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PATHOGENESIS OF BIOFILM-ISOLATED LISTERIA MONOCYTOGENES AND BIOFILMS CONTROL USING FOOD-GRADE NATURAL ANTIMICROBIALS
Foodborne pathogens form biofilms as a survival strategy in various unfavorable environments, and biofilms are known to be the frequent source for infection and outbreaks of foodborne illness. Therefore, it is essential to understand the pathogenicity of bacteria in biofilms and methods to inactivate biofilm-forming microbes from food processing environments, including school cafeteria or other community-based food production facilities, and to prevent foodborne outbreaks. Pathogen transmissions occur primarily through raw or under cooked foods and by cross contamination during unsanitary food preparation practices. Then, pathogens can form biofilms on the surface and become persistent in food production facilities and can be a source for recurrent contamination and foodborne outbreaks. In this study, our first aim was to use L. monocytogenes as a model pathogen to study how an enteric infectious pathogen isolated from biofilm modifies its pathogenesis compared to its planktonic counterpart. Both clinical and food isolates with different serotypes and biofilm-forming abilities were selected and tested using cell culture and mouse models. L. monocytogenes sessile cells isolated from biofilms express reduced levels of the lap, inlA, hly, prfA, and sigB and show reduced adhesion, invasion, translocation, and cytotoxicity in the cell culture model than the planktonic cells. Oral challenge of C57BL/6 mice with food, clinical, or murinized-InlA (InlAm) strains revealed that at 12 and 24 h post-infection (hpi), L. monocytogenes burdens are lower in tissues of mice infected with sessile cells than those infected with planktonic cells. However, these differences are negligible at 48 hpi. Besides, the expressions of inlA and lap mRNA in sessile L. monocytogenes from intestinal content are about 6.0- and 280-fold higher than the sessile inoculum, respectively, suggesting sessile L. monocytogenes can still upregulate virulence genes shortly after ingestion (12 h).
After learning biofilm isolated L. monocytogenes cells have similar virulence potential as the planktonic counterparts, our next goal was to effectively prevent or inactivate biofilms using food-grade natural microbials. Since L. monocytogenes cells are usually found in multi-pathogen biofilm in nature, I combined two food-grade broad-spectrum natural antimicrobials, chitosan nanoparticles (ChNP) and ε-poly-L-lysine (PL), as ChNP-PL nanoconjugates and tested its function on single or mixed culture biofilms of L. monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella enterica serovar Enteritidis, and Pseudomonas aeruginosa. ChNP- PL not only was able to significantly (P<0.05) prevent the biofilm formation but also inactivate pre-formed biofilms when analyzed by crystal violet staining and plate counting. In vitro cytotoxicity analysis (LDH and WST-based assays) using an intestinal cell line, indicated ChNP- PL to be non-toxic. In conclusion, our results showed ChNP-PL has strong potential to prevent the formation or inactivation of preformed polymicrobial biofilms of foodborne pathogens in food processing environment. Application of ChNP-PL could inhibit the colonization of foodborne pathogens, minimize cross-contamination during food production, and eventually reduce foodborne outbreaks.