Name: An In Vitro Bladder Model of Catheter-Associated Urinary Tract Infection (Video) | JoVE Journal
Uploaded: 2025-06-24T12:30:00.000+00:00
Duration: 7 min 57 s
Description: Learn an in vitro bladder model for studying catheter-associated urinary tract infections using Proteus mirabilis biofilms. This method evaluates antimicrobial treatments like chlorhexidine to improve patient outcomes.

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5 minutes

We thank Dr. Jonathan Nzakizwanayo and Dr. Anthony J. Slate for their shared expertise in developing this protocol. Work at the University of Bath was funded by The Medical Research Council GW4 Biomed DTP as a studentship to V.B. (MR/N0137941/1). B. V. J. is also supported by funding from the Dunhill Medical Trust (RPGF1906\171). Research by O.E.C. was funded by Kidney Research Northwest (RCN: 1144798). Figure 1 is created with Biorender.com.

The authors have nothing to disclose.

The catheterized urinary tract represents a complex biological niche that can be challenging to replicate in a laboratory environment. The in vitro bladder model was designed for the purpose of simulating CAUTI, and utilizes standard urinary catheters to replicate the sterile, closed drainage system used in clinical practice. The catheterized urinary tract is highly susceptible to infection, as is evidenced by urinary catheters being the single biggest risk factor for the development of CAUTI6,55. Commensurately, careful aseptic technique is critical to the success of running in vitro bladder models. Turbidity of AUM is a clear indicator of sterility, as contaminated or expired media will become cloudy over time, requiring all replicates to be discarded. Careful aseptic technique should also be utilized when inoculating and sampling the in vitro bladder model, as opening the central chamber to add in the prepared inoculum breaks the sterile, closed drainage system, making the in vitro bladder model susceptible to contamination. To avoid contamination of the central chamber, triple-lumen catheters can be utilized to sample the planktonic cell population (by clamping off the waste lumen and draining via the additional lumen). Some washout is expected at the 4-h timepoint, but troubleshooting may be required if no viable planktonic cells are enumerated at this point. If no viable cells are enumerated after 4 h the inoculum density or AUM formulation utilized may need to be optimized to better support bacterial colonization. Additionally, inoculum washout may indicate an issue with the media circuit; kinks within the tubing could prevent adequate drainage, resulting in the buildup of toxic metabolites which may impede bacterial growth, whilst any residual disinfectant used to flush through media tubing could have a bactericidal effect.
This protocol details the preparation of AUM as previously described by Nzakizwanayo et al., as it has previously been validated for use in the in vitro bladder model with prominent CAUTI pathogens, including Proteus mirabilis and Escherichia coli43,45,46,48. However, there is scope to modify the AUM formulation utilized in the protocol to select for the growth of other pathogens. Numerous diverse AUM formulations have been published and could instead be used in the media circuit28,56,57,58,59,60. Additionally, AUM in the media reservoir can be supplemented with nutrients or antibiotics to select for the growth of specific pathogens. Historically, in vitro bladder models have also been supplied with pooled human urine, however, the large volumes of media required to run an in vitro bladder model replicate can make collection of human samples impractical53,54. Furthermore, the composition of human urine can vary greatly between patient samples, affecting experiment reproducibility, as differences in the concentration of hormones, salts, and proteins as well as hydration status, have been shown to affect bacterial growth and biofilm formation38,39,61,62.
The above protocol details the inoculation of the in vitro bladder model with ~1010 CFU/mL replicating late-stage CAUTI. However, the inoculation site of the in vitro bladder model could be modified in future assays to better replicate the earlier stages of CAUTI, i.e.,&#160;initial colonization of the urinary catheter and ascension into the bladder. Manually contaminating Foley catheters with a known bacterial inoculum prior to the setup of in vitro models could provide insight into the early stages of CAUTI. Indeed, inoculum density has been modified in previous work to replicate earlier-stage infection; for example, Nzakizwanayo et al. inoculated in vitro bladder models with a lower starting CFU/ mL (ca. 103 CFU/mL) to replicate earlier-stage infection47. It is, however worth noting that altering inoculum density may result in extended time to blockage and requires additional validation prior to experimentation.
Additionally, whilst the protocol details inoculation of the in vitro model with bacterial monoculture, there is scope to inoculate models with fungal pathogens. Past work has suggested that up to 8% of CAUTIs result from infection with Candida spp., which are able to produce biofilms which are often recalcitrant to antifungal treatment27,63. Moreover, the in vitro bladder model can be modified to accommodate the study of polymicrobial CAUTI. Indeed, it has been suggested that up to 86% of CAUTIs are polymicrobial, and these infections are often associated with more severe symptoms and a higher rate of mortality than monospecies infection27,64,65,69. In vitro models can be inoculated with multiple species to study the formation of polymicrobial biofilms and the progression of CAUTI, but careful validation of polymicrobial communities and modification of inoculum density should be performed prior to inoculation. Previous studies have utilized the in vitro bladder model to monitor differences in biofilm formation in single species P. mirabilis CAUTI compared with dual species CAUTI with E. faecalis, highlighting the metabolic interplay between the strains, which resulted in enhanced biofilm formation compared with single species infection70.
