Listeria innocua Biofilm Assay Using NanoLuc Luciferase

Materials and Reagents

1. White flat-bottomed 96-well plates (Corning, catalog number: 3990)

2. 15 mL centrifuge tube, PP, screw cap (Brand, catalog number: 114818)

3. 1.5 mL microcentrifuge tubes (Eppendorf Safe-Lock, catalog number: EP0030123611)

4. Filtermax filter top (TPP, catalog number: 99505)

5. Presterile 50 mL disposable reservoirs (Biotix, catalog number: SR-0050-1SC)

6. Listeria innocua ŽM39 transformed with pMSP::Nluc (Jožef Stefan Institute collection) (Figure 1)

   
   

   
   Figure 1. Scheme of pMSP::Nluc plasmid. PnisA: nisin promoter. ErmR: erythromycin resistance marker. RepD-G: replication genes. NisRK: nisin response genes. ColE1: E. coli replication gene. Nluc: Nanoluc.




7. Tryptic soy broth (Merck, catalog number: 1.05459.0500)

8. Agar (Formedium, catalog number: AGA03)

9. Erythromycin (Merck, Sigma-Aldrich, catalog number: E5380)

10. Nisin (Merck, Sigma-Aldrich, catalog number: 72535)

11. Nano-Glo Luciferase Assay System (Promega, catalog number: N1120)

12. NaCl

13. KCl

14. Na₂HPO₄·2H₂O

15. KH₂PO₄

16. Phosphate-buffered saline (PBS) (see Recipes)

17. TSA (see Recipes)

Equipment

1. Incubator (Binder)

2. Shaker/Incubator (Braun Certomat H)

3. Luminescence plate reader (Tecan, model: Infinite M-1000)

4. Pipette set (Eppendorf, catalog number: Z683892)

5. Multichannel pipette (Eppendorf, catalog number: 2231300048; model: Research Plus)

6. Biosafety cabinet (Labcaire)

7. Deep freezer (Sanyo, model: MDF-U53V)

8. Spectrophotometer (Perkin Elmer, catalog number: L7110187; model: Lambda Bio+)

9. Centrifuge (Hettich, catalog number: 1706; model: Rotina 380R)

Procedure

1. Reviving glycerol stock of L. innocua

   1. Remove vial of permanent culture of Listeria innocua ŽM39 transformed with pMSP::Nluc from deep freezer and place it on ice.

   2. Aseptically transfer culture to TSA plates containing 10 µg/mL erythromycin with sterile loop.

   3. Incubate at 37°C for 24 h.

      
      

2. Overnight culture for biofilm inoculum or calibration curve culture preparation

   1. Prepare 5 mL of TSB medium with 10 μg/mL erythromycin in a 15 mL tube.

   2. Transfer biomass equivalent to three medium sized colonies from the TSA agar plate and inoculate it into the medium prepared above.

   3. Incubate with shaking (190 RPM) at 37°C for 18-24 h.

      
      

3. Setting up biofilm in microtiter plate

   1. Centrifuge overnight culture at 3,000 × g and 4°C for 10 min.

   2. Resuspend the pellet in 5 mL of TSB medium with 10 μg/mL erythromycin and 5 μL of nisin for Nluc induction (stock solution concentration: 1 mg/mL).

   3. Dilute the suspension until its OD₆₀₀ value corresponds to 1 × 10⁷ CFU/mL (see Note 1).

   4. Dilute bacterial suspension from Step C3 as required [e.g., 100-fold in TSB with 10 μg/mL erythromycin and 5 μL of nisin (stock solution concentration: 1 mg/mL) to obtain dilution corresponding to 1 × 10⁵ CFU/mL].

   5. Pipette 50 μL of the bacterial suspensions per well onto the microtiter plate in triplicate.

   6. Incubate at 30°C (see Note 2) for a selected time period (e.g., 4 h, 8 h, 24 h, 48 h, or 72 h).

   7. Wash biofilm three time using 80 µL of PBS. After last wash, add 50 µL of PBS to each well. Proceed with bioluminescence measurement.

