A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

The overall goal of this procedure is to enhance secondary bacterial pneumonia studies by providing a simple and noninvasive route of bacterial inoculation into the lower respiratory tract followed by bacterial recovery and purification of RNA for transcript analysis. This will be accomplished by first deeply anesthetizing a mouse and inserting a preloaded blunt-tipped bent needle into the trachea of a mouse that has been vertically suspended from the maxillary incisors. Second, following successful infection, bacterial colony-forming units are recovered by excision and homogenization of the lungs.

The resulting lung homogenate is serially diluted and plated on nutrient agar to enumerate colony-forming units. Lastly, RNA is purified from the lung homogenate. The advantages of this procedure is that it enables a controlled delivery directly into the lower respiratory tract.

That enables us to look at both the combinatorial and individual contributions of the pathogen towards the outcome of the disease. Recent advances within this field have demonstrated that bacterial gene regulation may have a large role in determining the outcome of infection. This technique benefits from the ability to recover both bacterial CFUs and then RNA.

Once the RNA is isolated, we can then assess the contributions of specific virulence genes towards the infection. In addition, this technique benefits from being nonsurgical. This allows for minimal stress to be placed on any animals used within this procedure and enables them to have an immediate recovery.

This procedure benefits from using common laboratory equipment without the use of any type of cannulas, guide wires or fiber-optic cables. Individuals new to this technique may struggle at first with the actual installation through the trachea. Once this technique is conceptualized, it becomes a simple and effective procedure to interrogate secondary bacterial pneumonia within a murine host.

Prior to beginning the procedure, prepare the workspace with the following supplies:an intubation platform, a sterile one mL syringe, sterile blunt-tipped forceps, sterile 21-gauge blunt-tipped needle, and two conical tubes to store the syringe and forceps. Begin the procedure by preparing the pathogen inoculums so that the desired final concentration is obtained in a 50 microliter volume. Next, using sterile gloves prepare a one to 1.5 inch 21-gauge blunt-tipped needle by bending it to an approximate 35 degree angle.

After fixing the needle to the syringe, draw a 100 microliter air cushion, followed by 50 microliters of the infectious agent. The 100 microliter air cushion ensures full delivery of the pathogen load once the syringe is depressed. Place the loaded needle and syringe in a location easily accessible by the dominant hand.

Remove and anesthetize the mouse from the anesthesia chamber and suspend the mouse on the intubation platform from the maxillary incisors. Using the blunt-tipped forceps, gently grasp and extend the tongue. Transfer the tongue from the forceps into the thumb and index finger of the nondominant hand.

Remain holding the tongue in the nondominant hand and pick up the preloaded syringe. Point the bent end of the needle away from the body. Insert the needle into the oral cavity.

At the base of the tongue gently angle the wrist to cause a slight push of the needle away from the body. This action ensures the needle will be inserted into the trachea and not the esophagus. Slowly guide the needle down into the trachea.

Often a slight tick is felt as the needle passes through the vocal fold. Pass the needle through the trachea until slight resistance is felt as the needle encounters the carina. Slightly lift the needle in the upward direction to suspend the needle above the carina.

This enables delivery into the primary bronchi. Fully depress the syringe plunger to deliver the infectious load. Remove the needle from the trachea and discard.

Remove the mouse from the intubation platform by depressing the rubber band, but maintain holding the mouse in the upright position. Obstruct the nasal airways by placing a finger directly over the nares. Hold this position for approximately one minute or until several deep breaths are observed.

This last step ensures the total inoculum volume is delivered into the lower respiratory tract. Return the animal to its cage and ensure there is a prompt recovery from the anesthesia. At selected time points postinfection the infected lungs can be excised to quantify and assess the bacterial contributions to the increased morbidities associated with secondary bacterial pneumonia.

Prepare a workspace with the following items:a scale and sterile weigh boat, dissection platform, a dissection kit containing scissors and forceps, and sterile gauze. For the purposes of this demonstration the excision of the lungs will be performed on a bench top. However, in order to maintain sterility throughout this procedure it is recommended that the lung excision be performed within a laminar flow hood.

Euthanize an infected mouse with CO2 or similar IACUC approved method of euthanasia. Place and secure the infected mouse on the dissection platform. Spray the mouse with ethanol to maintain sterility on the working surfaces.

Beginning at the umbilicus, use a pair forceps to lift the skin and make an incision towards the larynx. Grasping the skin on either side of the initial incision, pull the skin away from the body and cut through the tissue connections. It can be helpful to make lateral cuts across the skin to reveal more of the thoracic region.

Starting at the base of the xiphoid process, make a small incision with the intention of puncturing the diaphragm. This results in an increase in pressure in the thoracic cavity causing the lungs to retract. With the lungs retracted, continue the incision to free the diaphragm.

Remove the ribs by making an incision on either side. This will expose the thoracic cavity and the lungs. To excise the lungs grasp the base of the heart with the forceps and lift upward.

