Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

The overall goal of this procedure is to follow in real time a streptococcus pneumonia infection in aurin pneumonia or sepsis model. This is accomplished by preparing a streptococcus pneumonia infection dose after cultivation in the complex medium THY. The second step is the intranasal or intraperitoneal infection of mice with streptococcus pneumonia.

The final step is the quantification of bioluminescence, intensities of the mice, or of a selected region of mice to provide the total photon emissions. Ultimately, the bioluminescence intensities of a group of mice show the changes in the infection over time. In addition, analysis of individual images of bioluminescent mice is performed.

The main advantage of these technique over existing methods like determining the bacterial load in the lung or in different tissues, is that you can follow thecal infection using only one animal. The implications of this technique extend toward basic research because this technique can also be applied to evaluate the productivity of bacterial factors and anti-infective. This method can provide insight into this batch of promo Cox in mice.

It can also be applied to other systems such as determination of the bacterial load in the organs. Generally, researchers will find it easy to learn this method because using the bioimaging system is simple and software is available to prepare the images. I immediately had the idea to apply this method in infection experiments with bios and primo oxide when I was introduced to the bioimaging system.

With your demonstration of this method is crucial because of the required special handling of the animals, and since critical steps such as the inoculation are difficult to explain, Begin by preparing a stock of infectious bioluminescent pneumococci as explained in the text protocol. Then inoculate a tube containing 40 milliliters of FBS supplemented THY medium with the pneumococci, and start a culture at an OD 600 of 0.08. Incubate the pneumococci without agitation at 37 degrees Celsius and 5%carbon dioxide for three to four hours until the culture reaches an OD 600 hundred of 0.35 to 0.40.

Then harvest the pneumococci by centrifugation at 3, 750 times G for six minutes. Then discard the snat and resuspend the bacterial pellet. In approximately one milliliter of PBS at pH 7.4, supplemented with 0.5%FBS adjust the inoculum to the desired concentration in a suspension of 20 microliters of PBS with 10%FBS and 90 units of hyaluronidase.

Do not shake or vortex the pneumococci since this will trigger autolysis. Next, use an optical imaging system to verify the bioluminescence of the pneumococcal oulu Anesthetize a seven to 10 week old female outbred mouse by intraperitoneal injection of ketamine and xylazine. Test for proper depth of anesthesia by pinching the tail.

Once fully anesthetized, hold the mouse between the fingers and thumb with the nose upright. Then using a pipette with a long, narrow tip drop 10 microliters of the bioluminescent pneumococci onto each nostril. The mouse involuntarily inhales the bacteria.

Hold the mouse for one to two minutes upright to observe whether the mouse sneezes or keeps the inoculum.Complete. Inhalation of the infection dose must be ensured so that the mouse inhales the selected infection dose. Therefore, the mouse must breathe slowly and regularly.

Please avoid that the mouse sneezes and parts of the infection doses gets lost. Following successful inoculation. Mark the tail for later identification and place the mouse in a new cage.

Next, to assess virulence in a sepsis infection model, inject anesthetized mouse with 100 microliters of a pneumococcal strain intraperitoneal. After marking the tail, return the mouse to a cage image as controls. Inject another group of mice with PBS imaging.

The dissemination of the pneumococci is done via image analysis software. Air first, set the imaging mode to luminescent and then set the imaging parameters as follows. Set the exposure time to one minute.

A medium degree of binning and an FOV of 22.5 centimeters. Anesthetize the mice in the anesthesia chamber of the imaging equipment with a mixture of iso, fluorine and oxygen. When the mice are breathing slowly place them into the imaging chamber in the supine position with their noses inserted into tubes connected to the anesthesia system.

To activate the CCD camera on top of the chamber and to start bioluminescent optical imaging, press acquire in the imaging software immediately. A photograph of the mice in the closed chamber appears on the screen. After one minute of measurement, an overlay of the bioluminescence data and the photo is shown.

Now stop the anesthesia approximately 20 seconds before the end of exposure. Put the mice back into their cage and let them fully recover from anesthesia before the cage is returned to the rack. Continue to image the infected mice every eight to 10 hours in addition to monitoring their appearance, behavior, and weight.

Finally, use the image analysis software to determine the bioluminescence intensities per time point and group of mice by quantifying the total photon emission bioluminescent optical imaging of mice infected intranasally with D 39 lux or the isogenic mutant D 39 luxe delta MET Q are shown here. Imaging began 24 hours after infection and continued every eight hours until death or until 120 hours after infection in each group. Seven out of 10 mice developed severe signs of illness and died while the other three mice showed no bioluminescence and survived.

Note that the bioluminescence scene in two of the mice is actually spillover from the intense bioluminescence in the neighboring mice. Mice successfully infected with the bioluminescent wild type D 39 lux developed a severe lung infection, 32 hours post-infection, which progressed within eight to 16 hours to sepsis. All the mice that developed pneumonia and sepsis post-infection with D 39 lux had succumbed by 96 hours post-infection.

The loss of function of the methionine binding lipoprotein impaired significantly the virulence of the pneumococci. After 32 hours, only one mouse infected with the mEq mutant showed a weak lung infection compared to infections in all of the susceptible D 39 lux mice. However, after 48 hours, severe lung infections became apparent in the mice infected with the mutant.

As a result, these mice developed sepsis 72 hours post-infection and became more abundant later than 72 hours post-infection, demonstrating that in the acute pneumonia infection model, the deficiency of met Q attenuates the pneumococci. The values of the bioluminescence intensities of grouped mice are shown for the indicated time points. The quantification of the bioluminescent flux showed significant differences between wild type infected and met q infected mice 32 and 40 hours after infection.

Thus, the loss of function of met q Attenuates pneumococci and results in a significant delay of invasive infection, but does not result in a virulent lint bacteria Once mastered. This technique can be used to monitor 10 groups of four mice in one hour if it is performed properly. While attempting this procedure is important to ensure that the lum is complete and inhaled by the mice Following this procedure, other methods like co-infection in mice can also be performed in order to answer additional questions like slight attenuation and bacterial mutants After its development.

This technique paved the way for researchers in the field of infection biology to explore and visualize pneumococcal pneumonia or sepsis in mice. After watching this video, you should have a good understanding in how to infect mice and follow in real time pneumococcal infection using a bioimaging system. Don't forget that working with human pathogenic bacteria like promo cocy can be hazardous and precautions such as working under a class two safety bench should be taken while doing this procedure.