We underline the urgent need for increased surveillance and measures to contain foodborne bacterial infections, as emphasized by both the European Centre for Disease Prevention and Control and European Food Safety Authority (EFSA 2023). Campylobacteriosis was the most frequently reported zoonosis, with 137,107 cases in 2022. The treatment of infections associated with biofilms poses a particular challenge, as microbes in biofilms are up to 1000-fold more resistant to antibiotics than their planktonic counterparts (Rossi et al. 2021). Consequently, pathogenic biofilms contribute to persistent contamination and repeated infections and exacerbate the problem of antimicrobial resistance in the food industry (Paul et al. 2022). Biofilms function as multi-layered protective networks, and investigating the intricate and resilient nature of C. jejuni biofilms requires a comprehensive approach. Each cell within these communities plays a distinct role and is characterized by different properties that contribute to the overall function and resilience of the biofilm (Tram et al. 2020; Ma et al. 2022). The recurrent formation of biofilms is closely linked to the transition of bacteria to the VBNC state. This complex relationship emphasizes the importance of understanding the different roles and characteristics of individual cells within biofilms to effectively monitor and control persistent and resistant forms of C. jejuni.

In the current study, a plasmid construct containing the NanoLuc luciferase gene (pMW10_nLuc) was developed and successfully introduced into C. jejuni, resulting in a new bioluminescent C. jejuni NanoLuc strain. This method was validated by demonstrating increasing bioluminescence values with increasing cell densities. The limit of detection and limit of quantification were determined to be 9.12 (± 2.13) × 106 and 1.61 (± 0.48) × 107 CFU/mL, respectively. Although this method is effective, it is less sensitive than the Listeria innocua bioluminescence-based method described by Berlec et al. (2021). Nevertheless, the accuracy and precision of the method were within acceptable ranges: the relative error remained below 30% for most samples, and the precision (indicated by the coefficient of variation) was below 11% at higher concentrations and below 19% at lower concentrations. Crucially, the technique demonstrated reliable repeatability on consecutive days.

We used the C. jejuni NanoLuc strain together with bioluminescence assays to monitor the evolving state of biofilm after 4, 8, 24, 48, and 72 h. We compared the results of bioluminescence with the results of established biofilm quantification methods: crystal violet staining, plate counting, and resazurin fluorescence assays. We also used the bioluminescence method to monitor C. jejuni biofilm formation on both abiotic and biotic surfaces. Abiotic conditions were mimicked using polystyrene microtiter plates, whereas biotic conditions were mimicked by coating the same type of plates with different mucin concentrations. The process of initial cell adhesion and subsequent biofilm development showed similar patterns on both surfaces, with cells already adhering within the first few hours. This observation is consistent with the findings of Moe et al. (2010), who reported that the structural organization of Campylobacter biofilm becomes apparent already after 6 h.

After 4 and 8 h, the biofilm cell numbers on polystyrene surfaces determined by plate counting were similar or even lower than those determined by bioluminescence. With increasing incubation times from 24 to 72 h, cell numbers determined by bioluminescence measurements exceeded those determined by plate counting. The number of culturable cells identified using the plate counting method increased after 24 h and remained high for up to 48 h. After 72 h, however, the detectable number of culturable cells significantly decreased. These findings suggest that the critical period for C. jejuni biofilm formation is 24 h. After 48 and 72 h, the biofilm enters a mature or late phase, consistent with definitions that emphasize specific features related to the composition of extracellular polymeric substances (Ma et al. 2022) and that is characterized by cell death and dynamic processes of attachment and detachment, maintaining a relatively stable total cell number.

In our study, the differences in biofilm cell numbers between different mucin concentrations were less pronounced than expected. Notably, bioluminescence assays revealed that biofilms on mucin surfaces were more abundant than those on polystyrene, underscoring the important role of protein-glycan interactions in facilitating bacterial adhesion and invasion in the gastrointestinal tract (Alemka et al. 2012; Linden et al. 2008; Sabotič et al. 2023). After 72 h, the plate counting method revealed a decrease in culturable cells on mucin surfaces, similar to the observations on polystyrene surfaces. The data suggest that as biofilms mature (characterized by outer cells detaching and floating freely after two to 3 days), cells within the biofilm simultaneously transform into non-culturable states, including VBNC forms. However, these transformed cells remain detectable by bioluminescence assays, underscoring the effectiveness of this method in identifying different cell states during biofilm development.

