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Department of Food Science, Cornell University, Ithaca, NY 14853
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONLISTERIA MONOCYTOGENES AND...PATHOGENESIS AND ECOLOGY OF...POPULATIONS GENETICS,...CONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Key Words: dairy food safety • Listeria monocytogenes • ecology • population genetics
Abbreviation key: CDC = Centers for Disease Control and Prevention, MEE = multilocus enzyme electrophoresis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONLISTERIA MONOCYTOGENES AND...PATHOGENESIS AND ECOLOGY OF...POPULATIONS GENETICS,...CONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Changes in food production and distribution practices may affect the microbial ecology in food processing systems and the transmission of foodborne diseases. Similarly, changes in food consumption patterns, as well as in the human population (such as an increase in the number of elderly and immunocompromised people) can alter patterns of foodborne infections. Separately, or together, changing factors that affect our food system may furthermore contribute to the emergence or reemergence of newly recognized foodborne pathogens (Tauxe, 1997). Improved diagnostic methods also may lead to the discovery of pathogens that had been previously unrecognized as causes of foodborne infections. Continuous efforts to better understand and control transmission of foodborne pathogens in the dairy food system are thus key to assure safe dairy foods for the future.
Infections with foodborne pathogens are a consequence of the interactions of a given pathogen, the environment, and the host. While some foodborne pathogens may be predominantly associated with a single host (the human), a considerable number of foodborne pathogens have the ability to cause disease in a variety of hosts, often in farm animals as well as in humans. Highly host-adapted foodborne and waterborne pathogens (e.g., Vibrio cholerae, Salmonella Typhi) are generally more easily controlled than pathogens that have the ability to infect multiple hosts and/or have the ability to effectively survive and multiply outside a human host. Our capabilities for controlling multihost food- and milkborne pathogens are dependent on the thoroughness of our understanding of their ecology and pathogenesis in different hosts and nonhost environments. Only this basic knowledge will allow the development of science-based, effective, and economical food safety measures from farm to table. As reviewed below, my laboratory has chosen the foodborne pathogen L. monocytogenes as a model system to probe the pathogenesis and transmission of foodborne pathogens. We hope that our studies, together with ongoing work in many other laboratories, will ultimately provide a framework for the development and implementation of science-based food safety assurance strategies, not only for L. monocytogenes, but also for other pathogens.
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LISTERIA MONOCYTOGENES AND LISTERIOSIS |
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TOPABSTRACTINTRODUCTIONLISTERIA MONOCYTOGENES AND...PATHOGENESIS AND ECOLOGY OF...POPULATIONS GENETICS,...CONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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The Genus Listeria
In addition to L. monocytogenes the genus Listeria also includes Listeria ivanovii, Listeria seeligeri, Listeria innocua and Listeria welshimeri. All members of this genus are gram-positive nonspore-forming rods, closely related to the genera Lactobacillus and Streptococcus. The G + C % DNA content of L. monocytogenesis about 40%. Whereas L. monocytogenes causes both human and animal disease, L. ivanovii is predominantly associated with disease (specifically abortions) in sheep, and human clinical infections with this organism appear to be extremely rare (Cummins et al., 1994; Wesley, 1999). The other Listeria species are considered nonpathogenic. The pathogenic species L. monocytogenes and L. ivanovii are both hemolytic, as is the nonpathogenic L. seeligeri, while L. innocua and L. welshimeri are nonhemolytic. Listeria monocytogenes can be separated into 13 different serotypes (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7), all of which appear to have been associated with animal disease, although some of them are less commonly isolated from affected animals than others (Wiedmann and Evans, 2002). Generally serotypes 1/2a, 1/2b, and 4b appear to be most common among animal isolates, although serotype 4c is also occasionally found. There are also some indications that the frequency of different serotypes and molecular subtypes among human and animal clinical cases differs. Thus, certain subtypes may show at least some level of host specificity for humans and animals (Wiedmann et al., 1997).