A major advantage of using the in vitro bladder model to study CAUTI, is the ability to observe biofilm formation on the surface of a standard urinary catheter. The formation of biofilms in CAUTI is often initiated by the formation of conditioning films on the catheter lumen, comprised of organic components of urine to which uropathogens can adhere to, before proliferating and forming mature biofilms2,18,71,72,73. Standard assays used to study biofilm formation often utilize standard bacterial growth media rather than AUM and are often based on adherence to polystyrene microplates or pegs, i.e.,&#160;the Calgary device; however, past work has suggested that the nutrient environment, pH, and oxygen levels of the system, as well as physical properties such as flow and difference in surface structure (silicone vs plastics), can affect biofilm formation36,37,74,75,76,77,78. Additionally, culturing bacteria with extensive urease activity, such as P. mirabilis in a closed system in AUM, can result in the buildup of toxic metabolites, which can result in cell death78,79. This is mitigated in the in vitro bladder model as the AUM in the central chamber is constantly replaced with fresh media via the media circuit, allowing for the study of CAUTI biofilms under more clinically relevant conditions45. The day-to-day running of the in vitro bladder model and the endpoint selected for models largely depend on whether models are inoculated with urease-positive or negative organisms. Theoretically, in vitro models could run indefinitely if supplied with an adequate volume of AUM and if the sterility of the model is maintained; however, it is recommended that an endpoint is selected prior to experimentation, whether that be a blockage or an arbitrary number of days. It is generally recommended that in vitro models inoculated with urease-negative organisms are run for &#8804;7 days to simulate the clinical environment, as this is typically sufficient to observe biofilm formation in most organisms, whilst keeping in line with clinical interventions, as standard best practice indicates that catheters should be replaced once visibly infected1,2,3,47,48. Although urease-positive organisms are likely to produce crystalline biofilms, there is a great deal of variation within urease-positive strains in time to in vitro bladder model blockage, with some strains of Proteus mirabilis causing catheter blockage in under 10 h, and others taking over 30 h to block39,46. A standard urease test could be used prior to in vitro bladder model experimentation to determine whether to run models to blockage or a pre-defined endpoint80.
Perhaps the main limitation of using the in vitro bladder model to study biofilm formation is that biofilm measurements are an endpoint reading, as they necessitate the removal and dissection of the urinary catheter. Conversely, planktonic samples can be taken from the in vitro bladder model at any point during experimentation. Additionally, once biofilm samples are taken from the in vitro bladder models, numerous extra downstream techniques can be used to better quantify biofilm production. Past studies have utilized flame photometry to quantify the amount of calcium and magnesium present in crystalline biofilms, and more complex stains can be used to accurately identify components of the EPS matrix of urease-negative biofilms45,73,81,82,83,84.
This work also describes the application of the in vitro bladder model to study the effects of therapeutics aimed at treating CAUTI. The in vitro bladder model simulates important aspects of the CAUTI niche, such as the use of AUM, as well as clinically relevant biofilm formation and increased organic load seen in CAUTI. Therapeutics such as antibiotics can be added directly to the AUM tank to simulate clinically relevant drug bioavailability, and past studies have utilized repurposed drugs such as thioridazine and fluoxetine to demonstrate an antibiofilm effect81. Additionally, minimum inhibitory concentration assays and bacterial suspension tests are often used to infer the antimicrobial activity of biocides, though there is little evidence to suggest that assays correlate to real-world biocide susceptibility41,85,86,87,88. The in vitro bladder model is advantageous for the testing of biocidal products aimed at treating or preventing CAUTI, as the presence of the urinary catheter and more representative simulation of the CAUTI niche mean that products such as antimicrobial lubricant gels and irrigation solutions can be tested as per manufacturer&#39;s instructions. Past studies have also used the in vitro bladder model to determine the effectiveness of phage therapy, and demonstrated that phage treatment could extend P. mirabilis time to block the in vitro bladder model by over 100 h47. The use of a standard-sized Foley catheter in the in vitro bladder model also means novel, coated catheters can be assayed for antimicrobial activity. Past work by Slate et al. demonstrated that catheters coated in a theranostic ciprofloxacin coating could extend P. mirabilis time to blockage by ~60 h in an in vitro bladder model48. This highlights the potential application of the in vitro bladder model in the study of novel therapeutics aimed at treating CAUTI, allowing for a more accurate assessment of antimicrobials in a biologically relevant simulated CAUTI niche.