In this study, crystal violet staining indicated an increase in biofilm biomass over time due to the accumulation of extracellular polymers rather than an increase in cell numbers within mature biofilms. This observation is consistent with the challenges of counting individual cells in immature biofilms, which is labor-intensive and prone to bias and becomes even more complicated in mature biofilms due to their three-dimensional structure (Wilson et al. 2017).

We also measured fluorescence using resazurin, which quantifies cells that are metabolically active. After 48 and 72 h, fluorescence rates slightly decreased compared to that after 24 h. This subtle decrease implies that despite the decrease in cell number in the mature phase after the initial increase in cell number, the metabolic activity of the cells was not significantly changed. This trend is consistent with the observations made with different biofilm quantification methods over time, which vary depending on the initial inoculum concentrations (Supplementary Figure S3).

The negative trends observed with plate counting and resazurin fluorescence indicate a decrease in the culturability and metabolic activity of biofilm cells. This implies that methods relying on metabolic activity (e.g., resazurin assays) can provide inaccurate results for adherent cells (Klančnik et al. 2021) and thus potentially also biofilm cells. By contrast, the positive trends observed with NanoLuc and crystal violet staining indicate an increase in total cell count and biomass. Crystal violet and resazurin staining methods have relatively lower sensitivity and higher standard deviations compared to plate counting and bioluminescence methods, emphasizing the importance of using multiple techniques for a comprehensive quantitative analysis of biofilm formation. The differences in sensitivity and precision between methods indicate that no single method can cover all aspects of biofilm characteristics; instead, the combination of different techniques provides unique insights into biofilm behavior.

To simulate conditions similar to real food, chicken meat juice was used as the cultivation medium, replacing conventional MH broth. Bioluminescence and plate counting revealed significantly higher numbers of attached and biofilm-forming cells in chicken juice than those in MH broth. Chicken juice contains nutrient-rich particles that can form a protective layer on surfaces, facilitating the initial adhesion and subsequent biofilm formation of bacteria such as Campylobacter (Li et al. 2017). This synergy between the nutritive properties of chicken juice and its role in improving surface conditions highlights the complex interplay of factors that contribute to biofilm development on different materials (Brown et al. 2014; Li et al. 2017). In multispecies environments, the presence of other bacterial species can significantly influence the growth and biofilm formation of pathogenic bacteria, and this requires specific detection methods (Ica et al. 2012).

Our quantification of C. jejuni biofilm cells in a mixed culture with pathogenic S. enterica with the bioluminescence method revealed a higher number of cells compared to the plate counting method. This is an indication of the effectiveness of bioluminescence measurement in detecting biofilm formation in a competitive multispecies environment. This highlights the complex dynamics of bacterial interactions and their impact on biofilm formation and pathogen persistence.

The value of the NanoLuc bioluminescence method lies in its sensitivity and specificity as well as technical simplicity and speed. This combination enables the specific detection of C. jejuni cells expressing the NanoLuc enzyme, enabling holistic monitoring of biofilm formation. This capability is particularly critical for comprehensive studies of biofilm dynamics, as it provides insight into the behavior of C. jejuni under different conditions, supporting effective strategies for dealing with biofilms in both clinical and food safety contexts.

Table 3 shows a comparative analysis of the NanoLuc method under different experimental conditions. This includes an assessment of biofilm state (early/mature), mixed biofilms, biofilm thickness, surface type, and dynamic biofilms for all four methods: plate counting (CFU/mL), NanoLuc bioluminescence assay, crystal violet assay, and resazurin. Bioluminescence is a very sensitive and selective method with low background signal, making it suitable for use in complex biofilm environments. It can detect VBNC forms and damaged but not lysed cells and specifically identify organisms expressing the NanoLuc protein, which is advantageous for studying various microbial or multi-species biofilms altogether. When comparing methods for monitoring biofilms, NanoLuc bioluminescence stands out as an effective technique due to its ability to detect different phenotypic subpopulations within biofilms. This capability is particularly important for the study of Campylobacter as it allows the identification of combined culturable cells, VBNC forms and damaged but not lysed cells together. However, it should be emphasized that this method alone does not make it possible to differentiate between these various cell states that may be present in biofilms. In dead cells, the signal initially remains stable but is transient; the luminescence fades as soon as the cells are completely degraded and NanoLuc is inactivated.