Physiology and Ecology of Listeria
All Listeria spp. are catalase positive, oxidase negative, and esculin hydrolysis positive. The members of the genus Listeria are aerobic, but also grow under anaerobic conditions and can thus be classified as facultative anaerobes. Listeria monocytogenes has the ability to grow and survive under a variety of different conditions. For example, L. monocytogenes grows from close to 0 to 44°C and is thus considered psychrotolerant. All Listeria spp. are heat sensitive and pasteurization effectively kills listeria. When cultured at 20 to 25°C, Listeria is motile due to peritrichous flagella. In media, Listeria grows in a pH range from 4.4 to 9.6, with optimal growth at neutral pH. L. monocytogenes has a high level of osmotolerance, it can grow in media containing up to 10% (wt/vol) NaCl, and it can survive at even higher salt concentrations. Perhaps due to its considerable resistance to a variety of stress conditions, L. monocytogenes appears to be able to multiply and/or survive for extended time periods (up to more than 2 yr) outside mammalian hosts. Consequently it has been isolated from a variety of different environments (e.g., soil, surface water, sewage, vegetative materials, different food processing environments) (Gravani, 1999), and many authors thus consider this organism "ubiquitous."
Because L. monocytogenes represents a foodborne pathogen, a considerable numbers of studies and surveys on the presence of this organism in many raw and ready-to-eat foods have been published. Whereas the prevalence of L. monocytogenes varies tremendously by study, type of food surveyed, and other factors, this organism has been isolated from many different foods, occasionally at fairly high prevalences (>5 to 10%) (Farber and Peterkin, 1991; Farber and Peterkin, 1999). A summary of raw milk surveys for the presence of L. monocytogenes conducted in many different countries indicates that overall <5% of raw milk samples are positive for L. monocytogenes (Ryser, 1999b). Most recent surveys of ready to-eat foods in developed countries show L. monocytogenes prevalences considerably below 5%, however, including for dairy foods (Ryser, 1999a, 1999b). The ability of L. monocytogenes to survive and proliferate well in foods stored at refrigeration temperatures makes this organism a particular concern for the dairy food industry, since low initial contamination levels (possibly even at <1 cfu/25 g) may increase to numbers that could present a human health hazard if products are subject to extended refrigerated storage. Consequently, humans appear to be commonly exposed to L. monocytogenes by food ingestion. Based on the 2001 FDA/USDA Draft Listeria monocytogenes risk assessment (Food and Drug Administration and US Department of Agriculture, 2001), the average US consumer is likely to occasionally (possibly once a year) consume as many as 106 to 109 cfu of L. monocytogenes in a single serving. In spite of this apparent occasional high exposure, only 2500 human listeriosis cases occur annually in the United States. These data indicate that even exposure to high levels of this organism may not result in human disease, even when one considers that the at-risk population for human listeriosis (immunocompromsied people, pregnant women, etc.) represents only a fraction of the total US population.
Cellular and Molecular Pathogenesis
The cellular processes of L. monocytogenes infection have been well characterized, and genetic systems for gene replacement and for monitoring gene expression have been developed. In tissue culture models, the following stages of infection have been identified: 1) internalization of L. monocytogenes within the host cell; 2) bacterial escape from the host vacuole; 3) multiplication of the parasite within the host cell cytoplasm and its movement through the host cytoplasm by virtue of bacterially directed nucleation of host actin filaments; 4) bacterial movement to the host cell surface and extrusion of bacterial cells in pseudopod-like structures; 5) phagocytosis of these pseudopod-like structures by neighboring cells, followed by escape of the bacterium from the resulting double-membrane vacuole, thus allowing the cycle to repeat (Portnoy et al., 1992).