Urinary catheters are amongst the most commonly deployed medical devices, with an estimated prevalence of 19% in acute care facilities1,2,3. Catheters are primarily deployed to aid in urine output management in cases of bladder dysfunction, and are inserted directly into the bladder, allowing urine to void from the bladder via a central lumen2,3. Urinary catheters can either be used intermittently, where they are inserted into the bladder periodically to drain urine, or indwelling catheters can be utilized, where catheters are left in situ in the bladder to continuously drain urine3,4. Despite their widespread use, urinary catheters are the biggest risk factor for the development of urinary tract infections (UTIs), which account for an estimated fifth of healthcare-associated infections5,6. Indeed, the catheter itself often acts as the initial site of microbial colonization, providing a surface for uropathogens to adhere to and further colonize the urinary tract1,6,7,8. Virtually all patients undergoing long-term continuous catheterization (where the catheter is left in place for &#8805;28 days) will experience bacteriuria, with the risk increasing by ~5% for every day the catheter is in situ2,9,10,11. Commensurately, around 80% of UTIs are associated with the use of a urinary catheter6.
Catheter-associated urinary tract infections (CAUTIs) represent a significant burden to health services, and are often associated with more serious upper UTI symptoms (such as ureteritis and pyelonephritis) than uncomplicated UTIs, as well as an increased risk of secondary bloodstream infections1,7,12,13,14. Standard treatments for CAUTIs include antibiotic therapy, however, these infections are often recalcitrant to antimicrobial treatment as a result of the development of complex biofilm communities on the catheter surface15,16,17,18,19,20. Indeed, it is estimated that&#160;~80% of CAUTIs result from biofilm-forming urinary tract pathogens19. Biofilm formation is characterized by the attachment of bacterial cells to catheter surfaces and the development of communities embedded in an extensive matrix of extracellular polymeric substances (EPS)21,22,23,24. Production of the EPS is a defining feature of biofilm communities and is responsible for many of the emergent properties displayed by biofilm-associated cells, such as elevated resistance to antimicrobial agents and immune clearance21,22,24. The EPS can play a direct role in these phenotypes by physically shielding cells and protecting them from antimicrobials or immune effectors, as well as indirectly by facilitating the development of wider aspects of biofilm communities such as the physicochemical microenvironment of the biofilm and the formation of persister cells23,24,25,26. Furthermore, some uropathogens, including Proteus spp., which account for ~11% of CAUTIs, possess potent urease enzymes, enabling them to hydrolyze the urea present in urine and resulting in an overall increase in urinary pH2,27,28,29,30. This pH increase is accompanied by the precipitation of calcium and magnesium phosphate crystals, which can be incorporated into the growing biofilm, resulting in biomineralization and the formation of dense crystalline biofilm structures that can block catheters. These crystalline biofilms not only make treatment problematic, but can also initiate serious complications such as pyelonephritis and septicemia2,28,31,32,33,34.
The complex environmental niche in the catheterized urinary tract, and the important role of biofilm formation in these infections, pose particular challenges in the study of CAUTI pathogenesis. Simple and widely used laboratory models to study and quantify biofilm formation, such as 96-well plate-based assays, are inadequate for the study of biofilm formation in CATUI, particularly crystalline biofilms. For example, such models do not account for key aspects of the CAUTI niche, such as the complex nutrient environment provided by urine, how the catheter surface affects adhesion and subsequent biofilm production, and the continual flow of urine media35,36,37,38,39,40. Additionally, standard assays used to determine antimicrobial susceptibility of uropathogens often don&#39;t reflect real-world clinical susceptibility. Minimum inhibitory concentration assays and microbial suspension tests are frequently used to infer the antimicrobial susceptibility of CAUTI pathogens, but typically do not simulate important variables of the CAUTI environment, such as the effect the presence of a urinary catheter and any subsequent biofilm formation may have on antimicrobial susceptibility41,42,43.
This study details the setup and running of an in vitro bladder model that simulates the environment of the catheterized urinary tract, and replicates a standard catheter closed drainage system as used in clinical practice, first described by Stickler et al.44 in 1999 (Figure 1). This model utilizes standard-sized Foley catheters (which are currently available on the NHS supply chain), and artificial urine media (AUM) as previously described by Nzakizwanayo et al.45to simulate CAUTI and biofilm formation. The in vitro bladder model consists of a double-walled glass chamber that acts as the &#34;bladder&#34; and has an outlet through which a Foley catheter can be inserted into the central glass chamber. At the top of the central glass chamber, there is an inlet through which AUM can be supplied to the &#34;bladder&#34; at a biologically relevant flow rate using a peristaltic pump, which will flow through the catheter via the drainage eyeholes into a waste bag. Additionally, the outer chamber is connected to a water bath circuit, which allows the models to be kept at a constant temperature. The in vitro bladder model can be inoculated via the central chamber, allowing for the study of CAUTI pathogens in a biologically representative niche. Furthermore, as models are catheterized and constantly supplied with fresh AUM, the growth and population dynamics of both planktonic and biofilm-associated cells can be studied.
Past studies have utilized in vitro bladder models for several applications, including the fundamental study of biofilm formation in prominent CAUTI pathogens, including Proteus mirabilis and Escherichia coli 43,45,46,47,48,49. Additionally, the in vitro model has been utilized to evaluate the efficacy of products intended to control CAUTI or prevent blockage50,51,52. More recently, models have been used to determine whether adaptations and mutations linked to AMR impact biofilm formation and CAUTI pathogenesis in P. mirabilis43,46. The in vitro bladder model has also been used to study novel antimicrobial treatments aimed at treating CAUTI, including newly formulated theranostic catheter coatings and phage therapy treatments47,48. This protocol encompasses the setup and running of in vitro bladder models, the enumeration of viable planktonic and biofilm-associated cells from the model and urinary catheter surface respectively, and the measurement of urinary pH to monitor urease production. Protocols are also detailed for the assessment of antimicrobial products aimed at treating or preventing CAUTI, including bladder irrigation solutions and antimicrobial lubricant gels, which are currently available on the NHS supply chain.

