A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This method describes a new mouse model for studying the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. It more closely mimics what happens in humans. This model can be an important tool for identifying potential therapeutic targets against the secondary pneumococcal pneumonia in susceptible hosts.

This model reproduces the susceptibility of aging. Therefore, it can be used to understand what aspects of the host become defective with age and allow us to tailor treatment options for vulnerable aged hosts. Growing pneumococcal biofilm is challenging since the different strains take different amounts of time.

My advice is to try growing the biofilm prior to executing the animal study. Once the H292 cells are 100%confluent, wash the cells thrice with PBS. Then add 250 microliters of 4%paraformaldehyde per well to fix the cells and incubate for one hour on ice or overnight at four degrees Celsius.

Strake streptococcus pneumoniae strain of interest on blood agar plate and incubate overnight at 37 degrees Celsius and 5%carbon dioxide. The next day inoculate the bacteria from the plate into fresh, chemically-defined media or CDM plus Oxyrase by washing the bacteria off the plate by adding one milliliter of the CDM plus Oxyrase and gently lifting the bacterial colonies using the side of a one milliliter pipette tip, being careful not to scrape the agar. Dilute the bacterial culture in CDM plus Oxyrase to a starting OD 600 of 0.05.

Then grow the bacteria in a loosely capped 50 milliliter conical tube at 37 degrees Celsius and 5%carbon dioxide until OD of 0.2 is reached. Next vortex the bacterial culture tube. Seed 0.5 milliliters of the culture on the fixed H292 cells and add another 0.5 milliliters of CDM plus Oxyrase medium per well.

Add CDM plus Oxyrase to the control wells with no bacteria. Incubate the plate for 48 hours at 34 degrees Celsius and 5%carbon dioxide. Every 12 hours, following the initial seeding, gently replace the 0.5 milliliters of the medium with fresh CDM plus Oxyrase.

Be careful not to disrupt the forming of biofilm. Check the bottom of the plate for biofilm and look for increasing cloudiness as time goes on due to biofilm growth. After 48 hours, remove the supernatant and wash the biofilm twice with one milliliter of PBS.

Then resuspend the biofilm in one milliliter of fresh CDM and pipette up and down vigorously to lift the biofilm. For each bacterial strain, pool the culture from all the wells into a 50 milliliter tube. Mix well by gently tilting the tightly capped tube up and down several times.

Next, add 40%glycerol and CDM at equal volumes to achieve a bacterial suspension with a final concentration of 20%glycerol. Aliquot one milliliter into a micro centrifuge tube, flash freeze on dry ice, and save at minus 80 degrees Celsius. Thaw the biofilm grown aliquots on ice and spin at 1, 700 G for five minutes.

Carefully remove and discard the supernatant without disrupting the pellet. Then wash the pellet by resuspending it in one milliliter of PBS and spin it again. Remove the supernatant and resuspend the pellet in the volume needed to reach the desired concentration.

Inoculate the C57 black six male mouse intranasally with five times 10 to the sixth colony forming units or CFU by pipetting five microliters of the diluted inoculum into each naris. After inoculation, hold the mouse firmly, stabilizing the head until the volume is inhaled. Once the virus has thawed, dilute the virus in PBS to the desired concentration.

Place ophthalmic lubricant on the eyes of the mouse prior to anesthesia. Immediately infect the anesthetized mouse with 50 microliters of 20 plaque-forming units, or PFU, of influenza A virus or IAV intratracheally by using blunt tweezers to pull the tongue out of the mouth and pipetting the volume of liquid down the trachea. Following recovery, intranasally inoculate 10 microliters of 200 PFU of IAV using the inoculation method described earlier.

For lung collection, using dissection scissors, cut the sides of the exposed rib cage and gently pull the ribs up toward the head of the mouse to expose the heart. Insert a 25 gauge needle attached to a 10 milliliter syringe prefilled with PBS into the right ventricle and begin slowly perfusing. Look for bleaching of the lungs as an indicator of successful perfusion.

Flush slowly to avoid breaking the pulmonary tissue. Lift the heart with forceps and make a cut to separate the lungs and heart. Once separated, pick up all lobes of the lung with forceps and rinse in a dish with sterile PBS to remove any residual blood.

In a Petri dish, mince the lung into small pieces and mix well. Remove half of the lung mix to determine the bacterial CFU or viral PFU and place it in a round bottom 15 milliliter tube prefilled with 0.5 milliliters of PBS for homogenization. Remove the other half of the lung for flow cytometry and place it in a non-tissue culture treated 24-well plate with each well prefilled with 0.5 milliliters of RP10.

Leave at room temperature until processing. To homogenize the collected tissue clean the homogenizer probe by putting it in 70%ethanol and turning on the homogenizer at 60%power for 30 seconds. Repeat this step in sterile water for 10 seconds.

Homogenize each tissue for one minute. For enumeration of bacterial numbers, plate serial dilutions on blood agar plates. To calculate the total CFU use 10 microliters to plate and note the final volume in milliliters for each sample.

Plate the nasopharynx samples on blood agar plates supplemented with three micrograms per milliliter gentamycin to select the growth of S pneumoniae while inhibiting the growth of other microorganisms. Incubate overnight at 37 degrees Celsius and 5%carbon dioxide. For flow cytometry, add 500 microliters of digestion buffer to each well of a 24-well plate containing a lung sample.

Incubate the plate for 45 minutes to one hour. Next, using a P-1000 micro pipette, move the digested lungs and place them on the filter. Then use the plunger of a three milliliter syringe to mash the sample.

Biofilm growth S.pneumoniae inoculum results in consistent pneumococcal carriage restricted to the nasopharynx while avoiding systematic spread. The disease presentation in S pneumoniae IAV coinfected mice was dependent on the bacterial strain. While there was no significant difference in bacterial numbers of the nasopharynx among any of the strains, S pneumoniae, TIGR4, and D39, but not EF3030 disseminated to the lungs by 48 hours post IAV infection.

40%of the mice infected with S pneumoniae TIGR4 displayed bacterial dissemination to the lungs and of those, half of them became bacteremic. Mice infected with S pneumoniae D39 showed 100%dissemination to the lungs and half of that experienced bacteremia. In tracking the overall survival, regardless of the bacterial strain, the survival rate of coinfected mice was significantly lower than the mice singly challenged with S pneumoniae.

This model was also used to assess the presence of various immune cells in the lungs following IAV infection. The bacterial strains D39 and TIGR4 elicited a significant increase above baseline in the influx of inflammatory immune cells from the circulation, such as neutrophils and monocytes, while EF3030 did not. IAV infection alone elicited a significant increase above baseline in the influx of immune cells such as NK cells and gamma delta T-cells.

These antiviral responses were significantly blunted in mice intranasally infected with S pneumoniae prior to viral challenge. In singly-infected mice, the viral titers did not vary between the young and aged cohorts. Old mice displayed earlier and significantly more severe signs of disease and died faster, within 24 hours, whereas the young controls survived the infection better.

Separating carriage and disease into distinct steps allows us to examine both the immune response and the bacterial and or viral factors important at different phases of disease progression. We hope that this model will be adapted by other labs to study other polymicrobial interactions and host pathogen interactions during different phases of disease progression and across the various hosts.