Gene products essential for each step of the infection process have been identified. Six L. monocytogenes virulence genes (prfA, plcA, hlyA, mpl, actA, and plcB) are located together in one virulence gene cluster. This 8.8-kbp gene cluster is located at a defined position in all L. monocytogenes strains characterized to date, namely between the housekeeping gene ldh and prs. Additional L. monocytogenes virulence-associated genes (e.g., iap, inlA, inlB, inlD, inlE, inlF) are not physically linked to this virulence island (Dramsi et al., 1997; Portnoy et al., 1992). The specific functions for these genes in the Listeria infection process have been well defined. For example, internalin A (InlA), encoded by inlA, interacts with E-cadherin to allow L. monocytogenes to induce its own uptake by specific host cells (e.g., epithelial cells) thus facilitating step 1) of the infection process described above (Gaillard et al., 1991; Mengaud et al., 1996a; Mengaud et al., 1996b). ActA, the actA gene product, is a surface protein required for intracellular movement and cell-to-cell spread through bacterially induced actin polymerization (steps 3 and 4 of the infection process) (Brundage et al., 1993).
Listeria ivanovii and L. seeligeri contain virulence genes that are highly similar to those found in L. monocytogenes. As in L. monocytogenes, the virulence gene cluster in these species is also located between the two housekeeping genes ldh and prs. Whereas the organization of the L. ivanovii virulence gene cluster is very similar to that in L. monocytogenes, the L. seeligeri virulence gene cluster contains additional and differently organized genes (Kreft et al., 1999). Despite the presence of L. monocytogenes virulence gene homologues in L. seeligeri, this species is considered nonpathogenic and has been shown to be avirulent in the murine animal model. In tissue culture studies, L. seeligeri does not trigger its own uptake in nonphagocytic mammalian host cells and it is unable to escape from the vacuole when internalized by phagocytic cells (Kreft et al., 1999). Listeria innocua and L. welshimeri completely lack the virulence gene homologues found in L. monocytogenes, L. ivanovii, and L. seeligeri.
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PATHOGENESIS AND ECOLOGY OF FOODBORNE AND ZOONOTIC INFECTIOUS DISEASES |
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TOPABSTRACTINTRODUCTIONLISTERIA MONOCYTOGENES AND...PATHOGENESIS AND ECOLOGY OF...POPULATIONS GENETICS,...CONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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In a simplified model, transmission patterns of L. monocytogenes as well as of other foodborne pathogens may encompass all or some of the following steps and environments: 1) bacterial survival in the environment and in animal feeds (e.g., L. monocytogenes and pathogenic E. coli are common environmental contaminants); 2) bacterial survival inside nonvertebrate hosts (e.g., protozoans); 3) establishment of clinical or subclinical infections or carrier states in food animals; 4) shedding of the organism into animal products used for human consumption or secondary contamination of animal products; 5) bacterial survival and/or multiplication in non-host environments under food processing and distribution conditions; and 6) infection of human hosts, including survival of gastric passage and establishment of enteric or systemic infections (see Figure 1
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POPULATIONS GENETICS, TRANSMISSION, AND ECOLOGY OF LISTERIA MONOCYTOGENES |
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TOPABSTRACTINTRODUCTIONLISTERIA MONOCYTOGENES AND...PATHOGENESIS AND ECOLOGY OF...POPULATIONS GENETICS,...CONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Whereas the majority of human clinical infections occur as sporadic cases, human listeriosis can also occur in large epidemics. Most sporadic human listeriosis cases and large human foodborne listeriosis epidemics have reportedly been caused by L. monocytogenes serotype 4b (Farber and Peterkin, 1991; Wiedmann, 2002a). The 4b strains isolated from most epidemic outbreaks form two closely related homogenous groups (so called "epidemic clones") (Piffaretti et al., 1989; Wiedmann et al., 1997). Serotypes 1/2a and 1/2b are also responsible for significant numbers of sporadic cases of human illness, and a serotype 1/2a strain was responsible for a recent multistate human listeriosis outbreak in the US (Anonymous, 2000). Serotyping data collected by the CDC in 1986 showed that serotypes 1/2a (30%), 1/2b (32%), and 4b (34%) represented the majority of isolates from 144 human sporadic cases (Schwartz et al., 1989). Of 1363 human isolates collected in the UK, 15% were 1/2a, 10% were 1/2b, and 64% were 4b (McLauchlin, 1990). The remaining 10 currently recognized L. monocytogenes serotypes have been only rarely linked to human disease. This apparent association between a few specific L. monocytogenes strains and most cases of human listeriosis raises the intriguing challenge of identifying unique characteristics enabling these strains to be more effective than others in causing human disease. My research group has thus applied molecular subtyping methods to characterize L. monocytogenes isolates from different sources with the goal of defining L. monocytogenes subtypes and clonal groups and their associations with different hosts and nonhost environments. Phenotypic and phylogenetic analyses of selected L. monocytogenes strains and isolates are used to probe the functional and evolutionary basis of differences in host distribution and ecology of different clonal groups.