<table><tbody><tr><td>313 Rapid-Load Pumphead Extension</td><td>Watson-Marlow</td><td>313</td><td>Peristaltic pump - extra pumphead extension&amp;nbsp;</td></tr><tr><td>323S/D manual control variable speed pump</td><td>Watson-Marlow</td><td>323S/D</td><td>Peristaltic pump</td></tr><tr><td>3L Bed bags, sterile with lever taps&amp;nbsp;</td><td>Great Bear Healthcare&amp;nbsp;</td><td>GDW173</td><td></td></tr><tr><td>3mm inner diameter platinum cured silicone tubing</td><td>Fisher</td><td>10111801</td><td>Small AUM circuit tubing (connect pump tubing to models)</td></tr><tr><td>5mm inner diameter platinum cured silicone tubing</td><td>Fisher</td><td>10539201</td><td>Medium AUM circuit tubing (connect pump tubing to AUM reservoir)</td></tr><tr><td>ammonium chloride (NH&lt;sub&gt;4&lt;/sub&gt;Cl)</td><td>Fisher</td><td>11473713</td><td></td></tr><tr><td>anhydrous sodium sulfate (Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher</td><td>11498717</td><td></td></tr><tr><td>Azlon Tapered connector&amp;nbsp;</td><td>Fisher</td><td>10712807</td><td>Used to connect different lengths of tubing in AUM circuit</td></tr><tr><td>Bed bag hangers&amp;nbsp;</td><td>Great Bear Healthcare&amp;nbsp;</td><td>10705P</td><td></td></tr><tr><td>Bochem pinch cock tube clamps, 25 mm diameter</td><td>Fisher</td><td>11805101</td><td></td></tr><tr><td>calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher</td><td>10021681</td><td></td></tr><tr><td>Citric acid monohydrate&amp;nbsp;</td><td>Merck</td><td>C1909-500G</td><td>Used to remove crystalline biofilm from models post-experimentation&amp;nbsp;</td></tr><tr><td>Fisherbrand&amp;nbsp;Glass Graduated Class B Type 1 Pipettes</td><td>Fisher</td><td>11992168</td><td>Used to supply in vitro blader models with AUM via bored bung</td></tr><tr><td>gelatine</td><td>VWR</td><td>24360.233</td><td></td></tr><tr><td>Graphpad prism 10</td><td>GraphPad</td><td>V10.0</td><td>Analytical software used for graphing in vitro bladder model results and statistical analysis.&amp;nbsp;</td></tr><tr><td>Hanna HI 2211-02 pH benchtop meter</td><td>Phillip Harris&amp;nbsp;</td><td>PP00057414</td><td></td></tr><tr><td>&lt;em&gt;In vitro &lt;/em&gt;bladder model&amp;nbsp;</td><td>-</td><td>-</td><td>Bespoke equipment as previously described by Nzakizwanayo et al. (2019).</td></tr><tr><td>Instillagel 6 mL lubricant gel</td><td>mydoctorshop.co.uk</td><td>40-006</td><td>Lubricant gels used in section 7.1.; other suppliers are available.</td></tr><tr><td>Kartell Plastilab Uneven tapered connector</td><td>Fisher&amp;nbsp;</td><td>11914289</td><td>Used to connect tubing ofdifferent diameters</td></tr><tr><td>MacConkey agar without salt (dehydrated)</td><td>Fisher</td><td>10472643</td><td>For the enumeration of single colonies of swarming bacteria i.e. Proteus mirabilis</td></tr><tr><td>magnesium chloride hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher</td><td>10386743</td><td></td></tr><tr><td>monopotassium phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher</td><td>10573181</td><td></td></tr><tr><td>Nalgene Y-shaped connector</td><td>Fisher</td><td>10186070</td><td>Used to run multiple AUM circuits from one AUM reservoir (1-4 models per tank)&amp;nbsp;</td></tr><tr><td>potassium chloride (KCl)</td><td>Fisher</td><td>10375810</td><td></td></tr><tr><td>Pyrex Aspirator Bottle with Ground Glass Side Socket, Capacity: 5000 mL</td><td>Fisher</td><td>11347884</td><td>AUM reservoir</td></tr><tr><td>Rubber Stoppers With a Bore, Diameter Outer: 37mm, Red, Diameter: 37mm</td><td>Fisher</td><td>11517612</td><td>Used in AUM reservoir and to supply in vitro models with AUM</td></tr><tr><td>Size 14 French 100 % silicone Foley catheter with sterile syringe of water for inflation&amp;nbsp;</td><td>Great Bear Healthcare&amp;nbsp;</td><td>4833-0514</td><td></td></tr><tr><td>sodium chloride (NaCl)</td><td>Fisher</td><td>10735921</td><td></td></tr><tr><td>sodium oxalate (Na&lt;sub&gt;2&lt;/sub&gt;C&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher</td><td>10442211</td><td></td></tr><tr><td>Teknon Biocleanse concentrate biocidal cleaner 5L&amp;nbsp;</td><td>Fisher</td><td>12447580</td><td>Used to sterilize models post-experimentation</td></tr><tr><td>trisodium citrate (Na&lt;sub&gt;3&lt;/sub&gt;C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;)</td><td>Fisher</td><td>11434993</td><td></td></tr><tr><td>tryptone soya broth</td><td>Fisher</td><td>10198002</td><td></td></tr><tr><td>urea (CH&lt;sub&gt;4&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher</td><td>10404185</td><td></td></tr><tr><td>Uro-Tainer M NaCl 0,9%</td><td>B.Braun</td><td>FB99833</td><td>Saline bladder irrigation pouch used in section 7.2.&amp;nbsp;</td></tr><tr><td>Watson-Marlow Pumpsil 1.6mm inner diameter platinum cured silicone tubing</td><td>Fisher</td><td>12406280</td><td>Thick AUM circuit tubing (for use in perstaltic pump)</td></tr></tbody></table>