      
      

4. Preparation of calibration curve (see Note 3)

   1. Before the measurement (8 h), prepare 5 mL of TSB medium with 10 μg/mL erythromycin and 5 μL of nisin (stock solution concentration: 1 mg/mL) in a 15 mL tube.

   2. Transfer 100 µL of the overnight culture (prepared in section B) to the medium and incubate with shaking (190 RPM) at 37°C for 8 h.

   3. Immediately before the measurement, calibrate the OD₆₀₀ of the suspension to correspond to 5 × 10⁷ CFU/mL. This is the bacterial output suspension for the calibration curve.

   4. In 1.5 mL tubes, prepare five consecutive five-fold dilutions that will serve as a calibration curve.

   5. Transfer 50 μL of dilutions to the biofilm plate in duplicate.

      
      

5. Bioluminescence measurement

   1. Prepare the appropriate amount of substrate by mixing one volume of Nano-Glo Luciferase Assay Substrate with 50 volumes of Nano-Glo Luciferase Assay Buffer (e.g., 200 μL of Substrate in 10 mL of Buffer). Prior to use, incubate the substrate at 37°C to equal the temperature of the samples.

   2. Add 50 µL of substrate per well quickly with a multichannel pipette. Wait 3 min and then measure the bioluminescence for the first time using plate reader (e.g., Infinite M-1000, settings: Luminescence mode; Integration time, 1 s; Settle time, 150 ms; Figure 2).

   3. Measure bioluminescence at 10 min intervals three more times (see Note 4).

      
      

   
   Figure 2. Screenshot of microplate reader settings for measuring bioluminescence of Nluc.

   
   

6. Calculating concentration of bacteria in biofilms

   1. Use Excel to calculate logarithms of bacterial concentrations and logarithms of corresponding average bioluminescence and use them to plot the calibration curve. A representative calibration curve is shown in Figure 3.

   2. Use linear regression (Excel functions: Slope and Intercept) to calculate biofilm cell concentrations from measured bioluminescence.

      
      

   
   Figure 3. Representative calibration curve of Listeria innocua biofilm assay. LogC: logarithm of bacterial concentration. LogLI: logarithm of luminescence intensity.

Data analysis

Bioluminescence measurements are saved in Excel format, and Excel can be used to analyze the data. The coefficient of determination (R²) of the calibration curve is determined, and data are considered for analysis only if R² is greater than 0.95. All measurements are repeated in two biological replicates, each of which is repeated in three technical replicates.

Notes

1. Relationship between OD₆₀₀ and CFU number should be established in advance, by plating bacterial suspensions with different OD₆₀₀ (serial dilutions) and counting the numbers of colonies (CFU). Measured OD₆₀₀ values depend on the spectrophotometer used and are therefore not specified.

2. Listeria is cultured at 37°C for optimal cell growth. However, culturing at 30°C was suggested for the studies of biofilm formation (Lemon et al., 2007), due to higher motility of the cells.

   In time-course experiments of biofilm growth, each time point (or each plate measured) requires a new calibration curve, prepared from the 8 h culture that is inoculated from the overnight culture. Measurement after the first 10 min is usually selected for calculation; however, strength of the signal and reproducibility should be taken into account, and measurements after 20 min and 30 min can also be considered.

3. Careful pipetting is essential for reproducible results.

Recipes

1. PBS

   PBS is prepared by 10× dilution and sterile filtration of 10× PBS.

   10× PBS:

   80 g NaCl

   2 g KCl

   17.8 g Na₂HPO₄·2H₂O

   2.7 g KH₂PO₄

   Fill to 1 L with dH₂O.

2. TSA

   30 g TSB

   15 g agar

   Fill to 1 L with dH₂O and autoclave 15 min at 121°C. Pour the agar into Petri dishes in sterile conditions and leave the agar to solidify.

Acknowledgments

This study was funded by the Slovenian Research Agency (Grant Numbers P4-0127, J4-9327, J4-1771). This protocol was derived from Berlec et al. (2021).

Competing interests

The authors declare no competing interests.

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