Population Genetics and Evolution of Human, Food, and Animal Listeria monocytogenes Isolates
Initial studies by my research group and collaborators focused on probing the population genetics of L. monocytogenes using isolates from human and animals with clinical listeriosis as well as from contaminated food products. For more than 10 yr, our research group has developed a variety of collaborations to collect human clinical and animal L. monocytogenes isolates with the goal of establishing a phylogenetic framework for probing relationships among L. monocytogenes strains. We have developed a network for the collection of human and animal L. monocytogenes isolates that includes the health departments in different states (including New York State, New York City, Michigan, and Ohio) and the New York State Veterinary Diagnostic Laboratory at Cornell. Our collection already includes more than 700 human and more than 200 animal isolates and we currently obtain approximately 150 human and 20 to 30 additional animal L. monocytogenes isolates/year. Since 1997 we have also collected more than 700 isolates from selected food products and from environmental samples. This strain collection provides a unique resource for our research efforts to understand the population genetics and evolution of L. monocytogenes.
Molecular subtyping.
Most L. monocytogenes isolates in our strain collection have been characterized by the multiple genetic methods including automated ribotyping using rRNA probes and EcoRI digested chromosomal DNA and allelic analysis of the virulence genes hly, actA, and inlA using PCR-RFLP methods (Wiedmann et al., 1997). These genes encode a hemolysin involved in host cell vacuole lysis, ActA, and internalin A, respectively. Ribotyping is a system for generating DNA fingerprints in which the rRNA operons and surrounding regions are analyzed for polymorphic restriction sites by Southern hybridization. Fingerprints from various organisms differ by molecular weight patterns and relative band intensities. Isolates in our collection have specifically been characterized by a highly standardized and fully automated ribotyping system (RiboPrinter, Qualicon Inc., Wilmington, DE) that includes computerized data analysis (Bruce, 1996; Jeffers et al., 2001). Ribotype analysis specifically identified more than 100 L. monocytogenes EcoRI ribotypes that group into 11 ribotype subsets.
Current efforts are focusing on the development of a WWW-based molecular subtype and strain database to allow rapid exchange of strain characteristics and subtype information. A prototype database is already available at www.pathogentracker.net. We are also developing and implementing DNA sequencing-based subtyping methods for L. monocytogenes (Cai et al., 2002) to allow more definitive subtype differentiation. Multilocus DNA sequencing data for L. monocytogenes isolates will also provide us with the ability to further and more specifically probe the evolution of Listeria spp. and L. monocytogenes.