an in vitro bladder model of catheter-associated urinary tract infection

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

Catheter-associated urinary tract infections (CAUTIs) are among the most common healthcare-associated infections. Biofilm formation on urinary catheters is a key aspect in the pathogenesis of these infections, and frequently leads to blockage of catheters and serious complications such as pyelonephritis and septicaemia. Approaches to model and study biofilm formation in this setting are essential for both a fundamental understanding of the mechanisms underpinning the pathogenesis of CAUTI, as well as the development and robust pre-clinical evaluation of effective therapeutic strategies to control CAUTI and catheter blockage. This protocol describes an in vitro model of the catheterized urinary tract, which replicates the catheter closed drainage system as used in clinical practice, facilitating the study of CAUTI and catheter biofilm formation under representative conditions. Furthermore, this protocol details the application of the in vitro bladder model to evaluate the efficacy of antimicrobial catheter maintenance products, including products currently available on the NHS supply chain.

Successful operation of the in vitro bladder model is dependent on practicing good aseptic technique and careful observation of media turbidity throughout experimentation (Figure 3). Prior to inoculation, sterile AUM was pale yellow in color and transparent in the in vitro bladder model central chamber (Figure 3A). AUM in the media reservoir should remain clear throughout experimentation, as any turbidity changes that occur may indicate precipitation of salts from solution, or contamination of media. If AUM in the media reservoir or media circuit tubing becomes cloudy or shows a drastic change in color, replicates should be discarded and repeated. After inoculation, an increase in the turbidity of the AUM in the in vitro bladder model central chamber is clearly visible (Figure 3B). Changes in turbidity are often more pronounced in urease-positive organisms such as P. mirabilis, but should still be visible post-inoculation in urease-negative organisms. After AUM flow is initiated, a reduction in AUM turbidity is often observed as planktonic cells are washed out, and does not indicate a lack of viable cells in the model. Observing the media level in the central chamber can also be used to determine blockage time if utilizing urease-positive organisms (Figure 3C). As models are running, the catheter eyehole and tip should be completely visible to indicate proper drainage into the waste bags. Upon blockage, the level of AUM in the central chamber will rise above the catheter eyeholes and tip, indicating that the experiment is ready for any endpoint readings and termination. During experimentation, pressure effects in the in vitro bladder model media circuit can make models appear blocked. If a blockage is suspected, check that the AUM circuit, catheter, and waste bag tubing are taut before terminating the assay, as any potential air bubbles or kinks in the tubing can cause "airlocks" and prevent the catheter from draining.
During in vitro bladder model experiments, planktonic sampling can provide valuable insight into the growth dynamics within the model as well as the progression of CAUTI. Cell growth, pH, and biofilm formation can all be monitored throughout experimentation without necessitating the termination of models (Figure 4). If using urease-positive organisms, blockage time calculations can be utilized as a measure of the rate of biofilm formation and used to compare the properties of different bacterial strains (Figure 4). Comparison of a wild-type strain of P. mirabilis and an LPS mutant (mini-tn5 insertion into waaC) demonstrated an extension of time to blockage and thus reduced biofilm formation in the mutant (Figure 4). Urease-negative organisms do not typically block in vitro bladder models, and it is therefore recommended that a set endpoint is determined prior to experimentation. In some cases, urease-negative organisms can result in catheter blockage. For example, urease-negative strains of Klebsiella pneumoniae can produce extensive, mucoid biofilms, however, these typically take much longer than urease-positive biofilms to block drainage, so a set endpoint is still recommended.
Measurement of pH throughout bladder model experimentation can also provide insight into strain-to-strain differences, as well as the progress of CAUTI. If urease-negative organisms are utilized, pH in the in vitro bladder model central chamber should largely remain stable throughout experimentation, though small fluctuations in pH are expected as population dynamics change over the course of the experiment. Inoculation of in vitro models with urease-positive organisms results in a significant pH increase over time, with the highest pH usually being observed at the time of blockage, where pH readings as high as ~8-9 can be observed (Figure 4B).
