Unique Genetic and Physiological Attributes of Listeria Monocytogenes Implicated in Recent Outbreaks of Listeriosis in the United States

NON-TECHNICAL SUMMARY: Recent outbreaks of listeriosis in the United States involved ready to eat meats (hot dogs, turkey deli meats) that had become contaminated with highly unusual strains of Listeria monocytogenes serotype 4b. Current data indicate that the outbreak isolates are closely related members of the same clonal group (Epidemic Clone II). Unlike other Listeria strains, these isolates were highly resistant to Listeria-specific viruses isolated from processing plants at temperatures likely to prevail in the processing plant environment. In addition, several isolates harbored extrachromosomal elements (plasmids) that conferred resistance to cadmium and to disinfectants. These unusual characteristics may contribute to the ability of these organisms to contaminate the processing plant environment, and possibly to their virulence for humans. In this project we will characterize the role of temperature on biofilm formation and resistance of these organisms to disinfectants, cadmium, and Listeria-specific viruses. The role of plasmids on resistance to disinfectants and to cadmium or arsenic will be also investigated. The findings will contribute to further understanding of the ecology and adaptations of these problematic but currently poorly understood strains. Such understanding will be needed to develop effective, science-based strategies to reduce or eliminate the threat currently posed by establishment of these strains in the processing plant, and their contamination of ready to eat foods.

<P>

APPROACH: Objective 1: For analysis of the gene content and macroarray typing of the processing plant and other ECII isolates we will use used Listeria arrays following established methodology. This analysis will generate a binary score for each chromosomal DNA analyzed, indicating whether a gene is absent or present. These binary matrices will be analyzed with hierarchical clustering using the program J-Express and compared to the results obtained for 300 L. monocytogenes strains of the L. monocytogenes database at the Pasteur Institute (Paris, France). Objective 2: To characterize the mechanism underlying resistance of ECII bacteria grown at 20C to listeriaphage, we will construct transposon mutants of the 1998-99 epidemic strain H7550 that will be susceptible to listeriaphage attack when grown either at 20 or at 37C. The inactivated gene will be identified, and its role will be confirmed by construction of deletion mutants and by genetic complementation. Mutants will be characterized in terms of their biofilm formation (biofilm studies will be done by Dr. Hua Wang) and resistance to disinfectants, cadmium and arsenic. To determine the impact of the targeted gene on overall fitness of the organisms, mixed cultures (initiated with a 1:1 mixture of wildtype and deletion mutant) will be evaluated in terms of the relative survival of the organisms during growth, including during late stationary phage (nutrient depletion), and at 4C. Relative frequencies of wildtype and mutant will be determined by colony hybridizations using a probe internal to the deleted gene (which thus will hybridize with wildtype colonies, but not with those of the deletion mutant). Objective 3: To determine whether disinfectant resistance and biofilm formation is affected by growth temperature of the bacteria, we will determine resistance of bacteria grown at different temperatures (4, 20, 37C) to the quaternary ammonium compound benzalkonium chloride (BC). In addition, ability of ECII bacteria to form biofilms at these temperatures will be characterized bacteriologically and by means of epifluorescence and confocal microscopy, by Dr. Hua Wang. Objective 4: To evaluate stability of plasmids from ECII and other serotype 4b Listeria strains resistant to disinfectant and cadmium, we will determine whether the plasmids can be eliminated (plasmid curing) following repeated cultivation of the bacteria at 42C. Plasmid-free derivatives will be detected by their lack of resistance to cadmium and loss of plasmid will be confirmed by plasmid extraction and pulsed field gel electrophoresis. To determine whether the plasmids can be mobilized to plasmid-free recipients, bacterial conjugations will be performed and the transconjugants evaluated in terms of plasmid content and resistance to disinfectants and to heavy metals.

