Our research focuses on staphylococcus aureus infections with the aim of establishing an intercellular infection model. This model will provide valuable insights into the mechanism underlying intercellular infections and contribute to the development of strict age for their prevention and treatment. The latest progress in staphylococcus aureus intracellular infection is to study the infection mechanism to regulate the survival and the spread of bacteria within the cell, and will add to optimize the drug and the structural modification to improve more therapeutic effect.
These research field uses cell culture, cell emerging sequencing technology, high fuel oxy therms, gene editing technology, and other related technologies to study bacteria infection in cells. In contrast to traditional infection models, we have refined the experimental conditions specific to intracellular infections. After infecting cells with staphylococcus aureus individually, the bacteria infected cells are then inoculated into mice to establish intracellular infections.
This approach improves efficiency by minimizing the interference of the free bacteria, ensuring a more accurate retention of the intracellular infection process. Our method will focus on developing antibody drugs and advance to prevent and treat bacterial infections in the future. To prepare a 6%starch broth, add beef extract powder, tryptone, and sodium chloride to a glass container successively in the mass ratio of 3 to 10 to 5.
Add 100 milliliters of double distilled water and stir to dissolve the components. Heat the solution in the microwave. Then, add soluble starch at a mass ratio of 6 and stir the mixture until fully dissolved.
Autoclave the broth at 121 degrees Celsius for 30 minutes. Once sterilized, store the broth at four degrees Celsius until it forms a paste. To collect mouse peritoneal macrophages, using the left hand, grab the neck of the mouse and control its tail.
Then, flip the mouse so that its head is positioned downward and its abdomen is facing upward. Administer a three milliliter intraperitoneal injection of 6%starch broth into the mouse. Following 72 hours of injection, sacrifice the anesthetized mouse and disinfect it with 75%ethyl alcohol.
Using ophthalmic scissors, cut open the abdomen of the mouse to fully expose the peritoneum. Inject 10 milliliters of DMEM into the abdominal cavity through intraperitoneal injection. Gently rub the abdomen for one minute to lavage the cavity.
Then, use a syringe to collect the lavaged fluid into a 50 milliliter centrifuge tube. Centrifuge the lavage fluid at 300 G for five minutes, and discard the supernatant. Re-suspend the cell pellet in 10 milliliters of DMEM supplemented with 10%FBS, penicillin, and streptomycin.
Take 10 microliters of cell suspension and add it to the cells counter to count the cells. Dilute the cells to a concentration of 2 times 10 to the power of 6 cells per milliliter with DMEM FBS. Plate one milliliter of cell suspension per well onto a six well plate.
Incubate the plate at 37 degrees Celsius with 5%carbon dioxide for four hours. After incubation, remove the supernatant. Wash the cells twice with sterile PBS and culture them overnight with one milliliter of DMEM per well under the same conditions.
Incubate the peritoneal macrophages with staphylococcus aureus MRSA-252 for two hours. Remove the supernatant and wash the cells twice with PBS. Add one milliliter per well of complete DMEM medium containing 100 micrograms per milliliter gentamicin and incubate for two hours at 37 degrees Celsius in 5%carbon dioxide.
After incubation, wash the cells thrice with PBS. Using a cell scraper, collect the peritoneal macrophages into 50 milliliter centrifuge tubes. Centrifuge the samples at 1000 G for five minutes.
Add 10 micrograms per milliliter of lysozyme to the macrophages infected with intracellular MRSA-252, and incubate at 37 degrees Celsius for 10 minutes. After washing the cells twice with PBS, re-suspend them in one milliliter of PBS. Then, take out about 20 microliters of aliquot and add 0.1%Triton X-100 for five minutes to lyse the cells.
Perform serial dilutions of the lysed cell suspension with PBS and drop them onto a tryptic soy auger plate. Incubate the plate overnight at 37 degrees Celsius. The next day, count the bacterial colonies to calculate the intracellular MRSA-252 bacteria in peritoneal macrophages.
Place the mouse under an infrared physiotherapy lamp until the tail vein is dilated. Randomly divide the mice into four groups. Intravenously inject 3 times 10 to the power of 6 CFU Planktonic MRSA-252 into the tail vein of the mouse.
After 24 hours, sacrifice the anesthetized mouse and disinfect it thoroughly with 75%ethyl alcohol. Use tweezers with one hand to lift the abdominal skin and with the other hand cut the skin using ophthalmic scissors. Locate the kidneys in the abdominal cavity and carefully strip them completely.
Transfer the kidneys into a grinding tube containing one milliliter of PBS, and grind them until no solid tissue remains. Pour the homogenized tissue into the Eppendorf tube. Perform serial dilutions of the tissue homogenate using PBS.
Spot each dilution onto separate triptych soy auger plates and incubate them overnight at 37 degrees Celsius. Count the bacterial colonies the next day and analyze the data. Intracellular infection models of S.aureus were successfully established under optimized phagocytosis conditions and extended antibiotic treatment with some bacteria surviving within macrophages.
Macrophages infected with methicillin resistant S.aureus retained intracellular bacteria after two hours of antibiotic treatment when the supernatant no longer contained bacteria. In vivo, bacterial colonization assays in mice showed that vancomycin eliminated extracellular S.aureus, but failed to clear intracellular bacteria.