Enumeration of planktonic CFU/mL is an important component of in vitro bladder modeling, allowing operators to assess whether inoculation has been successful and to monitor the progress of experimentation. Measuring CFU/ mL at the end of experimentation can highlight any differences in bladder colonization between different bacterial strains. When comparing the mini-Tn5 mutant strain of P. mirabilis to the wild-type, a significant reduction in CFU/ mL at the time of blockage was observed (Figure 4C). Furthermore, enumeration of CFUs from biofilms on the catheter surface, and quantification of biofilm biomass can provide insight into the ability of different pathogens to colonize the catheterized urinary tract. Crystal violet stains were used to determine differences in biofilm formation between a wild-type P. mirabilis strain HI4320 and isolates passaged in chlorhexidine (Figure 5). Optical density measurements at 595 nm demonstrated that isolates passaged in chlorhexidine displayed a reduction in biofilm formation compared with the wild-type and controls passaged in the absence of chlorhexidine.
As the aim of the in vitro bladder model is to accurately replicate the CAUTI niche, models can also be utilized to study the effects of antimicrobial therapeutics aimed at treating CAUTI in a clinically relevant environment. For urease-positive organisms, comparing the blockage times of treated models with untreated models can provide useful insight into the effectiveness of treatment. Treatment of in vitro bladder models inoculated with P. mirabilis with a 0.02% chlorhexidine bladder irrigation solution significantly extended the time to blockage compared with models that were left untreated or treated with 0.9% saline (Figure 6A). Furthermore, the in vitro bladder model can be utilized to study the bactericidal activity of antimicrobial therapeutics. Monitoring changes in CFU/mL of planktonic and biofilm-associated cells following antimicrobial treatment can indicate whether treatments are effective. Treatment of in vitro bladder models inoculated with P. mirabilis demonstrated a &gt;7-log reduction in planktonic CFU/mL immediately after treatment, compared with untreated and 0.9% saline irrigated models when applied as per manufacturer's instructions (Figure 6B).
<img alt="Bioreactor setup diagram; schematic showing media and water flow, bioprocess methodology." class="xfigimg" src="/files/ftp_upload/67966/67966fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Schematic of the main components of the in vitro bladder model. (A) The bladder model (closed drainage system): (1) double-walled glass chamber simulating the bladder (2) media inlet, AU is supplied via peristaltic pump to the bladder model inner chamber (3) water inlet supplied via circulating water bath to outer chamber (4) water outlet (5) Foley catheter, inserted into inner chamber (6) drainage lumen connection point; connection point for waste bag/irrigation pouches (7) drainage bag collecting urine outflow. (B) Schematic depicting setup of the in vitro bladder model and direction of flow of media and water through the model. Artificial urine is stored in a media reservoir (1) at room temperature and supplied to the model via a peristaltic pump (2) at a constant flow rate of ~ 0.75 mL min-1 for the duration of the experiment. Media flows into the central chamber of the double-walled glass vessel representing the bladder (3) and drains out via the eyehole of a Foley catheter (4) into a drainage bag (5), completing the sterile closed drainage system. The in vitro bladder model is kept at a constant temperature of 37 °C via a circulating water bath (6), which flows through the outer chamber of the double-walled glass vessel. Dashed lines represent silicone tubing and arrows represent the direction of flow. <a href="https://www.jove.com/files/ftp_upload/67966/67966fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Balloon catheter diagram, deflated setup with drainage and inflation lumens for medical procedure." class="xfigimg" src="/files/ftp_upload/67966/67966fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Sectioning of urinary catheters to process biofilm-associated cells. Example points on a urinary catheter for dissection for downstream biofilm quantification. (A) Catheters are first sectioned latitudinally in the 8 cm region proximal to the tip/eyeholes (where most biofilm formation is observed). 