The transition of Campylobacter to the VBNC state is triggered by several stress factors that occur during food production and processing. These conditions often lead to the accumulation of organic and inorganic matter, which favors the development of resistant biofilms and enables bacteria to survive in hostile environments. This represents a significant challenge for both clinical management and public health efforts, as emphasized by Carrascosa et al. (2021). Recognizing the importance of VBNC states in the analysis of Campylobacter is therefore critical, as these states have a profound impact on detection methods and research results. As such, advanced detection methods such as NanoLuc bioluminescence should be incorporated into studies on the complex dynamics of bacterial biofilms, particularly in the context of mitigating public health risks associated with foodborne pathogens.

In contrast to NanoLuc, plate counting is an indirect method that focuses exclusively on live culturable cells. Crystal violet and resazurin also offer direct detection, with crystal violet detecting all components of the biofilm, and resazurin targeting living metabolically active cells. NanoLuc combined with measurements of culturable cells by plate counts and metabolic activity by resazurin provides absolute cell quantification, in contrast to the relative quantification of crystal violet.

One of the downsides od this methosd is C. jejuni requiring genetic modification to express the NanoLuc enzyme. In complex samples, other luminescent signals or quenching effects could interfere with the detection of the specific bioluminescent signal of NanoLuc-expressing C. jejuni, requiring additional controls. In terms of operating costs and time, a plate reader is required for NanoLuc as well as crystal violet and resazurin tests. Conversely, plate counting is more time-consuming but does not require specialized equipment.

Our findings have important implications for food safety and public health, particularly for controlling the spread of Campylobacter in poultry. By contaminating chicken meat with luminescently labelled Campylobacter, we could track the transmission of the pathogen through the entire food processing chain—from poultry to equipment and packaging to consumers’ kitchens. This study highlights the potential for biofilm formation at various stages of food processing, with the bacteria persisting in a viable but non-culturable (VBNC) state. Using molecular methods, we could monitor the presence and persistence of these bacteria, providing important insights into contamination sites. These findings can help to develop better hygiene practices and intervention strategies to reduce foodborne infections.

The developed technology has proven useful in various applications, including cell biology, the study of microbial interactions, cell signaling, protein–protein interactions, gene regulation, and protein stability (England et. al, 2016; Shang et. al, 2023; Chen et. al, 2015; Russo et. al, 2022). These applications can be specifically tailored to the C. jejuni strain developed in this study.

In summary, the NanoLuc bioluminescence assay represents an advance in monitoring and understanding C. jejuni biofilms by enabling non-invasive assessment of bacterial communities without the need for physical cell manipulation. Its specificity, sensitivity, and technical simplicity emphasize its potential as a tool for specific C. jejuni detection and holistic biofilm monitoring, which could improve our understanding of biofilm development, structural integrity, and resistance mechanisms. By allowing in situ application, this method preserves the natural conditions of biofilms, which is essential for studying their natural development and resistance to cleaning and disinfection. In addition, the advantages of the assay, including direct detection, absolute cell quantification, minimal time required, and high sensitivity, represent a significant alternative or complement to conventional biofilm quantification techniques. Its ability to quantify all cell types within biofilms, including VBNC cells, underscores its importance for public health, particularly regarding C. jejuni infectivity and survival strategies. However, the limitations of the approach, such as detection thresholds, the need for genetic modification, and the relative cost, point to the importance of an integrated methodology. Future studies should aim to validate the NanoLuc assay under different real-life conditions and explore its applicability in vivo to expand its utility in infection studies and clinical diagnostics. The NanoLuc bioluminescence assay shows promise to improve our understanding of biofilms, with significant implications for public health, food safety, and beyond.