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

Abstract

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 × 10⁴ and 3.0 × 10⁴ colony forming units (CFU)/mL, respectively, with a dynamic range of 1.0 × 10⁴ to 5.0 × 10⁷ 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.

Keywords: Listeria innocua; NanoLuc; biofilm assay; bioluminescence; luciferase.

FIGURE 1

Bioluminescence intensity (logLI) as a function of concentration (logC) of L. innocua expressing Nluc (i.e., containing plasmid pMSP:Nluc; red) or control L. innocua (i.e., containing empty plasmid pMSP3545; black).

FIGURE 2

Monitoring of biofilm formation on polystyrene plates over 72 h using the Nluc bioluminescence assay (A), CFU counting assay (B), crystal violet staining (C), and resazurin fluorescence (D). Bars of the same color indicate biological repeats, each performed as three technical repeats. Different concentrations of L. innocua were inoculated to trigger biofilm formation (1.0 × 10⁷ CFU/mL, red; 1.0 × 10⁵ CFU/mL, yellow; 1.0 × 10³ CFU/mL, gray). Error bars: standard deviation; horizontal dashed blue lines, limit of detection (Nluc) or minimal reliable signal (crystal violet, resazurin). FI, fluorescence intensity; n.d., no CFU on agar plates (i.e., <1 log CFU/mL).

FIGURE 3

Correlations of L. innocua concentrations in biofilms determined with Nluc bioluminescence assay and the CFU counting assay (A), the crystal violet (CV) staining assay (B), and the resazurin fluorescence intensity (FI) (C). The Pearson’s coefficients (r) are also shown.