1 cm sections are removed with a scalpel, generally avoiding the region containing the deflated catheter balloon due to difficulties in processing reproducibility (1-3). (B) Once catheters are initially sectioned into 1 cm pieces, another longitudinal cut is made down the central lumen to expose any biofilm present for further biofilm quantification, i.e., CFU/mL enumeration of crystal violet staining. Dashed lines represent dissection points. <a href="https://www.jove.com/files/ftp_upload/67966/67966fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Column chromatography setup with buffers, proteins eluting, purification process steps." class="xfigimg" src="/files/ftp_upload/67966/67966fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Stages of an in vitro bladder model experiment. Images of the in vitro bladder model central chamber, throughout different stages of experimentation. (A) AUM in the in vitro bladder model central chamber prior to inoculation. Media in the central chamber is completely transparent, indicating successful sterilization, and the catheter tip and eyeholes are visible above the line of media, indicating the catheter is successfully draining. (B) Media in the in vitro bladder model central chamber following inoculation with P. mirabilis (4 h post-inoculation). Successful inoculation is indicated by the increased turbidity of the media. The visibility of the catheter tip and eyeholes indicate that AUM is successfully draining through the catheter, and blockage has not occurred. (C) An example of a blocked in vitro bladder model. Inoculation with P. mirabilis and the subsequent biofilm formation and mineralization have resulted in the blockage of catheter eyeholes, preventing drainage and causing the level of media in the central chamber to rise above the catheter tip. <a href="https://www.jove.com/files/ftp_upload/67966/67966fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bar graphs comparing WT and Mutant: blockage time, pH, CFU/mL, statistical analysis included." class="xfigimg" src="/files/ftp_upload/67966/67966fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Endpoint measurements for an in vitro bladder model run until blockage. Example endpoint measurements at the time of blockage from an in vitro bladder model inoculated with a clinical strain of P. mirabilis, RS47 (WT), and a mutant strain with attenuated biofilm formation, RS47-2 (Mutant). (A) Blockage time (h) of WT and mutant strains of P. mirabilis. (B) pH of in vitro bladder models at the time of blockage. (C) CFU/mL of planktonic cells in in vitro bladder model chamber at the time of blockage. All data represent the mean of five biological replicates. Error bars show the standard error of the mean (SEM). *p ≤ 0.05; **p ≤ 0.01. This figure has been modified from Clarke et al.46. <a href="https://www.jove.com/files/ftp_upload/67966/67966fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bar graph showing OD_595nm absorbance results of a photometry experiment with statistical analysis." class="xfigimg" src="/files/ftp_upload/67966/67966fig05.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: Biofilm-associated cell measurements from the in vitro bladder model. Quantification of P. mirabilis biofilm formation, comparing wild-type strain HI4320 to adapted populations passaged with or without chlorhexidine (CHD) via crystal violet staining and measurement of optical density (OD) at 595 nm. WT: P. mirabilis HI4320; 1, 3, 5: passage controls with no CHD exposure; 2, 4, 6: HI4320 passaged in 4 µg/ mL CHD. All data represent the mean of five biological replicates. Error bars show SEM. *p ≤ 0.05; **p ≤ 0.01. <a href="https://www.jove.com/files/ftp_upload/67966/67966fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bar chart showing blockage time and CFU/mL; untreated vs treated samples, statistical significance." class="xfigimg" src="/files/ftp_upload/67966/67966fig06.jpg" data-altupdatedat="1788304762000" data-alt="Figure 6"> 
Figure 6: Measurements from in vitro bladder models following antimicrobial treatment. Planktonic samples were taken from an in vitro bladder model inoculated with P. mirabilis, following irrigation with a 0.02% chlorhexidine antimicrobial solution (100 mL) or a 0.9 % saline control. (A) Blockage time of in vitro bladder models following irrigation. (B) Planktonic CFU/mL immediately following irrigation treatment. All data represent the mean of five biological replicates. Error bars show SEM. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001. <a href="https://www.jove.com/files/ftp_upload/67966/67966fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This protocol details an in vitro model of the catheterized urinary tract, which can be used to study both planktonic and biofilm-associated bacterial cell populations in simulated catheter-associated urinary tract infections. This model can be further utilized to study the efficacy of antimicrobial products aimed at controlling urinary tract infections.

