Campylobacter jejuni, a widespread pathogen found in birds and mammals, poses a significant risk for zoonosis worldwide despite its susceptibility to environmental and food-processing stressors. One of its main survival mechanisms is the formation of biofilms that can withstand various food-processing stressors, which is why efficient methods for assessing biofilms are crucial. Existing methods, including the classical culture-based plate counting method, biomass-staining methods (e.g., crystal violet and safranin), DNA-staining methods, those that use metabolic substrates to detect live bacteria (e.g., tetrazolium salts and resazurin), immunofluorescence with flow cytometry or fluorescence microscopy, and PCR-based methods for quantification of bacterial DNA, are diverse but often lack specificity, sensitivity, and suitability. In response to these limitations, we propose an innovative approach using NanoLuc as a reporter protein. The established protocol involves growing biofilms in microtiter plates, washing unattached cells, adding Nano-Glo luciferase substrate, and measuring bioluminescence. The bacterial concentrations in the biofilms are calculated by linear regression based on the calibration curve generated with known cell concentrations. The NanoLuc protein offers a number of advantages, such as its small size, temperature stability, and highly efficient bioluminescence, enabling rapid, non-invasive, and comprehensive assessment of biofilms together with quantification of a wide range of cell states. Although this method is limited to laboratory use due to the involvement of genetically modified organisms, it provides valuable insights into C. jejuni biofilm dynamics that could indirectly help in the development of improved food safety measures.

Biofilms serve as a bacterial survival strategy, allowing bacteria to persist under adverse environmental conditions. The non-pathogenic Listeria innocua is used as a surrogate organism for the foodborne pathogen Listeria monocytogenes, because they share genetic and physiological similarities and can be used in a Biosafety Level 1 laboratory. Several methods are used to evaluate biofilms, including different approaches to determine biofilm biomass or culturability, viability, metabolic activity, or other microbial community properties. Routinely used methods for biofilm assay include the classical culture-based plate counting method, biomass staining methods (e.g., crystal violet and safranin red), DNA staining methods (e.g., Syto 9), methods that use metabolic substrates to detect live bacteria (e.g., tetrazolium salts or resazurin), and PCR-based methods to quantify bacterial DNA. The NanoLuc (Nluc) luciferase biofilm assay is a viable alternative or complement to existing methods. Functional Nluc was expressed in L. innocua using the nisin-inducible expression system and bacterial detection was performed using furimazine as substrate. Concentration dependent bioluminescence signals were obtained over a concentration range greater than three log units. The Nluc bioluminescence method allows absolute quantification of bacterial cells, has high sensitivity, broad range, good day-to-day repeatability, and good precision with acceptable accuracy. The advantages of Nluc bioluminescence also include direct detection, absolute cell quantification, and rapid execution.

Graphic abstract:

Engineering Listeria innocua to express NanoLuc and its application in bioluminescence assay.