Background

Campylobacter jejuni, a pathogen found in the gastrointestinal tract of both domestic and wild birds and mammals, is a major cause of zoonosis worldwide [1]. However, it is sensitive to environmental stressors outside its hosts. To this end, C. jejuni forms biofilms as a survival strategy. Biofilms are characterized by a unique bacterial growth state and act as a protective shield that enables bacterial cells to resist certain environmental stressors—such as temperature fluctuations, desiccation, pH changes, and oxidative stress—commonly found on food preparation surfaces, food products, and water reservoirs [2–5]. Bacteria adapt to environmental stressors by, for example, adopting the nonmotile coccoid form and entering a viable but nonculturable state (VBNC) [6–8]. Viable but nonculturable cells cannot divide in vitro but maintain their membrane integrity and metabolic activity [9], which hinders their detection in quality control tests (a critical aspect of food safety) and may explain the persistence of campylobacteriosis in developed countries despite strict hygiene standards [1]. Studies suggest that bacteria in the VBNC state not only remain viable but also have the potential to recover and cause infections [10,11]. To assess biofilms, conventional cultivation methods and colony counts are generally used and usually complemented with biomass estimation using crystal violet staining [12,13]. The metabolic activity of biofilms can be assessed colorimetrically by monitoring the conversion of tetrazolium salt to formazan [14]. Simultaneously, ATP can be measured using BacTiter-Glo or resazurin fluorescence [8,12,13]. Advanced techniques such as transmission electron microscopy, scanning electron microscopy, and immunofluorescence with flow cytometry or fluorescence confocal laser scanning microscopy provide detailed information on the heterogeneity, cell localization, and structure of biofilms [15–17]. Additionally, PCR-based methods are used [12]. However, most of these methods are expensive, unspecific, technically demanding, and unsuitable for cell quantification. Therefore, new methods that can efficiently and rapidly detect C. jejuni cells would be valuable for advancing research related to food safety and possible applications in the food industry. The reporter protein NanoLuc offers several advantages, such as small size (19 kDa), high temperature stability (Tm = 60 °C), activity over a broad pH range (6–8), and the absence of post-translational modifications or disulfide bonds, which ensures uniform distribution within cells. Its most outstanding feature is its highly efficient bioluminescence. The reaction involves the NanoLuc enzyme, which is constitutively expressed, and the Nano-Glo substrate, commonly referred to as luciferin. Upon addition of the substrate in the presence of oxygen, NanoLuc (as luciferase) catalyzes the oxidation of luciferin and generates oxyluciferin. This molecule releases light in its excited energy state when it returns to its ground state [18,19]. No ATP is required for the luminescence reaction, which helps to minimize background luminescence. This reduction in the background signal ensures that the luminescence signal more accurately reflects the metabolic activity of the cells without external interference [20]. NanoLuc has already been successfully used to monitor and quantify Listeria innocua biofilms [21], and we have adapted the protocol to assess C. jejuni growth and biofilm formation. The protocol provides a comprehensive approach to biofilm monitoring and can be used as an important complement or alternative to conventional biofilm monitoring methods.