Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

This protocol is significant for creating and determining mind-body desired mutations in the genome of Pseudomonas aeruginosa, as well as the testing the effect of mutations on virulence reduction in a reproducible mouse model. The key advantage of this technique is a chrome validation and a reproducibility of mouse infection model. Bacteria are constantly working, continually changing.

To slow down this process, we use a frozen stops, bag them, hang them on multiple rungs on modification before testing them in the mouse model. Someone unfamiliar with these methods will most likely struggle with the development and selection of possible crossover recombinant to test. Furthermore, the handling of the mice for infection will be difficult for someone unfamiliar with animal work.

This method can be applied to testing other pathogens and their mutants, as well as virulence infect in mice. Compared to current models, procedures with reproducibility plus clone validation before and after infection can benefit other investigators in the field. Visual demonstration of this protocol provides insight to extensive procedures that may be difficult to understand or comprehend through reading alone.

Start by growing single crossover, recombinant colonies in Pseudomonas Isolation Broth, or PIB. Inoculate and streak 10 microliters of each culture onto pre-warmed PIA plates, supplemented with 10%sucrose. Then incubate the plates overnight at 37 degrees Celsius.

On the next day, remove the plates from the incubator and inspect them for growth. The sucrose resistant colony should be double crossover recombinants. Use sterile toothpicks to patch at least 20 colonies onto pre-warmed plates of PIA, PIA supplemented with 10%sucrose, and PIA supplemented with carbenicillin.

Incubate the plates overnight and examine them for growth on the next day. True double crossover recombinants will be carbenicillin-sensitive and sucrose-resistant. Screen 10 to 20 colonies for deletion, using colony PCR.

Pick up the growth of a suspected double crossover recombinant with a sterile toothpick and suspend it in 50 microliters of PBS. Boil the suspension at 100 degrees Celsius for 10 minutes. Centrifuge it for three minutes at 13, 000 times G and place it on ice.

Then, perform PCR according to manuscript directions. Once PCR is finished, perform Agarose gel electrophoresis on the products. Smaller amplification products indicate colonies in which the region of interest has been deleted.

On the morning of injections, thaw the cryovials of bacterial cells at four degrees Celsius for three to four hours. Keep the vials on ice after thawing and inject the mice within two hours. Transfer the contents of each cryovial to a new two milliliter tube and centrifuge it at 4, 500 times G for 10 minutes.

Discard the supernatant and re-suspend the cell pellet in one milliliter of PBS. Repeat the centrifugation and re-suspend the cells in PBS to a final concentration of 2.5 times 10 to the ninth colony forming units per milliliter. Take three samples from the final suspension of each strain to validate concentration, genotype, and phenotype.

For each strain, aliquot 1.5 milliliters of the cell suspension into a two milliliter tube and prepare the PBS for control injections. Gather the mice and materials needed for injections in a sterile animal surgical room and wipe all surfaces with sanitizing wipes prior to starting. Wear two pairs of latex gloves to limit the risk of puncture, if bitten, as well as a lab coat, safety glasses, and face mask.

Remove the mouse from the cage and weigh it, marking the tail with permanent marker for post-injection tracking. Open a new one milliliter syringe with a 27 gauge needle and draw up 200 microliters of sterile PBS. Grab the mouse behind its ears, using the thumb and forefinger, and pinch to create a skin fold at the nape of the neck.

Then, secure the tail into the palm using the pinky to hold the mouse flat and immobile. Insert the needle at a 30 degree angle into the peritoneal cavity to the left or right of the midline. Slightly lift the needle to ensure that it was not inserted into organs.

Then, slowly inject the PBS and withdraw the needle. A bolus at the injection site is typical. Place the needle in the designated sharps disposal container and move the mouse to a separate cage.

Repeat the procedure with the next mouse and after all mice from one cage are injected, move them back to their original cage. After injecting the control group, inject the test groups using the same procedure. Once all injections are complete, return the mice to the housing room and clean the work area with sanitizing wipes.

To image the animals, prepare the imaging system by setting the camera parameters and heating the stage. Set the oxygen flow to 1.5 liters per minute and isoflurane to 3.5%and move the mouse to the anesthetic chamber, then to the temperature stabilized stage, following anesthesia. Position the mouse on its back with arms outstretched and fit a nose cone for administration of 2.5%isoflurane during imaging.

Close the door and take bioluminescent images and x-rays of the mouse. When imaging is complete, return the mouse to its cage and monitor it. It should regain consciousness within three to five minutes.

The targeted genomic deletions were confirmed by colony PCR with specific primers that amplified the region of interest. Colonies with the genomic deletion yield a shorter PCR band compared to wild type colonies. Intraperitoneal injection of the attenuated strain of P aeruginosa, PGN five, resulted in 0%mortality, equivalent to mortality observed with E.coli BL 21.

Injection of the parent strain, however, was fatal to 80%of the mice. Infection progression was tracked using bioluminescence-marked parent and attenuated strains. The attenuated strain remained localized at the injection site until bioluminescence faded, which likely coincided with clearance of infection.

The method presented only examined the mortality produced by the strain. More in depth immunological and toxicological aspects could be utilized to determine the dynamics of infection and the end effect the infection has that does not result in death. The most important thing in the procedure is the extensive validation done, between various steps from initial identification to animal testing.

Missing certain validation steps could potentially lead to wrong result, as the strains tested may undergo mutation and selection, or become contaminated. With the development of these two techniques, we think this will allow our researchers in the field of infection immunology to more effectively investigate how powerful package interaction leads to the extreme phenotype, sepsis, and mortality. Different nutrients'impact on variants can be compared through size dosing of bacteria.