L. monocytogenes phylogenetic framework.
Initial analysis of the molecular subtyping data described above allowed us to separate L. monocytogenes strains into three distinct genetic lineages, designated I, II, and III (Wiedmann et al., 1997). Some or all of these lineages may represent different L. monocytogenes subspecies. Each lineage can be separated into three to four ribotype subsets. Ribotype subsets represent groups of similar ribotypes that share a common ribotype fragment (Bruce et al., 1995). Within each ribotype subset, we found between 5 and 20 distinct genetic subtypes as defined by EcoRI ribotypes and virulence gene PCR-RFLP data. Specific virulence gene alleles are generally unique to a specific lineage (Wiedmann et al., 1997). Subtype characterization by a variety of additional molecular subtyping methods performed by other groups (including multilocus enzyme electrophoresis, pulse-field gel electrophoresis; amplified fragment length polymorphism, and DNA sequencing of selected L. monocytogenes genes) also confirmed lineages I and II as the two primary divisions of L. monocytogenes (Piffaretti et al., 1989; Rasmussen et al., 1991; Mazurier et al., 1992; Brosch et al., 1994; Wiedmann, 2002a). Relatively few lineage III strains have been characterized to date as strains classified into this group represent a rare subgroup of L. monocytogenes that appears to be primarily associated with animal hosts (Wiedmann et al., 1997). In addition to our data (Wiedmann et al., 1997), Rasmussen et al. (1995) also reported that DNA sequencing data for selected virulence genes provided evidence for the existence of a third L. monocytogenes lineage.
Phylogenetic analyses of partial DNA sequences of the actA virulence gene from 22 isolates also cluster the strains into three main groups that are identical to the three lineages defined by ribotyping and virulence gene PCR-RFLP analyses. Serotype characterization of L. monocytogenes strains was also consistent with our division into three lineages. Genetic lineage showed a direct correlation with serovar clusters: lineage I contained serotypes 1/2b, 3b, and 4b; lineage II contained serotypes 1/2a, 1/2c, 3a, and 3c; lineage III contained serotypes 4a and 4c (Nadon et al., 2001). Ongoing work in my laboratory now uses sequencing of multiple L. monocytogenes genes to further probe the phylogeny of this species. I anticipate that results from these experiments will allow us to further refine our understanding of the population structure of L. monocytogenes and particularly of the strains grouped into lineage III.
In summary, through application of molecular tools, we have been able to define distinct L. monocytogenes lineages and clonal groups, and also have found strong evidence suggesting a linkage disequilibrium among the genetic and phenotypic characteristics examined (Wiedmann et al., 1997). We did not find any indications for horizontal gene transfer among the genetic loci investigated. Our preliminary data thus establish a phylogenetic framework for further investigation of virulence differences and habitat specialization among L. monocytogenes clonal groups.
Subtype characterization of human outbreak strains.
In additional studies, we used ribotype analysis to further probe the phylogenetic characterization of human epidemic L. monocytogenes strains (Jeffers et al., 2001). The majority of human listeriosis cases appear to be sporadic in nature and, therefore, are associated with a variety of different strains. Relatively few large epidemic listeriosis outbreaks have been reported. Listeria monocytogenes strains associated with these epidemic outbreaks are of particular interest as they may represent a clonal group or groups with a unique transmission potential. It is known for other bacterial pathogens that specific clonal "epidemic" groups are responsible for the majority of human cases of specific infectious diseases. Confirming previous reports, we found that two L. monocytogenes ribotypes were associated with the majority of human epidemic outbreaks reported before 1998. Specifically, ribotypes DUP-1038 and DUP-1042 were associated with at least six and four human epidemics, respectively (Jeffers et al., 2001). Both of these clonal groups represent serotype 4b strains and are grouped into lineage I. Recent listeriosis outbreaks in the United States (since 1998) appear to be caused by different strains. For example, the strain responsible for a multistate listeriosis outbreak in the United States in the fall of 1998 represents another lineage I ribotype (DUP-1044A) (Anonymous, 1999), and the strain associated with a multistate listeriosis outbreak in the United States in 2000 (Anonymous, 2000) is grouped into our lineage II.