<P>

PROGRESS: 2007/09 TO 2008/08<Br>
OUTPUTS: We have characterized a library of strains from the turkey processing plant environment in regard to their resistance to disinfectant and to heavy metals (cadmium arsenic), as well as in regard to their molecular fingerprints. Bioinformatics analyses were performed to determine prevalence of these systems in the chromosome or in mobile elements (plasmids) of Listeria. We investigated stability of cadmium resistance and disinfectant resistance phenotypes (and underlying genetic determinants) in several genetically distinct strains from the processing plant environments, as well as in strains from specific outbreaks of listeriosis. We have analyzed the strains from the turkey processing plants in terms of the prevalence of mutated internalin A genes, which harbor premature stop codons. The impact of internalin A premature stop codon mutations in environmental fitness of Listeria (especially in regard to survival and growth in raw and pasteurized milk) was assessed. The resistance of strains form the processing plant environments to Listeria specific phages was also determined. Phages were isolated from environmental samples derived from the turkey processing plants, and screened in terms of their ability to infect and kill Listeria strains from these environments. Strains from major outbreaks were extensively characterized in terms of their susceptibility to phage, to determine the suitability of phage as possible biocontrol for such strains. The genetic mechanisms underlying resistance to phage among strains from major outbreaks were investigated. The impact of growth conditions on freeze thawing tolerance of Listeria was also investigated. We genetically characterized two genomic regions that appears to undergo marked divergence in L. monocytogenes, and that harbor unique sequences in strains from different epidemic clones. We assessed the potential of specific sequences for accurate epidemic clone detection and monitoring. <Br>
PARTICIPANTS: The project provided numerous opportunities for training in bacteriology, bacterial genetics and physiology, subtyping and determinations of susceptibility to antimicrobials, disinfectant, and heavy metals for five graduate students (Cheng Ying, Savitri Mullapudi, Sangmi Lee, Reha Azizoglu, Sheea White); one postdoctoral scientist (Driss Elhanafi); two undergraduate research interns (Simone Wilson, Jach Byrd); nine collaborating scientists (Nan Faith and Chuck Czuprynski, University of Wisconsin; Nattawan Promadej, university of Hawaii, currently at the Centers for Disease Control and Prevention; Q. Zhang and J. Luchansky, USDA-ARS; Brien Nudeck, University of Tennessee; Kate Sperry, Leslie Wolf and Justin Edwards, North Carolina Department of Health, Raleigh, NC); one visiting scientist (Natalia Markelova, Moscow State university, Russia). In addition, the laboratory manager, Robin Siletzky and the PI Sophia Kathariou participated in the project. <Br>
TARGET AUDIENCES: Target audiences for the research project and findings thereof include individuals associated with food industry (especially managers of food processing plants) who will be interested in knowing the prevalence of disinfectant and phage resistance among Listeria strains from the environment of the processing plants. A second target audience would include individuals from regulatory agencies, e.g. agencies approving and monitoring use of phage for Listeria biocontrol and regulatory agencies involved in the formulation of improved risk assessments for Listeria in the food supply; accurate data on prevalence of potentially attenuated strains in the processing plants will be needed for improved risk assessments. A third target audience would include members of the scientific community, who would be interested in the population structure and genomic attributes of the organisms, the implications of the findings for the pathogens' evolution and environmental adaptations, the genetic basis for epidemic clone emergence and niche adaptation, and horizontal gene transfer of resistance determinants in Listeria. A fourth target audience would be members of the food safety community, who would be interested in multiple aspects of the findings related to the adaptations of the pathogens and their survival in the processing plants and in specific commodities; Listeria continues to be associated with significant disease burden in the United States and to pose serious economic threats to the ready to eat food industry. <Br>
<Br>
IMPACT: 2007/09 TO 2008/08<Br>
Our bioinformatics analysis revealed three different cadmium resistance gene cassettes in Listeria. We succeeded in identifying genes responsible for resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) on the large plasmid harbored by certain strains. The prototype for this plasmid is pLM80, identified during the genome sequencing of a strain implicated in the 1998-99 outbreak of listeriosis traced to contaminated hot dogs. Analysis of disinfectant resistance among Listeria strains from the environment of turkey processing plants revealed that all strains resistant to BC were also resistant to the heavy metal cadmium. A novel finding was that strains resistant both to BC and to cadmium had a higher likelihood of harboring one specific cadmium resistance cassette than strains which were only resistant to cadmium, but susceptible to BC. Serial passages of the strains under conditions that would lead to plasmid curing suggested that the resistance determinants were unusually stable in the majority of the environmental strains. However, loss of cadmium and BC resistance following such passages provided evidence of plasmid harborage. A number of the strains from the processing plants harbored internalin A genes with premature stop codons. Sequence analysis provided accurate information of the prevalence of the different stop cordon mutations among these strains, and identified several novel mutations, including in serotype 4b bacteria which previously were never reported to harbor such mutations. Comparative fitness studies assessed survival and growth of strains harboring such mutations in laboratory media and in raw as well as pasteurized milk. A high proportion of strains of serotype 1/2a and 1/2b were found resistant to wide host range phages isolated from the processing plants, as well as to other tested wide host ranges. In contrast, strains of serotype 4b were largely phage susceptible. Strains from one of the major epidemic clones were uniformly resistant to the phages under certain growth conditions, suggesting that resistance would be likely in the environment of the processing plants and in foods. These findings raise suggest great caution in possible uses of phage as biocontrol: such use might eliminate other strains, while leaving problematic epidemic strains unharmed. The genetic basis of phage resistance among the epidemic strains was investigated, and a novel gene cassette was identified using transposon mutagenesis. Analysis of freeze thawing tolerance following growth under different conditions revealed that cryotolerance was significantly enhanced following growth at 37C, a temperature that also permits virulence gene expression in Listeria. Genetic analysis of two regions that have unique genetic content in strains from major epidemic clones revealed possible roles of the genes in survival under specific stress conditions (e.g. low temperature and high salt; high temperature and nutrient depletion). DNA macroarray analyses identified sequences that can be used for unambiguous detection of epidemic clone II strains from clinical and environmental sources.

