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Molecular Mechanisms for Interactions Between Listeria Monocytogenes and Produce

Kathariou, Sophia; Mandrell, Robert; Gorski, Lisa; Miller, Webb
North Carolina State University
Start date
End date
The long term goal of the research is to characterize the mechanisms mediating produce colonization with Listeria monocytogenes, and to design novel control strategies.

To contribute to this long term goal, the following specific objectives will be pursued: Objective 1: To assess relative produce colonization ability of a panel of strains of L. monocytogenes, including representatives of different lineages and sources.
Objective 2: To identify and characterize genes of L. monocytogenes required for produce colonization (adherence, survival, growth).
Objective 3: To assess role of the genes on biofilm formation by L. monocytogenes on stainless steel and produce surfaces.

More information
Non-Technical Summary:
Listeria monocytogenes is a major cause of severe foodborne illness and death in the United States. Even though several high-profile outbreaks of listeriosis have involved deli meats and soft cheeses, current epidemiological trends suggest that Listeria -contaminated produce is associated with significantly higher disease burden than previously recognized. A multistate outbreak in 2008-09 was attributed to soybean sprouts, and in 2010 an outbreak with high mortality was due to chopped celery. This past summer and fall one of the largest known outbreaks of listeriosis (133 cases, 28 deaths) involved contaminated cantaloupe. Listeria is also responsible for numerous recalls. However, in spite of the evident public health and economic burden of Listeria-contaminated produce, only rudimentary knowledge is available on mechanisms and attributes mediating Listeria's ability to adhere to and colonize produce. Under Objective 1, produce dip inoculation will be employed to assess produce adherence and colonization ability of a carefully chosen panel of strains. Strains with high adherence and colonization potential will be chosen to identify and characterize genes required for adherence and colonization, in Objective 2. In Objective 3 we will employ molecular, bacteriological and microscopy tools to characterize biofilm formation on stainless steel and on produce surfaces, taking into account the presence of produce-associated microbiota. This component of the study is especially relevant for Listeria, a bacterium which is notorious for its ability to persistently colonize the food processing plant environment. Such environmental contamination has indeed been implicated in the recent cantaloupe outbreak of listeriosis as well as in the previous celery-associated outbreak. The study will provide baseline data critical for novel targeted strategies to reduce food safety threats associated with Listeria-contaminated produce, including threats associated with persistent colonization of produce-processing facilities and with bacterial adherence and growth on the produce itself.

Objective 1. A carefully chosen panel of 48 strains will be employed to determine the relative ability of the bacteria to survive and grow on lettuce and on soybean sprouts, cantaloupe and celery at different temperatures. Bacteria will be enumerated from the inoculated produce incubated at different temperatures.
Objective 2. We will employ two different Listeria monocytogenes strains, and two mariner mutant libraries for each strain. A pool of non-adherent mutants will be obtained from the rinsate of inoculated produce, along with an adherent pool which will remain on the surface. The mariner-based transposon-Listeria junction fragments in each pool will be processed for 454 sequencing and genes with different representation in each pool will be identified. The involvement of selected genes in colonization of produce will be characterized by deletion construction and genetic complementation analysis.
Objective 3. Mutants and their wild type parental counterparts will be analyzed in biofilms on stainless steel and on produce surfaces. Stainless steel biofilms will be constructed with pure cultures as well as with Listeria in the presence of produce-derived microbiota. Viable L. monocytogenes will be enumerated by quantitative real-time PCR (qPCR) with propidium monoazide. For biofilm assessments on produce, L. monocytogenes will be inoculated onto produce and inoculated surfaces will be screened by confocal laser scanning microscopy.