Association of L. monocytogenes Subtypes and Clonal Groups with Different Hosts
We have used the phylogenetic framework described above to probe for possible host specificities and virulence differences among L. monocytogenes clonal groups using human and animal clinical isolates. Specifically, we assessed whether different clonal groups as defined by lineage, ribotype subset, and ribotype differed in their likelihood to cause human sporadic or epidemic listeriosis cases or animal listeriosis. An initial evaluation of linkages between independent phylogenetic and virulence markers in 195 human and animal L. monocytogenes isolates was published in 2001 (Jeffers et al., 2001). Interestingly, the clinical histories of the L. monocytogenes isolates suggested differences in pathogenic potentials among the three lineages when the association between classification to lineage and the isolate source (human or animal) was evaluated. We found a statistically significant predominance of human isolates compared with animal isolates among lineage I strains as well as a significant predominance of animal isolates in comparison to human isolates among lineage II and lineage III strains (Jeffers et al., 2001). Specifically, only one human sporadic isolate and no human epidemic isolates were classified to lineage III, while 11% of animal isolates were assigned to this lineage. This suggests that strains in this lineage may have reduced virulence for humans and/or that they have limited transmission potential from animal to human hosts. Lineage I contained the majority of human outbreak and sporadic isolates, but only 42% of the animal isolates. These preliminary findings led us to hypothesize that lineage I strains or a subset of lineage I strains have a greater pathogenic potential for humans as compared to strains in the other two lineages. This hypothesis is also supported by the findings of Vines et al. (1992) who reported that perinatal listeriosis is caused more frequently by strains equivalent to those in our lineage I than by strains in lineage II. A report by McLauchlin (McLauchlin, 1990) also found that serotypes 1/2b and 4b (grouped in lineage I) represent the majority (74%) of human isolates (n = 1363), and thus further supports our findings. In conclusion, we and others have accumulated strong evidence that L. monocytogenes clonal groups differ in their association with human and animal hosts.
Phenotypic Characterization of L. monocytogenes Strains and Subtypes
Phenotypic characterization of selected isolates from our collection has been initiated to probe the functional basis of possible virulence and transmission differences among different L. monocytogenes clonal groups and subtypes. Initial tissue culture virulence characterization using a plaque assay in mouse L cells indicate that lineage II strains may show reduced plaquing efficiency and/or plaque size as compared to lineage I and III strains (Wiedmann et al., 1997), which may be indicative of reduced virulence for lineage II strains. These preliminary tissue culture data are consistent with data from our group and from others (detailed in the proceeding section), which indicate that lineage I strains (which includes serotypes 1/2b and 4b) appear to be the predominant strains responsible for human listeriosis cases and outbreaks, while lineage II strains (serotypes 1/2a and 1/2c) appear to be the most common strains isolated from contaminated foods (Norton et al., 2001b). Further experiments are in progress to confirm our current working hypothesis that lineage I (or a subset of strains within this lineage) represents a subgroup with increased human virulence as compared to lineage II, while lineage II shows an increased ability for environmental survival and multiplication as compared to lineage I. Because lineage III strains represent about 10% of isolates from animals with clinical symptoms, but are considerably less frequent among human clinical isolates and among food isolates (Norton et al., 2001b), these strains may be characterized by a limited transmissibility from animals through foods (and/or environmental; sources) to humans, possibly either due to limited environmental survival and growth capabilities or due to limited human virulence.
Ecology of L. monocytogenes
Survival of L. monocytogenes outside human and animal hosts plays an important role in the transmission of this organism in the dairy food system. While it is conceivable that L. monocytogenes can enter the dairy food chain at many and possibly even at nearly any point, agricultural environments and food processing plant environments may be of particular importance as sources for the introduction of L. monocytogenes into the dairy food system.
Listeriosis infections in farm animals and particularly in cattle are often linked to consumption of contaminated silage. In addition, animal listeriosis cases sometimes occur in animals that are not fed silage and environmental sources have been speculated to be responsible for at least some of these cases. The agricultural environment thus may serve not only as an important source for contamination of silage, but may also be a direct source of animal infection in some cases.