Place the scissors behind the lungs and begin to make small incisions through the tissue connections while continuing to lift the heart upward. Once the lungs have been excised, place them onto a sterile gauze and remove the heart by lifting it away from the lungs and cutting the remaining tissue connections. Transfer the lungs to the pre-tared weigh boat and record the mass.

After the lungs have been weighed, transfer the lungs into sterile phosphate-buffered saline and temporarily store them on ice. Now that the lungs have been excised and weighed, the bacteria can be recovered to determine their role towards pathogenesis. In preparation for bacterial recovery, the following supplies will be needed:a tissue grinder, sterile RNase-free PBS, buffer RLT supplemented with beta-mercaptoethanol, and 1.5 mL RNase-free microcentrifuge tubes.

Begin by first adding one mL of sterile RNase-free PBS to the tissue grinder. Once filled, store the tissue grinder on ice. Next, prepare a series of sterile RNase-free serial dilution tubes to quantify the bacterial load recovered from the infected lungs.

Transfer the lungs into the prepared tissue grinder and thoroughly homogenize the tissue by pressing down on the pedestal while rotating. Open the tissue grinder and aliquot 100 microliters of the homogenized sample into the first of the prepared serial dilution tubes. Serially dilute the samples and plate on nutrient agar to enumerate the colony-forming units.

CFUs can be recorded as CFUs per mL or CFUs per mL, per milligram of lung tissue. After the 100 microliter aliquot has been removed, replace the grinding pedestal and centrifuge the sample at 4, 000 rpm for 10 minutes at four degrees Celsius. When the centrifugation is complete, remove the supernatant from the pelleted sample.

The supernatant can be saved and stored at minus 80 degrees Celsius for further analysis at a later date. Re-suspend the homogenized tissue pellet in 700 microliters of RLT beta-mercaptoethanol and transfer the sample to a sterile RNase-free 1.5 mL centrifuge tube. At this point the sample can be stored at minus 80 degrees.

The RNA can be purified from the RLT lung homogenate slurry by loading the sample into a two mL microcentrifuge tube containing 1 millimeter silica beads and processed via a bead beater for 20 seconds at six meters per second. RNA is then immediately purified using an adaptation of the RNeasy method as reported in Voyich, et al. Methods in Molecular Biology"2008, and described in the manuscript accompanying this video.

Following purification, the RNA yield can be quantified using a spectrophotometer. Once quantified, it is beneficial to dilute the resulting RNA into several aliquots of 50 nanograms per microliter. This enables the RNA from the sample to be used for multiple analyses without being subject to multiple freeze-thaw cycles.

The use of this technique enables the installation of a controlled pathogen load directly into the lower respiratory tract. The efficiency of this delivery system can be demonstrated through the installation of a 1%Coomassie brilliant blue solution. The top row displays a pair of uninfected lungs to serve as a control, while the bottom row displays a pair of lungs that have received 50 microliters of the 1%Coomassie brilliant blue dye through an intratracheal installation.

Use of this dye demonstrates how the intratracheal installation provides an even distribution of the pathogen load into and throughout the left and right lungs. Once infected, the pathogen load can be recovered and analyzed at various time points post-intratracheal installation by excision and homogenization of the lung tissue. To demonstrate the efficiency of this recovery technique two groups containing three mice each were infected with a low and high dose of Staphylococcus aureus.

The bacterial input was plated on nutrient agar to enumerate CFUs. One hour following the intratracheal installation, the mice were euthanized, lungs excised and homogenized in a tissue grinder. The homogenized tissue was serially diluted and plated on nutrient agar to enumerate CFUs.

The RNA isolated from the homogenized lung tissue can be examined by reverse transcriptase PCR and used to quantify the relative transcript abundance of target genes. To ensure minimal background DNA contamination it is recommended that a PCR reaction, without the addition of reverse transcriptase, be run alongside any qRT-PCR reactions. In addition to bacterial loads, this technique can be extended to the installation and isolation of viral loads.

Following homogenization of the lung tissue, viral RNA can be isolated and used to measure the transcript abundance of viral genes or construct a standard curve for viral quantification. The use of this system provides a highly efficient and reproducible system to study secondary bacterial pneumonia. Once mastered, this procedure can be used in large-scale experiments.

Working in batches to euthanize mice, the intratracheal installation can occur within 30 seconds per mouse. After this is completed, moving on to the excision of the lungs, this can be completed in approximately two to three minutes per mouse. While the methods discussed here had been within the context of a secondary bacterial infection, they can be extended towards any procedure where a controlled delivery and recovery of an inoculum is necessary.

In addition to the methods described here, this procedure can be supplemented by the addition of a bronchoalveolar lavage prior to the excision of the lungs. While this often results in a decreased recovery of the pathogen load, it benefits from obtaining data, such as cytokine analysis, lactate dehydrogenase activity, and the identification of different cellular populations. To gain a more complete understanding of disease pathogenesis it becomes important to interrogate both host and pathogen contributions to disease.

The purpose of this video was to provide a simple and efficient method for exploring secondary bacterial pneumonia within a murine host.