Campylobacter jejuni Biofilm Assessment by NanoLuc Luciferase Assay

Materials and reagents

Biological materials

1. C. jejuni 81-176 (GenBank accession number NC_008787.1) transformed with pMW10_nLuc (GenBank accession number OR958835) (Figure 1)

Figure 1. Scheme of the pMW10_nLuc plasmid. KanR, kanamycin resistance gene; porA, promotor; RBS, ribosome-binding site; nLuc, NanoLuc gene; TT, transcription terminator; BamHI and XbaI, restriction sites.

Reagents

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

2. Phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: P3813)

3. Mueller-Hinton broth (MHB) powder (BD Difco, catalog number: 275730)

4. Mueller-Hinton agar (MHA) powder (BD Difco, catalog number: 225250)

5. Karmali Campylobacter selective supplement (Oxoid, catalog number: SR0167E)

6. Ethanol, 99.8%, for HPLC, absolute (Thermo Scientific, catalog number: 445740010)

7. Kanamycin sulfate from Streptomyces kanamyceticus (BioReagent, Sigma-Aldrich, K1377)

Solutions

1. PBS solution (see Recipes)

2. MHB medium/MHA agar (see Recipes)

3. MHA Karmali agar (see Recipes)

Recipes

1. PBS solution

One pouch of PBS

1 L of purified water

Mix thoroughly. Autoclave at 121 °C for 15 min. Avoid overheating.

2. MHB medium/MHA agar

38 g of MHB powder or 21 g of MHA powder

1 L of purified water

Mix thoroughly. Heat with frequent agitation and boil for 1 min to completely dissolve the MHB or MHA powder. Autoclave at 121 °C for 15 min. Avoid overheating. Pour the MHA agar into sterile Petri dishes.

3. MHA Karmali agar

19 g of MHA powder

500 mL of purified water

One vial of Karmali Campylobacter selective supplement

1 mL of sterile distilled water

1 mL of 99.8% ethanol

Aseptically add 2 mL ethanol/sterile distilled water in a 1:1 ratio to the vial containing the Karmali Campylobacter selective supplement and mix gently to ensure that the contents dissolve completely. Add to 500 mL of the prepared MHA agar cooled to 50 °C (see Recipe 2). Mix well and pour into sterile Petri dishes.

Laboratory supplies

1. White flat-bottomed 96-well plates (Thermo Fisher, Microlite, catalog number: 7417)

2. 50 mL centrifuge tube (Corning, Mini Bioreactor, catalog number: 431720)

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

4. Pre-sterile 50 mL disposable reservoirs (Biotix, catalog number: EP0030123611)

5. Microplate sealing tape (Corning, catalog number: 3345)

6. Pipette tips 0.1–10 μL (Eppendorf, catalog number: 0030000811), 2–200 μL (Eppendorf, catalog number: 0030000889), 50–1,000 μL (Eppendorf, catalog number: 0030000927)

7. Inoculation loops (Golias, catalog number: EZ08)

Equipment

1. Incubator (Thermo Scientific)

2. Orbital shaker (Thermo Scientific)

3. Luminescence plate reader (BMG LabTech, FLUOstar Omega)

4. Pipette set (Eppendorf, catalog number: 05-403-151)

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

6. Biosafety cabinet (Nuaire)

7. Deep freezer (Haier, model: TwinCool)

8. Spectrophotometer (Bio-Rad, model: SmartSpec 3000, catalog number: 4006168)

9. Centrifuge (Thermo Scientific, model: LEGEND X1R, catalog number: 75004261)

Procedure

A. Reviving glycerol stock of C. jejuni

1. Remove a vial containing a stock culture of C. jejuni, previously transformed with the pMW10_nLuc plasmid via triparental mating [22], from -80 °C and place it on ice. For handling C. jejuni, see General note 1.

2. Using a sterile loop, aseptically transfer half a loop of culture to Karmali-selective plates and incubate the plates at 42 °C under microaerophilic conditions (85% N₂, 10% CO₂, and 5% O₂) for 24 h. Microaerophilic conditions are ensured by blowing the gas mixture into an airtight box, which is then placed in the incubator.

3. Using a cotton swab, respread the colonies grown on Karmali agar to MHA agar containing 30 μg/mL of kanamycin. Incubate the plate at 42 °C under microaerophilic conditions for 24 h.

B. Overnight culture for calibration curve preparation

1. Prepare 15 mL of MHB medium with 30 μg/mL of kanamycin in a 50 mL centrifuge tube.

2. Using a sterile inoculation loop, inoculate a few colonies from the MHA agar plate into the medium prepared above. Vortex the tubes.

3. Incubate at 42 °C under microaerophilic conditions (85% N₂, 10% CO₂, and 5% O₂) with gentle shaking (80 rpm) for 16–20 h.

C. Establishing biofilms in microtiter plates

1. Using a sterile loop, take a few colonies from the MHA agar plate and resuspend them in MHB medium until they reach OD₆₀₀ = 0.1 (approximately 10⁷ CFU/mL, see General note 2).

2. Transfer 100 μL of this suspension into 9.9 mL of MHB medium to achieve a final concentration of approximately 10 CFU/mL. This will serve as the initial culture.

