Campylobacter jejuni are known as the main cause of gastroenteritis in humans worldwide. In nature, they are predominantly found in single-species and multi-species biofilms, which allow them to survive in unfavorable environmental conditions. Under stress, they enter a viable but non-culturable (VBNC) state, making it impossible to accurately quantify them using classical methods, such as colony-forming unit (CFU) counting. Cells in the VBNC state retain their virulent potential, so it is essential to develop a method that enables rapid and reliable quantification of all living C. jejuni cells in a biofilm sample. The aim of this master's thesis was to establish and optimize a method for quantifying C. jejuni bacteria in single- and multi-species biofilms using ddPCR in combination with the reagent PMA, which selectively penetrates damaged cell membranes, covalently binds to DNA, and prevents its amplification during PCR. Special emphasis was placed on optimizing PMA treatment. It was found that PBS buffer is not suitable for preparing dilutions and washing biofilms when combined with PMA. The results showed that double treatment improves PMA efficiency. Double treatment with 25 µM PMA provides the best results in reducing dead cell signals without affecting the signal of live cells, as confirmed by qPCR and ddPCR methods. In
general, more C. jejuni are detected with the ddPCR method than with the CFU method, suggesting the transition of cells into the VBNC state and the sensitivity of the method
itself. Using the ddPCR-PMA and CFU methods, we demonstrated that the ability of C.jejuni to form biofilms improves in mixed biofilms with Pseudomonas fragi. A major issue in interpreting the results was contamination with C. jejuni DNA, which was found to be present on laboratory surfaces and in pipettes, and appeared as background during sample quantification. Before conducting further experiments and repeating some optimization steps, it would be necessary to eliminate the contamination.
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