The role of contaminated raw animal-based agricultural products (e.g., milk, meat) as a direct source of L. monocytogenes contamination of ready-to-eat food products is likely to be minimal since commercially applied heat treatments generally kill L. monocytogenes effectively enough to provide an appropriate margin of safety. While, infected animal and agricultural environments appear to rarely be a direct cause of human infections, there have been cases in which a direct link has been observed. Specifically, animal sources can play an important role in animal-derived food products that are not processed before consumption (e.g., raw milk). In addition, manure from infected or shedding animals may represent a source of food contamination. For example, an outbreak involving 42 human cases in Nova Scotia in 1981 was linked to the consumption of coleslaw. This coleslaw was produced from cabbage harvested from fields fertilized with untreated sheep manure that had been obtained from a farm with a history of ovine listeriosis (Schlech et al., 1983).
Contaminated raw meat and milk as well as agricultural environments and/or animal fecal materials may also serve as an indirect source of food contamination by contributing to contamination of the food processing plant environment. Increasing evidence indicates that the processing plant environment may be one of the most important sources of food product contamination with L. monocytogenes. We thus have focused research efforts on developing a better understanding of the ecology of L. monocytogenes in agricultural environments and in food and dairy processing plants to better understand the contributions of these environments to processed food product contamination with L. monocytogenes. Further, it is possible that these environments serve as reservoir for this organism. Application of molecular subtyping methods that have been developed over the last 10 to 15 yr provides an opportunity to study of the distribution of L. monocytogenes in different environments and to track the sources and spread of specific L. monocytogenes subtypes throughout the food chain, allowing insight into the ecology of different L. monocytogenes clonal groups (Wiedmann, 2002a).
Listeria monocytogenes in the agricultural environment.
To better understand the ecology of L. monocytogenes, we have initially applied molecular subtyping methods to track the spread and distribution of different L. monocytogenes subtypes on farms with listeriosis outbreaks. These studies have found a considerable diversity of L. monocytogenes subtypes in feed (primarily silage) samples as well as in other environmental samples collected on outbreak farms. Interestingly, in some outbreaks multiple distinct L. monocytogenes subtypes were shown to be responsible for the animal cases (Wiedmann et al., 1996), while in others a single subtype was responsible for all cases observed (Wiedmann et al., 1997). These findings might indicate that listeriosis epizootics can differ with regard to the causative strains and their spread. In some epizootics, a single, possibly highly virulent, strain may be responsible for infection of all involved animals, whereas in others distinctive strains (possibly with lower virulence and/or transmission potential) may be responsible for individual infections (Wiedmann et al., 1997). In many of the listeriosis outbreaks in farm animals investigated by our group, a considerable proportion, and sometimes all, of the L. monocytogenes subtypes found on a given farm were only isolated from environmental samples and not from infected animals. These findings could indicate that some environmental isolates may have a limited ability to cause disease and thus further supports that L. monocytogenes strains may differ in their virulence characteristics.
Ongoing research in my laboratory focuses on developing a broader understanding of the ecology of L. monocytogenes in farm environments (including farms without listeriosis cases) as well as in pristine and urban environments. Initial findings indicate that while L. monocytogenes is indeed found in many environments, including urban and pristine environments, it is considerably more frequently isolated in farm environments (Sauders et al., 2002). Further subtype and phenotypic analyses and virulence characterization of the L. monocytogenes isolates from these environments will provide a better understanding of the ecology of this foodborne pathogen outside human and animal hosts.
Listeria monocytogenes in food and dairy processing environments.
