Avaliação da invasão bacteriana de células cardíacas na cultura e colonização cardíaca em camundongos infectados usando listeria monocytogenes

The overall goal of this procedure is to provide efficient methods of assessing microbial infection of cardiac cells using in vivo and in vitro models of heart colonization and cardiac cell invasion. This is accomplished by first growing bacterial cultures and collecting, washing and resus, suspending the bacterial cells in phosphate buffered saline. Next cardiac cell, monolayers grown on glass cover slips are infected with a bacterial suspension, or the bacterial suspension is inoculated into mice using tail vein injections.

Then the cardiac cells are incubated in the presence of Gentamycin to kill extracellular bacteria followed by cell lysis to recover. Bacteria or infected animals are monitored for wellbeing two to three days post infection then sacrificed for isolation of target organs. Finally, cell and organ suspensions are diluted and plated using a spot plating method to determine the numbers of viable bacteria associated with cells or target organs.

Ultimately, bacterial colonies are counted on auger medium to determine the efficiency of cardiac cell invasion for cells grown in tissue culture or the efficiency and extent of cardiac infection in animals. Though this method can be used to investigate the potential for different clinical and environmental isolate lister monocytogenes to cause invasive cardiac disease. It can also be applied to other microbial pathogens capable of targeting cardiac tissue, or the protocol can be modified to investigate other organs or tissues of interest The day before performing the invasion assay prepare 24 well plates of H nine C two cells and inoculate sterile BHI broth with the desired strains of listeria as described in the text protocol.

After Resus suspending bacterial strains to be tested in PBS load at least 20 microliters of the PBS bacteria solution into the wells of the 24 well plate. Gently rock the plate to evenly spread the sample in each well and incubate the plate at 37 degrees Celsius, 95%humidity and 5%carbon dioxide for 45 minutes. After the incubation, return to the biosafety cabinet and aspirate the media from each well using a pipette with suction.

Then add one milliliter of warmed PBS to each well to wash the cover slips. Finally, the PBS is aspirated from the wells using a pipette with suction. Next, add one milliliter of DMEM containing Gentamycin to each well the gentamycin will kill only those bacteria that remain outside of the cells.

Place the plate back into the incubator and allow it to incubate for an additional hour. Then under the hood, use tweezers to remove each cover slip from its well and place it into a previously prepared 14 milliliter tube containing one milliliter of sterile ddh two o. Dip the tweezers into ethanol and flame in between Cover slips to prevent contamination.

After briefly vortexing each tube, remove an aliquot from each and transfer to the wells of a 96 well plate. Then prepare a serial dilution of each sample from one to 10 to one to 1000. Then on prewarm dry lb plates, spot each dilution onto the auger after the spots have dried, place the plates in a 37 degree Celsius incubator overnight the following morning.

Count the number of colonies in each spot and use the series to calculate the number of bacteria per cover slip for each strain assessed to inoculate L monocytogenes into mice. Place one animal into a harness in order to restrain the animal during injection. Use an alcohol pad to clean the injection site and dilate the tail vein.

Then with a one milliliter syringe and a 27.5 gauge needle, gently inject the tail vein of the mouse with 200 microliters of the desired culture. Use a paper towel to stop any bleeding that may occur from the injection before placing the animal into a fresh cage. After sacrificing the animals up to 72 hours later, according to the text protocol under the biosafety cabinet, secure the animal onto a dissection block and use 70%ethanol to saturate it.

Make a Ys shaped incision on the abdomen and thorax that extends from the vaginal opening up to the xiphoid process, progressing to the axilla of each arm. Pull back the skin. Remove the needles from the legs and use them to hold.

Open the dissection and cut gently through the peritoneum, exposing the intestines, stomach, liver, and spleen. Cut the hepatic vein and coronal ligament at the top of the liver and remove additional ligaments beneath the liver that connected to the first section of the small intestine and the musculature of the back. Place the liver in five milliliters of sterile DD H2O in a 50 milliliter tube.

Next, cut away the vasculature and ligaments of the spleen and remove it before placing it into a separate tube containing five milliliters of DDH two o. Now locate the diaphragm and gently cut along the rib cage in order to visualize the thorax cut through the xiphoid process and sternum up to the neck to open the thorax, exposing the heart and lungs. Using forceps gently grab the heart by its apex and lift it to create tension in the aorta and pulmonary vessels.

Then cut the vessels to free the heart and place it into a separate tube of water. After preparing a homogenizer, according to the text protocol, homogenize one liver for at least two minutes or until no visible portions of the organ remain. Use tweezers to remove any large debris from the probe.

Once all organ samples have been homogenized, prepare serial dilutions of one to 10 to one to 10, 000 for the liver and spleen samples, and one to 10 to one to 1000 for the heart spot plate, the dilutions onto prewarm LB auger plates. Undiluted samples can be plated on separate plates to ensure that organs were sufficiently infected after the plates dry, place the plates in the incubator at 37 degrees Celsius. Use perfil to wrap the 96 well plates of diluted samples and place in a minus 80 degrees Celsius freezer the following day.

Count the number of colonies and use the dilutions to calculate the total number of bacteria per organ. Shown here is an example of a tissue culture-based assay comparing the ability of strain 1 0 4 0 3 s to invade heart cells with that of strain zero seven PF 0 7 7 6 more than twice as many zero seven PF 0 7 7 6. Bacterial CFUs were recovered from infected H nine C two cardiac cells following gentamicin treatment compared to cells infected with 1 0 4 0 3 s.

This figure shows the recovery of bacteria from the livers, spleens and hearts of infected mice at three days post infection, infection with atropic strain zero seven PF 0 7 7 6 or strain 1 0 4 0 3 s results in comparable numbers of bacteria recovered from the livers and spleens of infected mice. However, mice infected with zero seven PF 0 7 7 6 are more likely to yield detectable numbers of bacteria from the heart and to exhibit greater bacterial burdens in this organ Following this procedure. Other methods such as fluorescence based microscopy and organ histology can be used to answer additional questions such as the efficacy of bacterial cell to cell spread and tissue monolayers, or the dynamics of immune cell recruitment to sites of infection.