Identifying competitive exclusion microorganisms against Listeria monocytogenes from biological soil amendments by metagenomic, metatranscriptomic, and culturing approaches

Listeria monocytogenes, ubiquitous in the environment, has been increasingly recognized as the etiological agent causing foodborne disease outbreaks associated with a broad range of food products, with fresh produce as especially vulnerable for contamination at both farm and processing environments. Therefore, fully understanding the ecology of L. monocytogenes in these environments is the key to reduce or eliminate produce contamination with this pathogen. With great advance in next generation sequencing technology, both structure and functional capabilities of complex microbial communities can now be fully characterized. In our previous CPS-funded project, we successfully isolated competitive exclusion (CE) microorganisms against Escherichia coli O157:H7 from dairy and poultry composts as biological soil amendments. In considering of compost as a rich source of microorganisms with a diversity of microbial species, we propose to discover those potential compost-adapted CE microorganisms against L. monocytogenes using both metagenomic and metatranscriptomic approaches along with culturing methods. The first objective of our proposed study is to analyze the microbial community structure of compost products using16S rRNA and 18S rRNA sequencing in the presence and absence of L. monocytogenes (ca. 5~7 logs CFU/g). Briefly, the extracted high quality DNA from different compost samples (dairy- and poultry-wastes based) (n = 30) collected during composting process will be subject to phylogenetic marker analysis based on the sequencing of 16S rRNA for bacteria and 18S rRNA for eukaryotes to profile the microbial communities of these compost samples. The second objective is to conduct functional metatranscriptomics analysis of L. monocytogenes interactions with indigenous compost microflora to identify CE microorganisms with strong antagonistic activities against the pathogen. Following high throughput sequencing of high quality rRNA-depleted RNA from above selected composts, high-quality reads will be annotated and categorized based on the SEED database, and analyzed statistically with various metagenomics and metatranscriptomics tools. The impact of L. monocytogenes will be assessed by comparing the microbial functional profiles of composts with and without the inoculation of the pathogen and identifying changes in the abundances of genes associated with specific functional pathways using correlation-based analyses. For the third objective, we’ll use various modified culturing methods to isolate CE microorganisms including some viable but non-culturable species, and verify antagonistic activities of those CE cultures (ca. 5~8 logs CFU/g) against a cocktail of 3 L. monocytogenes strains in the inoculated composts by growth inhibition experiments. Findings from this project will reveal the structure and functional capabilities of indigenous microflora in a variety of compost samples, identify and isolate potential CE microorganisms for future studies on biological control of L. monocytogenes in various ecosystems, such as soil, water, and produce processing plants. Ultimately, our study will provide biological tools to effectively control L. monocytogenes contamination in produce growing and processing environments.