Because L. monocytogenes is effectively inactivated by commercial heat treatments (pasteurization) used in the dairy industry, the food processing environment seems to represent a major source of finished product contamination. Whereas L. monocytogenes has been reported to be regularly isolated from food and dairy processing environments (Cox, 1989; Pritchard et al., 1995; Sutherland and Porritt, 1996; Gravani, 1999), a better knowledge of the spread and survival of L. monocytogenes in food processing environments and the contributions of different environmental sources to finished product contamination is necessary to understand the transmission of L. monocytogenes. Whereas many food processors only test for the presence of Listeria spp. in the processing environment, detection and isolation of L. monocytogenes followed by molecular subtype characterization provides an opportunity to study the molecular ecology of L. monocytogenes in food processing plants. A thorough understanding of the molecular ecology of foodborne pathogens is necessary for the design of rational and science-based methods and approaches for the control of L. monocytogenes and other foodborne pathogens.
In a pilot study using three smoked fish processing plants, we showed that specific L. monocytogenes subtypes persisted in the environment of a given processing plant (Norton et al., 2001a). In addition to these persistent, plant-specific, subtypes, we also observed a considerable diversity of transient L. monocytogenes subtypes. Interestingly, the persistent L. monocytogenes subtypes were the major cause of finished product contamination. Through a collaborative research project on the ecology of L. monocytogenes in Mexican-style cheese processing plants, we also showed a similar pattern of strain distribution in dairy processing plants, including persistence of plant-specific subtypes over time. In one of the three plants studied, the persistent L. monocytogenes subtype was also responsible for finished product contamination (Kuaye et al., 2001). These findings are consistent with other reports that used bacterial subtyping methods to show the persistence of specific L. monocytogenes subtypes in a variety of food processing environments including those for smoked fish, poultry, meat and dairy foods (Lawrence and Gilmour, 1995; Rorvik et al., 1995; Nesbakken et al., 1996; Autio et al., 1999;). Whereas some studies found that persistent strains may have an increased capability to adhere to inorganic surfaces (Lunden et al., 2000), others were not able to discern an association between strain persistence and a strain’s ability to form biofilms (Djordjevic et al., 2002). Further research will be necessary to determine whether specific phenotypic and genetic strain characteristics are responsible for persistence in a processing plant environment.
While further confirmation of the association between persistent stains and finished product contamination in other dairy and food processing plants will be important, our results indicate that control and elimination of persistent L. monocytogenes subtypes in processing plants may allow for considerable reduction of finished product contamination. Transient L. monocytogenes strains on the other hand may only be minor contributors to finished product contamination. Molecular subtyping thus provides an important tool to track in-plant sources and spread of bacterial contaminants and to better control finished product contamination by foodborne pathogens (Wiedmann, 2002a, Wiedmann, 2002b).
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONLISTERIA MONOCYTOGENES AND...PATHOGENESIS AND ECOLOGY OF...POPULATIONS GENETICS,...CONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONLISTERIA MONOCYTOGENES AND...PATHOGENESIS AND ECOLOGY OF...POPULATIONS GENETICS,...CONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Received for publication August 12, 2002. Accepted for publication December 6, 2002.
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TOPABSTRACTINTRODUCTIONLISTERIA MONOCYTOGENES AND...PATHOGENESIS AND ECOLOGY OF...POPULATIONS GENETICS,...CONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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 K. K. Nightingale, Y. H. Schukken, C. R. Nightingale, E. D. Fortes, A. J. Ho, Z. Her, Y. T. Grohn, P. L. McDonough, and M. Wiedmann Ecology and Transmission of Listeria monocytogenes Infecting Ruminants and in the Farm Environment Appl. Envir. Microbiol., August 1, 2004; 70(8): 4458 - 4467. [Abstract] [Full Text] [PDF]
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T. J. Ward, L. Gorski, M. K. Borucki, R. E. Mandrell, J. Hutchins, and K. Pupedis Intraspecific Phylogeny and Lineage Group Identification Based on the prfA Virulence Gene Cluster of Listeria monocytogenes{dagger} J. Bacteriol., August 1, 2004; 186(15): 4994 - 5002. [Abstract] [Full Text] [PDF]
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