Preparation of Agar Bead Embedded Mycobacterium abscessus to Inoculate Immunocompetent Mice Intratracheally

We are developing a mouse model of chronic mycobacterium abscesses lung infection to test antibiotic efficacy in preclinical studies. The first challenge is to overcome the intrinsic resistance of mice to mycobacterium abscesses. The second challenge is to generate reproducible batch of agar beat embedded bacteria for intratracheal infection. We want to share a detailed protocol of agar beat preparation and infection that can be easily implemented by other labs to test new drugs in mice.

Infecting with agar beat embedded bacteria instead of free bacteria, partially overcomes the ability of immunocompetent mice to spontaneously clear the infection. This model provides a robust preclinical tool for evaluating new antibiotics against mycobacterium abscessus, facilitating discovery and development of much needed antibiotics against pulmonary infections.

[Instructor] To begin, obtain exponentially growing mycobacterium abscessus suspension cultures adjusted to an optical density of 30. Transfer 27 milliliters of molten triptych soy auger pre-equilibrated at 50 degrees Celsius into a tube containing three milliliters of bacterial suspension. Mix the suspension by pipetting. Carefully pipette the bacteria auger mixture into a flask containing 60 milliliters of mineral oil in a flask. Place the flask in a secondary container and immediately begin stirring at medium speed on a magnetic stir using a magnetic stirring bar. Ensure that a visible vortex forms in the oil auger mixture and continue stirring for six minutes at room temperature. Place ice into the secondary container to allow the suspension to cool. Then transfer the slurry mixture into 250 milliliter centrifuge tubes. Centrifuge the suspension at 3,700 G for six minutes at four degrees Celsius for the first five cycles, and resuspend the auger bead pellet with DPBS. Also, assemble a funnel cell strainer, connector ring, and a 50 milliliter tube into a single unit. After the 10th wash, resuspend the pellet and dilute the bead suspension four times. Add the diluted sample material onto the strainer to filter the bead suspension. Slowly, pull the piston of a 30 milliliter syringe attached to the connector ring to create suction and enhance filtration. Then centrifuge the diluted suspension at 1000 G for two minutes to concentrate it. After pulling the bead pellets into a 50 milliliter tube, store them at four degrees Celsius for up to one week, but prepare fresh beads for each batch of experiments. To begin, restrain sedated CD one mice using a custom designed three-dimensional, printed-tilted stand. Secure the mouse by its incisors using a suture string attached to the central rod of the stand. Positioning the mouse in a ventral recumbent posture with its head raised. Attach a metal 24 gauge animal feeding needle to a glass syringe filled with mycobacterium abscessus auger bead suspension. Place nylon clips or stoppers on the plunger to ensure a controlled inoculum volume of 50 microliters per mouse. Now, position the metal gavage needle vertically above the mouse. Carefully insert the needle at the top and slide it further down into the trachea. Remove the nylon clip or stopper and slowly dispense 50 microliters of mycobacterium abscessus auger bead suspension into the lungs. Let the mouse rest for five minutes. Then repeat the inoculation procedure a second time to ensure deep and reproducible deposition of the full inoculum into the lungs. Auger beads containing mycobacterium abscessus expressed mCherry red fluorescent protein and varied in size from approximately 70 to 250 micrometers. The bacterial burden across six independent bead preparations showed high-reproducibility. Bacterial enumeration before and after passing through the glass syringe showed no significant loss of bacterial burden. The inter-tracheal inoculation method was reproducible across three independent bead preparations as shown by lung CFU counts at 24 hours post-infection. Infection reproducibility between three independent operators was confirmed as lung CFU counts at 24 hours post-infection remained consistent across all groups.