2012/02 TO 2013/02
OUTPUTS: We characterized the ability of a panel of Listeria monocytogenes strains, representing different serotypes (1/2a, 1/2b and 4b) to grow and persist on fresh produce stored at different temperatures (4, 8 and 25 degrees C) for up to 21 days. The panel included three different strains from the 2011 cantaloupe outbreak, a strain from the 2010 celery outbreak, and a strain isolated from celery and previously found to excel in produce colonization ability. Bacteria were inoculated on the rind, on freshly exposed flesh, in freshly extracted cantaloupe juice and freshly extracted celery juice. The impact of surface washing of the rind on removal and subsequent growth of the inoculated bacteria was tested. We pursued the construction of mariner-based transposon mutant libraries in four different strains of L. monocytogenes (two of serotype 1/2a and one each of serotype 1/2b and 4b) from produce (one strain) and produce-related outbreaks (three strains). Several transposon mutants and their wildtype parental counterparts were characterized for their ability to colonize produce. Mutants earlier identified based on their reduced susceptibility to disinfectant and other antimicrobials were also characterized for adherence on produce. We examined the ability of the cantaloupe outbreak-derived strains to serve as recipients of disinfectant resistance genes in conjugations with non-pathogenic listeriae that may serve as reservoirs for such genes. In consultation with co-PIs Gorski and Miller we developed a protocol to pilot-test the mutant library screening on produce.
PARTICIPANTS: Project involved participation of PI Kathariou and co-PIs Gorski, Mandrell and Miller. Kathariou had oversight of the overall project design and implementation including construction and characterization of transposon mutant libraries and assessing Listeria'a ability to grow on produce (cantaloupe rind, flesh and extract, celery extract). Gorski was responsible for implementing the lettuce seeding model to assess Listeria survival and test mutant libraries. Mandrell and Miller were responsible for pilot trials of the novel, sequencing-based protocol for identification of putative mutants. R. Siletzky participated as lab manager and research technician in project. S. Ratani (Masters student), Rakic-Martinez (predoctoral student) and C. Parsons (predoctoral student) participated as graduate students. K. Berger and R. Poe participated as undergraduate research assistants.
TARGET AUDIENCES: Target audiences include the produce industry which is interested in pre-harvest reductions of Listeria, especially since the 2011 cantaloupe outbreak. Listeria-contaminated produce is subject to recall with adverse economic repercussions. Discontinuation of production of certain types of produce due to implication in disease adversely impacts the economic vitality of this sector of US agriculture and compromises the well being and sustainability of farming communities. The food safety and public health sector are relevant target audiences, since their mission includes reductions in prevalence of human listeriosis, for which contaminated produce has emerged as a major vehicle. Graduate students participating in the project were served by enhanced knowledge and expertise in food safety-related research, including research on molecular genetic and bacteriological attributes mediating Listeria's ability to adhere to, survive and persist on fresh produce. Technical staff participating in project was served by development of additional expertise. The scientific community is served by acquisition of further knowledge on contamination of fresh produce by Listeria.
PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

IMPACT: All tested Listeria monocytogenes strains were able to grow on cantaloupe inoculated on the rind, freshly exposed flesh, and in juice from cantaloupe or celery. Data were statistically analyzed with SAS using the linear mixed effects model. L. monocytogenes populations increased by approximately 10fold following 21 days incubation at 4 or 8C, and by approximately 100fold following 7 days incubation at 25C. Increases were higher on the rind than on the flesh or in the juice, with statistically significant differences after 7 days of incubation at 4C and 72 hr at 25C. No significant differences were noted among the three different strains from the cantaloupe outbreak, but certain other strains exhibited enhanced potential to grow on the inoculated produce. Rinsing of the inoculated rind resulted in temporary reductions of the L. monocytogenes population but the remaining bacteria resumed growth more rapidly than those on inoculated rind in the absence of rinsing. Mariner-based transposon mutant libraries were constructed in four strains, including the celery-derived strain F8027 previously reported to excel in produce colonization potential and three strains from recent (2010, 2011) listeriosis outbreaks involving cantaloupe and celery. A panel of mutants with selected phenotypes (cold sensitive; catalase-negative; non-hemolytic; deficient in purine biosynthesis ; reduced tolerance to cadmium; altered phage susceptibility) were tested for growth on inoculated rind, flesh and extract together with their wild type parental counterparts and found to exhibit intact ability to grow and persist on inoculated cantaloupe. Mutants earlier identified based on their reduced tolerance to disinfectants and other antimicrobials were found to be intact in their produce colonization potential. The cantaloupe outbreak-derived strains were found to serve as recipients of disinfectant resistance genes in conjugations with non-pathogenic listeriae that may serve as reservoirs for such genes. An optimized scheme for screening the entire mutant library for produce colonization was developed in consultation with co-PIs Gorski and Miller. This scheme involves the use of cantaloupe rind inoculations with the mutant library to serially enrich for transposon mutants unable to colonize the produce. Non-adherent mutants in the rinsate will be utilized as inoculum in successive inoculations. The mutant library screening in this cantaloupe rind model will allow pilot testing of the method prior to testing by co-PIs Gorski and Miller on the more demanding lettuce seedling model.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens
Chemical Contaminants
Natural Toxins