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Identification of Listeria Monocytogenes Genetic Clusters having Increased Pathogenic Potential


The ultimate objectives of this project are to improve our understanding of the virulence mechanisms of Listeria monocytogenes, and to obtain background information needed for the development of a science-based strategy for managing L. monocytogenes infections (which also would include developing tools for rapid and accurate detection of "highly virulent" L. monocytogenes strains in foods and clinical samples). As an initial step to address these objectives, we propose to develop a state-of-the-art, multilocus sequence typing (MLST) protocol for molecular characterization of L. monocytogenes strains, and to use this methodology, in combination with the current "gold standard" pulsed field gel electrophoresis (PFGE) approach, to determine whether we can identify a subgroup(s) of L. monocytogenes strains significantly associated with human listeriosis. <P>
During the course of these studies, we will also (i) determine the relative prevalence of major known Listeria virulence genes in the strains in various genetic clusters, and (ii) delineate the phylogenetic relationships between environmental and clinical L. monocytogenes isolates. The specific aims will include: (i) develop a MLST approach for L. monocytogenes, (ii) characterize L. monocytogenes strains from food, environmental and clinical samples by PFGE and MLST, and (iii) screen L. monocytogenes strains in various genetic clusters for the presence of known and putative virulence markers.

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NON-TECHNICAL SUMMARY: Listeria monocytogenes is a major bacterial pathogen responsible for foodborne illnesses, and several large outbreaks of listeriosis have occurred during the past few years (several of them involved fatalities). Thus, the development of appropriate strategies for the detection and elimination of L. monocytogenes contamination of foods is one of the top priorities for improving food safety and public health. In this application, we propose to develop a state-of-the-art multilocus sequence typing protocol for L. monocytogenes, and to use this methodology, in combination with other, well-established approaches, to characterize a large collection of L. monocytogenes strains from food, environmental, and clinical sources. During the course of these studies, we will (i) determine whether a subgroup(s) of L. monocytogenes strains significantly associated with listeriosis can be identified, and - if that is the case (as expected) - we will identify the genetic substitutions responsible for grouping the L. monocytogenes strains.

APPROACH: The project is specifically designed to address the issue of differences in the virulence of L. monocytogenes strains, by (i) determining whether a cluster of L. monocytogenes strains significantly associated with listeriosis can be identified by sequence analysis of various housekeeping and virulence-associated genes, and (ii) delineating the phylogenetic relationships between clinical and environmental L. monocytogenes strains. During the studies, the MLST approach for characterizing L. monocytogenes strains will be developed, and a large collection of clinical and environmental L. monocytogenes strains will be characterized using this methodology in combination with the well-established PFGE approach. In addition, our clinical and environmental L. monocytogenes strains collections will be screened for the presence of various known and putative virulence markers, using DNA hybridization, PCR, and in silico (analysis by computer) analyses. Various state-of-the-art data analysis programs will be used to analyze the results. The data generated during the project should provide important information for the future elucidation of the virulence mechanisms of L. monocytogenes, and for the development of advanced strategies for dealing with L. monocytogenes infections.


PROGRESS: 2001/12 TO 2004/06<BR>
Listeria monocytogenes is an important foodborne pathogen responsible for approximately 2,500 cases of listeriosis in the United States each year (of which ca. 500 are fatal), and the annual cost of foodborne listeriosis in the United States has been estimated to exceed 2.3 billion dollars. The bacterium is of particular concern from the standpoint of food safety because of its ability to grow at refrigerator temperatures, and the high fatality rate associated with L. monocytogenes infections. The FDA and the USDA's FSIS have established a zero tolerance policy for the species in ready-to-eat foods. The policy's establishment was based on the limited information concerning L. monocytogenes virulence traits available at the time, including the assumption that all L. monocytogenes strains are equally pathogenic for humans. However, recent data strongly suggest that some L. monocytogenes strains/subgroups of strains have an increased potential to cause human disease. The mechanisms responsible for the increased virulence of those strains are unknown at the present time. During the USDA-funded project to study genetic clustering of various L. monocytogenes strains, we analyzed genomic sequences of several housekeeping genes of L. monocytogenes using a multilocus sequence typing (MLST) approach, and we identified several genetic clusters which contained exclusively clinical (and presumably highly pathogenic) L. monocytogenes strains, or strains of 4b serotype predominantly associated with human listeriosis. Moreover, we found that the distribution of various hly and actA amino acid-based allele types (ATs) in the genetic clusters was also not random, and that some hlyO and actA ATs were strongly associated with the 4b serotype strains and/or with the clinical strains. Those data suggested that certain allelic versions (tentatively designated Highly Virulent ATs) of the virulence genes hly and actA are associated with increased virulence of L. monocytogenes. Our split decomposition analysis revealed evidence of recombination within and between various clonal groups of L. monocytogenes. MLST differentiated most of the L. monocytogenes strains better than did PFGE, and the discriminating ability of PFGE was better than that of serotyping. Several strains with different serotypes were found, by MLST and PFGE, to have very closely-related genetic backgrounds, which suggested possible antigen switching among them. This observation suggests that conclusions about the pathogenic potential of L. monocytogenes strains (based solely on their serotypes) must be interpreted with caution, and that strains belonging to nonpathogenic L. monocytogenes serotypes (i.e., other than serotypes 1/2a, 1/2b, and 4b) should not be considered avirulent or as having reduced virulence solely based on their serotype classification. Our observation also suggests that conclusions about the genetic relatedness of strains based on their serotypes may be misleading, and that genetic typing methodologies (e.g., MLST) must be used to determine the true genetic background of, and the relationships among, various L. monocytogenes isolates.
IMPACT: 2001/12 TO 2004/06<BR>
The results of our USDA-supported studies advanced knowledge concerning L. monocytogenes' genetic make-up and the evolutionary mechanisms involved in the diversification of that important foodborne pathogen. On a more applied level, these data should enhance the development of advanced tools for the rapid and specific identification of highly virulent L. monocytogenes strains or clusters of strains. Our findings about the superior discriminating ability of MLST, compared to that of PFGE and serotyping, has important practical implications. PFGE (alone or in combination with serotyping) is currently the method of choice for investigating foodborne outbreaks of listeriosis and for tracing the outbreak-causing strain to the source of contamination - which has important legal and financial ramifications. However, our data demonstrate that some L. monocytogenes strains undistinguishable by PFGE are not necessarily the same isolates and, in some cases, they are not even closely-related. Therefore, MLST may be of value for improving the differentiation of L. monocytogenes strains isolated during foodborne outbreaks of listeriosis, and for tracing the outbreak-causing strains to their sources. Furthermore, the data obtained during our studies can lead towards several important areas of investigations to better understand the pathogenic traits of L. monocytogenes - studies that can be invaluable for designing science-based strategies for significantly reducing the occurrence of foodborne listeriosis.

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