3. Pipette 200 μL of the initial culture to each well, using three separately prepared samples (biological replicates) with each biological replicate in three wells (technical replicates). This results in nine wells per condition. Cover the plates with sterile microplate sealing tape.

4. Incubate the plates under microaerophilic conditions (85% N₂, 10% CO₂, and 5% O₂) for different time periods (e.g., 4, 8, 24, 48, or 72 h).

5. After incubation, carefully rinse the biofilms in the microtiter plates three times with 100 μL of PBS. After the last wash, add 50 μL of PBS to each well. Proceed with bioluminescence measurements as described in section D. Thaw the Nano-Glo luciferase buffer (from Nano-Glo Luciferase Assay System) and gently mix the Nano-Glo luciferase substrate by pipetting. Allow both the reagents and the cultures in the microtiter plates to equilibrate to room temperature before proceeding with the experiment.

D. Calibration curve preparation

1. Adjust the overnight culture (prepared in section B) using sterile MHB medium to obtain an OD₆₀₀ corresponding to 1.50 × 10⁸ CFU/mL (see General note 2). This will be the bacterial output suspension for the calibration curve.

2. Prepare five distinct dilutions (0.8, 0.6, 0.4, 0.2, 0.1) in 1.5 mL tubes to create a calibration curve using the prepared bacterial output suspension (1.50 × 10⁸ CFU/mL) as the starting concentration (1.0 or 100%). Prepare each dilution by adding the appropriate volumes of bacterial suspension and sterile growth medium, e.g., tube 0.8: mix 800 μL of bacterial suspension with 200 μL of sterile growth medium, etc.

3. Transfer 50 μL of dilutions to the unused wells of the microtiter plate with biofilms in duplicate.

4. Use sterile MHB medium as a blank, which is placed in three wells. The mean value is subtracted from each measurement.

E. Bioluminescence measurement

1. Thaw the Nano-Glo luciferase buffer and gently mix the Nano-Glo luciferase substrate by pipetting. Allow both the reagents and the cultures in the microtiter plates to equilibrate to room temperature before proceeding with the experiment.

2. Prepare the appropriate amount of substrate by mixing one volume (e.g., 100 μL) of Nano-Glo luciferase assay substrate with 50 volumes (e.g., 5 mL) of Nano-Glo luciferase assay buffer.

3. Quickly add 50 μL of substrate to each well containing the culture and wait 3 min. Then, measure bioluminescence using the plate reader (e.g., Omega FLUOstar) (settings: luminescence mode; integration time, 1 s; settle time, 150 ms).

F. Calculating the concentration of bacteria in biofilms

1. Use Excel to plot the calibration curve with relative light units on the y-axis and CFU/mL on the x-axis. A representative calibration curve is shown in Figure 2.

Figure 2. Representative calibration curve of the Campylobacter jejuni biofilm assay. RLU, relative light units; CFU/mL, colony-forming units per mL.

2. Use linear regression (Excel functions: slope and intercept) to calculate biofilm cell concentrations from measured bioluminescence (see General note 3).

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 > 0.95. All measurements are repeated in three biological replicates, each of which is repeated in three technical replicates.

Example for the calculation of the biofilm cell concentration based on the measured bioluminescence (RLU): To calculate biofilm cell concentration from RLU, first create a calibration curve using known CFU/mL dilutions (see section F). For example, the curve equation is y = 0.0002x + 36.49 (k = 0.0002; n = 36.49; x = CFU/mL value; y = RLU value). By rearranging the equation, the CFU/mL is calculated from the RLU value of the samples as in Table 1.

Table 1. CFU/mL calculated from the RLU value of the samples

Validation of protocol

This protocol was validated in Čukajne et al. [23], DOI: 10.1007/s00253-024-13383-0.

General notes and troubleshooting

General notes

1. Biosafety level 2 (BSL-2) practices must be followed when handling Campylobacter jejuni, which is classified as a Risk Group 2 pathogen. This includes the use of personal protective equipment such as lab coats and gloves, working in Class II biosafety cabinets to prevent exposure to infectious aerosols, and strictly limiting access to the laboratory to trained personnel. Decontamination of biological waste must be ensured prior to disposal, usually by autoclaving [24].

2. The relationship between OD₆₀₀ and CFU number should be established in advance by plating bacterial suspensions with different OD₆₀₀ values (serial dilutions) and counting the colonies. Measured OD₆₀₀ values depend on the spectrophotometer and other experimental conditions used and are therefore not specified. In our settings, for example, OD₆₀₀ = 1 corresponds to ~1.50 × 10⁸ CFU/mL.

3. In time-course experiments of biofilm growth, each time point (or each plate measured) requires a new calibration curve prepared from the overnight culture. Therefore, an overnight culture should be prepared one day in advance. By including a calibration curve at each time point, we can account for any variations between plates, ensuring accurate measurements despite the high sensitivity of the method.

Acknowledgments

This study was funded by the Slovenian Research Agency (grant numbers J4-3088, J4-4548, J7-4420, P4-0116, and BI-US/22-24-073). This protocol was derived from Čukajne et al. [23], DOI: 10.1007/s00253-024-13383-0. The authors acknowledge Dr. Eva Lasic for editing and reviewing the manuscript.

Competing interests

The authors declare no competing interests.

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