Sekundarni povzetek: |
Campylobacter jejuni, a leading cause of bacterial gastroenteritis worldwide, presents a significant challenge due to its ability to form tenacious biofilms in the food processing industry. These biofilms enable its survival in adverse environments, posing a grave threat to food safety and public health. Understanding biofilms is essential for tackling bacterial resistance and the development of effective infection control strategies. In master's degree thesis, we addressed the pressing need for novel, rapid, specific, and reproducible methods to detect and quantify bacteria within biofilms which is particularly crucial for C. jejuni, which can survive harsh conditions in a viable but non-culturable state. We designed the pMW10_nLuc plasmid with a constitutive NanoLuc luciferase system. The plasmid was successfully transformed into C. jejuni, introducing a precise and consistent method for quantifying bacteria through bioluminescence. Our innovative approach allowed us to quantify planktonic cells and assess bacterial adhesion and biofilm formation on polystyrene and mucin-coated surfaces. We compared our method to conventional quantification techniques, including colony counting, resazurin-based live cell quantification, and crystal violet staining of biofilm biomass. To establish specificity, we validated our method in mixed cultures with Salmonella enterica strain. Its applicability was demonstrated in vitro, using bovine feces, and with chicken juice as a food model. Furthermore, we employed our method to quantify planktonic cells and evaluate adhesion/biofilm formation after treating cells with the antibiotic ciprofloxacin and three natural anti-adhesive substances (epigallocatechin gallate, carvacrol, thymol). In summary, our introduced method represents a significant advancement in the rapid and comprehensive quantification of adhered C. jejuni cells within biofilms. Additionally, we successfully generated mutant strains featuring Cj0794 and Cj0736 gene knockouts, shedding light on their roles in C. jejuni biofilm formation. |