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Studies of biofilm formation by bacteria are crucial for understanding bacterial resistance and for development of novel antibacterial strategies. We have developed a new bioluminescence biofilm assay for  Listeria innocua , which is considered a non-pathogenic surrogate for  Listeria monocytogenes. L. innocua  was transformed with a plasmid for inducible expression of NanoLuc luciferase (Nluc). Concentration-dependent bioluminescence signals were obtained over a concentration range of more than three log units. This biofilm assay enables absolute quantification of bacterial cells, with the necessary validation. For biofilm detection and quantification, this “Nluc bioluminescence” method has sensitivity of 1.0 × 10 4  and 3.0 × 10 4  colony forming units (CFU)/mL, respectively, with a dynamic range of 1.0 × 10 4  to 5.0 × 10 7  CFU/mL. These are accompanied by good precision (coefficient of variation, <8%) and acceptable accuracy (relative error for most samples, <15%). This novel method was applied to assess temporal biofilm formation of  L. innocua  as a function of concentration of inoculant, in comparison with conventional plating and CFU counting, the crystal violet assay, and the resazurin fluorescence assay. Good correlation ( r  = 0.9684) of this Nluc bioluminescence assay was obtained with CFU counting. The limitations of this Nluc bioluminescence assay include genetic engineering of bacteria and relatively high cost, while the advantages include direct detection, absolute cell quantification, broad dynamic range, low time requirement, and high sensitivity. Nluc-based detection of  L. innocua  should therefore be considered as a viable alternative or a complement to existing methods.

Expression of NanoLuc Luciferase in Listeria innocua for Development of Biofilm Assay