples taken from employee contact surfaces (aprons, hands, and door handles), and 12.3% of the samples taken from other nonfood contact surfaces. Listeria spp. were isolated from environmental samples in each of the four plants, whereas L. monocytogenes was not found in any of the environmental samples from one plant. Overall, the L. monocytogenes prevalence in the plant environment showed a statistically significant (P < 0.0001) positive relationship with the prevalence of this organism in finished product samples. Automated EcoRI ribotyping differentiated 15 ribotypes among the 83 L. monocytogenes isolates. For each of the three plants with L. monocytogenes-positive environmental samples, one or two ribotypes seemed to persist in the plant environment during the study period. In one plant, a specific L. monocytogenes ribotype represented 44% of the L. monocytogenes-positive environmental samples and was also responsible for one of the two finished product positives found in this plant. In another plant, a specific L. monocytogenes ribotype persisted in the raw fish handling area. However, this ribotype was never isolated from the finished product area in this plant, indicating that this operation has achieved effective separation of raw and finished product areas. Molecular subtyping methods can help identify plant-specific L. monocytogenes contamination routes and thus provide the knowledge needed to implement improved L. monocytogenes control strategies.