The overall goal of this non-surgical intrabronchial infection procedure is to establish a robust bacterial pneumonia in immunocompetent rodents. This method adds value for the discovery and development of antibacterials by allowing efficacy of potential treatments to be assessed in an immunocompetent model of severe pneumonia. The main advantage of this technique, is that it establishes a robust and reproducible lung infection with many isolates including those that typically require the animals to be neutropenic or isolates that do not produce a viable infection with other methods.
Generally, individuals new to this procedure will struggle. Proper intubation is a blind procedure that requires learning the right feel to intubate the trachea instead of the esophagus. Proper intubation is different for each scientist.
The exact angle of the cannula may not be the same for everyone. Feeling for the ridges of the trachea is the key. Demonstrating this technique, will be Cindy Mininger, a scientist from our laboratory.
To begin, prepare saline bacterial suspensions from overnight broth cultures, as indicated in the text protocol, or from agar plates, as demonstrated here. Harvest overnight growth from agar plates by using a sterile loop to scrape colonies from the surface. Transfer the harvested material into five milliliters of sterile saline until a cloudy, opaque suspension is obtained, and gently vortex the culture until homogeneous.
Serially dilute then and plate an aliquot from each dilution according to the text protocol. After preparing the agar and assembling the necessary tools for the infection process, according to the text protocol, aliquot nine milliliters of agar into a sterile tube and then place the tube into the water bath until the agar equilibrates to approximately 42 degrees Celsius. Add one milliliter of the previously prepared saline bacterial suspension into the tube of agar.
Then, invert the tube several times to mix and return it to the water. Place a clean, disposable mat onto the work surface close to the water bath. To prepare the work surface and intubation device, first sterilize the metal cannula and polyethylene tubing according to the text protocol and then transfer it in its glass storage tube to the work surface.
Then, remove the cap from the storage tube and slide the handle end of the cannula to the open end of the tube. Using sterile forceps, remove one length of polyethylene tubing from the beaker and insert it through the inside of the sterile metal cannula, making sure it moves freely. Place a guide mark at a predetermined location with indelible pen.
Next, fit a sterile disposable 25 gauge needle onto the free end of the polyethylene tubing by sliding the needle several millimeters into the tubing. Then, use a sterile saline to fill a fresh disposable one milliliter syringe. Attach the syringe to the needle fitted onto the polyethylene tubing and flush the entire volume of saline through the tubing.
Discard the used syringe in the sharps container. Then, fill a new sterile disposable one milliliter syringe with the agar-based inoculum from the water bath. Reattach the 25 gauge needle and tubing and flush agar through the tubing by depressing the plunger just until the tubing is completely filled with agar.
After anesthetizing the rat according to the text protocol, place the animal in a supine position on the disposable mat with the head facing right and the tail facing left. Next, insert the free end of the metal cannula into the animal's mouth. Turn the cannula so that the free end is angled upward and gently advance it into the trachea, carefully bypassing the laryngeal structures with a slight twisting motion.
Confirm insertion into the trachea, as opposed to the esophagus, by gently sliding the cannula slightly forward and back several times while using the left forefinger to palpate the tracheal rings. To ensure intubation into the trachea, and not the esophagus, move the cannula back and forth slightly and feel for the ridges. If the cannula slides smoothly down the throat and the ridges cannot be felt, remove the device and try again.
When the cannula reaches the bifurcation where the trachea splits into the left and right bronchi, make a slight twisting motion towards the animal's left side to ensure that the cannula is inserted into the left bronchus. A common error is not placing the inocula deep enough into he lung which can block the airways and inhibit breathing. When the cannula is turned to the proper angle, it should slide easily into the left bronchus and advance smoothly to the appropriate depth.
Advance the metal cannula until the end is halfway to three quarters down the left lung, using the previously placed guide mark to confirm that the appropriate depth has been reached. With the metal cannula in place, advance the polyethylene tubing several millimeters using the previously placed guide mark on the tubing to ensure it is advanced only far enough to exit the end of the metal cannula and not puncture the lung. With both cannulae in place, use the attached syringe to instill 100 microliters of agar suspension deep into the large lobe of the left lung.
Withdraw several millimeters of the tubing and gently remove the intact intubation device. Set it back into the glass storage tube and move the animal into a fresh cage to recover. Prepare the work surface and cannula as just demonstrated for rats, using the appropriately-sized equipment for mice.
Use a sterile disposable 1 milliliter syringe and sterile 25 gauge needle to fill the glass micro-injection syringe with sterile saline. Then, flush the saline through the reassembled device. Next, use a new sterile disposable one milliliter syringe and sterile 25 gauge needle to fill the micro-injection syringe with the agar inoculum.
Then, reassemble the device and flush agar through the tubing just until the tubing is completely filled with agar. After anesthetizing the mouse according to the text protocol, position the animal in the same orientation as demonstrated for the rat, and use steady but gentle pressure to intubate the trachea in a similar manner as with the rat. Avoid using force as the technique is more delicate in mice.
Ensure the cannula is inserted into the trachea by feeling for the tracheal rings. Slightly turn and advance the cannula into the left bronchus, inserting deep into the lung using previously placed guide marks. With the metal cannula in place, advance the polyethylene tubing several millimeters and instill 20 microliters of agar suspension.
Shown here is a representative example of colonies growing on an agar plate after serial dilution and triplicate plating of a lung homogenate sample. As demonstrated in this graph with S.pneumoniae, an increase in bacterial burden of several log base ten CFU above baseline controls is typically observed in infected lungs for most bacterial isolates, even at 96 hours post-infection, and variability between animals is low. In this experiment, two compounds were evaluated against quinolone susceptible or quinolone resistant isolates of S.pneumoniae to illustrate that this lung infection model can be used during optimization of a lead chemical series to support structure activity relationships.
Each symbol represents CFU determined from the lungs of one rat. Shown here are E max models created using dose-ranging efficacy data in immunocompetent mice infected in the lung with isolates of S.pneumoniae or H.influenzae to determine pharmacokinetic, pharmacodynamic targets for a novel polypeptide deformylase inhibitor. Circles represents the mean bacterial burden from four to five mice per group treated with varying doses of compound.
The open circles represent free area under the curve and filled circles represent the area under the concentration time curve over the minimum inhibitory concentration. Once mastered, this technique can be completed in about 30 seconds per animal, with up to five or six animals inoculated per syringeful of agar inoculum. After watching this video, you should have a good understanding of how to perform this non-surgical intrabronchial infection method and be able to utilize it for establishing lung infections with many different bacterial isolates.
energetic instrumetnal
