An In Vitro Bladder Model of Catheter-Associated Urinary Tract Infection

- Our research is focused on catheter-associated urinary tract infections. We use in vitro models like the bladder model to better understand the pathogens that cause CAUTIs.

The in vitro bladder model provides a controlled environment to test the practical applications of diagnostic tools and therapeutic interventions for use in CAUTI treatment.

The catheterized urinary tract is a complex environmental niche with important parameters such as the catheter surface and the nutritional environment of urine, which make it difficult to replicate in vitro.

A key aim of our research is to find ways to control biofilm formation on medical devices like urinary catheters to improve outcomes for patients. We're also using our models to study the development of antimicrobial resistance in bacterial communities to answer questions about how this arises and what we can do to prevent it.

[Narrator] To begin, insert a Foley catheter into the central glass chamber of the in vitro bladder model, and secure the catheter in place after inflating the balloon using the syringe of sterile water provided. Then, attach the waste bag to the catheter port before connecting the in vitro bladder model device to the AUM reservoir. Now, attach the in vitro bladder models to clamp stands. Connect the media circuit tubing to the peristaltic pump and secure the waste bags in the stands positioned below the models. Connect the outer chamber of the bladder model to a circulating water bath using silicone tubing. Set the water bath to heat to 37 degrees Celsius and turn on the tank circulators. Sterilize the in vitro bladder model using 70% ethanol. Remove the stopper at the media inlet to make the inner chamber accessible. Using a serological pipette, remove 10 milliliters of AUM from the bladder model volume. Inoculate the in vitro bladder models through the central chamber using the entire normalized 10 milliliter suspension and mix thoroughly. Using the pipette, remove 10 milliliters of the inoculated sample from the central chamber and aliquot them into a 15 milliliter universal tube for later assays. After decontaminating the external surfaces, replace the stopper and briefly resume media flow to ensure that the AUM level in the central chamber reaches the eye hole, then stop the flow. Following a one hour incubation, switch on the media flow using the peristaltic pump to begin running the in vitro bladder models. After decontaminating the bladder model stopper, remove it to access the central chamber. Using a serological pipette, gently mix the contents of the central chamber, avoiding the catheter tip and balloon. Then, remove one milliliter from the sample using a pipette and aliquot it into a 1.5 milliliter microcentrifuge tube for serial dilutions. Measure the pH of the remaining sample using a pH meter. Disconnect the waste bag from the catheter and drain any residual urine from the model by deflating the balloon. Remove the catheter from the bottom of the model using sterile forceps. Now, place the removed catheter onto a sterile board and using a sterile scalpel, cut the catheter at the required points for sectioning. Using sterile forceps, dip the catheter section three times into sterile PBS to remove non-adherent cells and incubate the prepared catheter section in 0.25% trypsin at 37 degrees Celsius for 30 minutes. After incubation, sonicate the solution containing the catheter for 10 minutes. Using the aseptic technique, insert the end of the syringe containing the lubricating agent into the neck of the bladder model and inject five milliliters of gel. Rotate the model to ensure the base is evenly coated with gel. To insert a Foley catheter into the model, push the catheter tip through the gel and secure it after filling the balloon as normal. Connect the model to the rest of the setup. Then, fill the model lumen with urine until it starts to drain through the catheter eye hole. As soon as urine begins to drain, stop the pump to prevent loss of gel before inoculation. At the point of switching on the pump after inoculations, plate out dilutions for colony forming unit counts. If using an individual irrigation pouch, disconnect the waste bag from the catheter and use the cap provided with the pouch to seal the waste bag. Immediately attach the irrigation solution pouch to the catheter, and using light pressure, squeeze the product into the central lumen following the manufacturer's instructions for 15 minutes. Once the recommended treatment time is completed, allow the residual solution to drain from the inner chamber via the catheter eye hole into the irrigation pouch, then disconnect the pouch. Sterilize the catheter drainage port, and the waste bag connection port with 70% ethanol before reconnecting them. To terminate the experiment, turn off the water bath flow and disconnect the media circuit and water bath circuit tubing from the in vitro bladder models. After removing the stoppers, collect the entire volume of residual urine, drain the waste bags, and discard any residual media. Then, disconnect the catheter waste bags from the catheter port and discard them. Finally, using a syringe, slowly deflate catheter balloons before removing catheters from the models. After inoculating the bladder model, the blockage time was significantly extended in the mini-Tn5 mutant strain of Proteus mirabilis compared to the wild type strain indicating reduced biofilm formation. Planktonic colony forming units per milliliter at the time of blockage were significantly reduced in the mini-Tn5 mutant strain compared to the wild type. Crystal violet staining demonstrated that Proteus mirabilis isolates passaged in chlorhexidine exhibited a significant reduction in biofilm biomass compared with those passage without chlorhexidine. Treatment of in vitro bladder models with 0.02% chlorhexidine irrigation significantly extended the time to blockage and reduced the planktonic colony forming units per milliliter compared with untreated and saline-treated controls.