<P>

PROGRESS: 2006/09/01 TO 2007/08/31<Br>
OUTPUTS: The objectives of this project were to compare genome content between 1998-99 and 2002 outbreak strains and isolates with ECII genetic markers obtained from two different processing plants; to further investigate the impact of temperature on resistance of ECII bacteria to listeriaphage from the processing plant environment, and to elucidate the underlying mechanism; to determine possible impact of temperature on resistance of ECII bacteria to disinfectants and on formation of biofilms; and to characterize stability and self-mobilization of the ECII plasmid pLM80, and to determine the possible role of this plasmid and selected plasmid-borne genes in resistance to disinfectants and biofilm formation. We have provided conclusive evidence that the 1998-99 and the 2002 outbreak strains were members of the same clonal group. In addition, we identified and characterized intriguing differences, localized on the large plasmids harbored by strains from the two outbreaks. We identified and validated a novel probe (chromosomal locus) that appears to be highly discriminatory for identification of ECII strains. We confirmed that all ECII strains, regardless of source, demonstrate the unusual temperature-dependent resistance to listeriaphage from the processing plant environment. We have also partially characterize these phages, and found that they are highly similar to P100 (GRAS status listeriaphage). To characterize the genetic mechanism underlying the observed resistance to phage, we constructed and screened mutant libraries of Tn916E, Tn917, and, mariner transposon. Two mutants were identified (one Tn916E, one mariner), and are being characterized. We have identifying a novel gene cassette mediating resistance to BC. We have confirmed the location of this cassette, and a cassette mediating resistance to cadmium, on large plasmids. Plasmid mobilizations, curing, and biofilm assays are continuing. <Br>PARTICIPANTS: Individuals who worked on this project included three graduate and two undergraduate students, one postdoctoral scientist, and our laboratory manager. The project included collaborations with the Centers for Disease Control and Prevention, the North Carolina Department of Health, and USDA-ARS. It also involved collaborations with scientists from several universities, including Ohio State University, the University of Wisconsin, University of Tennessee, Michigan State University, and Virginia Tech. The project provided opportunities for academic and research training essential for the three students pursuing their Ph.D. (two students), as well as for the postdoctoral scientist and the undergraduate students. <Br>TARGET AUDIENCES: Our target audiences are: The Public Health and Food Safety sector, specifically stakeholders involved in the monitoring and prevention of human illness due to Listeria, and in reduction of this pathogen's burden in foods, especially those that are highly processed, ready to eat, cold-stored food commodities. The food processing industry, especially those sectors dedicated to production of foods that are at risk for Listeria contamination. Consumers groups, who need information on food safety issues associated with specific commodities for which Listeria contamination may pose special risks, such as contaminated soft cheeses, hot dogs, deli meats, and other highly processed specialty products.
<Br><Br>
IMPACT: 2006/09/01 TO 2007/08/31<Br>
Listeria continues to be troublesome pathogen, due to its high case fatality rate, its ability to colonize the environment of food processing plants, often persisting there for several years, and its ability to grow in the cold. In the course of the project we have discovered that the disease burden of ECII strains (apparently sporadic cases) has increased in years subsequent to the 2002 outbreak. Prompt identification and effective monitoring of these troublesome strains will contribute to prevention of outbreaks and reduction of disease burden. Our findings related to listeriaphage resistance and disinfectant resistance will be valuable for understanding of the ecology of these organisms, and for their reductions and control in the processing plant environment.