Measurement of the In Vivo Burden of Bacteria Expressing Luciferase in an Insect Larva

NOTE: All work described below is to be carried out in a CL2 laboratory within a class 2 microbiological safety cabinet (MSC) following local health and safety guidelines.

1. Preparation of M. bovis BCG lux for Infection

1. Defrost a frozen 1.2 mL glycerol (15%) stock of Mycobacterium bovis Bacillus Calmette-Guérin (BCG) lux, the Montreal vaccine strain transformed with the shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi encoding the luciferase enzyme.
2. Inoculate 15 mL of Middlebrook 7H9 broth containing 0.2% glycerol, 10% albumin, dextrose, catalase (ADC) enrichment, and 50 µg/mL hygromycin with a defrosted 1.2 mL aliquot of BCG lux, in a labeled 250 mL Erlenmeyer flask.
3. Place the flask in a sealed biosafety container and incubate at 37 °C in an orbital shaker incubator at 220 rpm for 72 h (or until the culture reaches the mid-log phase of growth).
4. Check the growth of BCG lux culture by preparing 1:10 dilutions of the culture in luminometer tubes using phosphate-buffered saline (PBS, pH 7.4, 0.01 M phosphate buffer, 0.0027 M potassium chloride, and 0.137 M sodium chloride) in duplicate. Vortex, and load the luminometer tubes into the luminometer and measure the bioluminescence (relative light unit [RLU]/mL) using n-decyl aldehyde as the substrate (1% v/v in absolute ethanol).         
   NOTE: The ratio of RLU/colony forming units (CFU) was previously determined to be 3:1 when BCG lux was grown in vitro in Middlebrook 7H9 broth.
5. Centrifuge the culture at 2,175 x g for 10 min at room temperature to pellet the cells and discard the supernatant into an appropriate disinfectant with known mycobactericidal activity. Dispose of all culture waste with disinfectants appropriate for mycobacteria following local guidelines.
6. Wash the cell pellet twice in PBS containing 0.05% polysorbate 80 (PBS-T) to prevent bacterial clumping.
7. Following the final wash, decant waste supernatant, resuspend the mycobacterial cell pellet in PBS-T, and dilute the mycobacterial suspension to the desired cell density using RLU measurements.
8. Prepare ten-fold serial dilutions of the inoculum in 24-well plates using PBS-T. Plate out 10 µL onto Middlebrook 7H11 agar plates (0.5% glycerol, 50 µg/mL hygromycin, 10% oleic acid, albumin, dextrose, catalase [OADC]) in duplicate to enumerate inoculum CFU counts.

2. Preparation of G. mellonella Larvae

1. Purchase last instar larvae from appropriate sources and maintain the larvae in the dark at 18 °C upon arrival and use within 1 week of purchase. Alternatively, larvae can be self-reared following the protocol of Jojāo et al. For purchased larvae, discard any dead, discolored, or pupating larvae prior to storage.     
   NOTE: Pupating larvae are morphologically distinguishable from the last instar larvae.
2. Identify and select healthy larvae for experimentation, based on uniform cream color with little to no discoloration (melanization), size (2−3 cm in length), weight (approximately 250 mg), displaying a high level of motility, and possessing the ability to right themselves when turned over.
3. Carefully count the healthy larvae (minimum of 20−30 per group) into a Petri dish (94/15 mm) lined with a layer of filter paper (94/15 mm) using blunt-end tweezers to minimize contamination and store at room temperature in the dark until use.

3. Infecting G. mellonella with BCG lux

1. Minimize the clutter within the MSC to reduce the risk of contamination and needle stick injury.
2. Prepare the injection platform by taping a circular filter paper (94 mm) to a flat raised surface. Soft or hard surfaces can be used and are entirely dependent on user preference, e.g., pipette box lids or a nylon scouring sponge.
3. Sterilize a 25 µL microsyringe (25 G) by aspirating 3 volumes of 70% ethanol and further rinse with 3 volumes of sterile PBS-T.
4. Aspirate 10 µL of BCG lux inoculum (prepared in section 1) or PBS-T into the sterilized 25 µL microsyringe. Use a separate syringe for PBS-T negative control.
   NOTE: Resuspend the BCG lux inoculum following 10 injections to ensure uniform cell suspension.
5. Use tweezers to pick up one larva and place it onto the injection platform.
6. On the platform, flip the larva onto its back and immobilize it by securing the head and tail with tweezers. Locate the last left proleg counting down from the head of the larva and carefully insert the tip of the needle (5−6 mm) at a 10−20° angle to the horizontal plane.     
   NOTE: Short and narrow tweezers allow for easy immobilization with minimum larval stress. Pay attention not to over penetrate which may puncture the gut and cause non-BCG lux specific melanization or death.
7. Count the infected larvae into a Petri dish lined with a layer of filter paper, a single 90 mm Petri dish can accommodate up to 30 larvae.
8. Store the Petri dish containing the larvae in a vented or non-sealed dark box inside an incubator at 37 °C with 5% CO₂.

4. Monitoring the Survival of G. mellonella Following Infection

1. Over the time course, monitor the survival of the larvae every 24 hours. Larvae are considered dead when they fail to move in response to touch.

5. Measuring the In Vivo Burden of BCG lux in G. mellonella

1. At each time point, randomly select five infected larvae previously prepared in section 3 and gently sterilize the larval surfaces using cotton bud swabs soaked in 70% ethanol.         
   NOTE: This step is important when plating larval homogenate for mycobacterial CFU enumeration as non-sterilized larvae can lead to contamination.
2. Place the larvae individually into 2 mL lysing matrix tubes containing 800 µL of sterile PBS.
3. Homogenize the larvae using a homogenizer for 60 s at 6.0 m/s.
4. Centrifuge the lysing tubes at 3,500 x g for 5 s to remove homogenate from the lids, and carefully decant the homogenate into sterile luminometer tubes individually.
   NOTE: For CFU enumeration (step 5.7), ensure to reserve 100 µL of homogenate in a sterile 1.5 mL reaction tube.
5. Recover any remaining homogenate by washing the lysing matrix tubes with 1 mL of PBS-T and pipette into the corresponding luminometer tubes.
6. Vortex the luminometer tubes and measure the bioluminescence of the homogenates previously described in step 1.4.
7. Prepare ten-fold serial dilutions of the homogenate in 24-well culture plates using PBS-T. Plate out 10 µL of the dilution onto Middlebrook 7H11 agar plates containing 0.5% glycerol, 50 µg/mL hygromycin, 10% OADC, and 20 µg/mL piperacillin, to determine the RLU/CFU ratio of BCG lux following in vivo infection.
   NOTE: Piperacillin eliminates native G. mellonella microbiota with minimal growth inhibition of BCG lux.