Catheter-associated urinary tract infections (CAUTIs) are among the most common healthcare-associated infections. Biofilm formation on urinary catheters ...

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Research

JoVE Journal

Immunology and Infection

Source: O'Brien, V. P., et. al., Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice. J. Vis. Exp. (2020)This video demonstrates the use of scanning electron microscopy to visualize bacterial colonization and host–pathogen interactions in the bladder epithelium of a pre-infected mouse model.

Preparation of Mouse Bladder Tissue for Visualization of Bacterial Infection

Source: Conover, M. S., et al., J. Establishment and Characterization of UTI and CAUTI in a Mouse Model. J. Vis. Exp. (2015)This video demonstrates the procedure for assembling and implanting a transurethral catheter in a female mouse, followed by bacterial inoculation to establish catheter-associated urinary tract infection and investigate biofilm-mediated host-pathogen interactions.

Catheter-Associated Urinary Tract Infection in a Mouse Model

Source: Conover, M. S. et. al., Establishment and Characterization of UTI and CAUTI in a Mouse Model. J. Vis. Exp. (2015)This video demonstrates the detection of intracellular bacterial communities (IBCs) in mouse bladder tissue infected with β-galactosidase-expressing Escherichia coli. Following tissue fixation, permeabilization, and staining with X-gal, IBCs appear as blue puncta under a dissecting microscope, confirming bacterial invasion during urinary tract infection.

Visualizing Intracellular Bacterial Communities in an Infected Mouse Bladder

In this video, we describe the technique for the intravesical administration of uropathogenic bacteria to generate a urinary tract infection model in mice. This model can be used to understand the spread of uropathogenic bacterial infection within the urinary system and the mechanisms that determine UTI susceptibility.

Generating Urinary Tract Infection Model: A Technique to Administer Uropathogenic Bacteria in Mouse to Develop Urinary Tract Infection Model

This video demonstrates the establishment of uropathogenic Escherichia coli infection in a female mouse bladder through transurethral catheter instillation. The injected bacteria successfully establish a urinary tract infection within the bladder, providing a valuable platform for studying healthcare-associated diseases related to the urinary tract.

Transurethral Catheterization and Bacterial Inoculation to Generate a Urinary Tract Infection Model in Male Mice

Establishment of a Urinary Tract Infection Model in Female Mice

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work’s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Establishment and Characterization of UTI and CAUTI in a Mouse Model

The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes.

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Learn an in vitro bladder model for studying catheter-associated urinary tract infections using Proteus mirabilis biofilms. This method evaluates antimicrobial treatments like chlorhexidine to improve patient outcomes.

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

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