All experiments were performed using a mixed culture of the following bacterial strains: Escherichia coli (ATCC® 11229), Proteus mirabilis (ATCC® 14153, DSM 778) and Staphylococcus aureus (ATCC® 6538). An overnight culture plate (Nutrient agar, OXOID, Germany) of each bacterium was washed away in 10 mL NaCl peptone and transferred to a sterile flask with glass beads. This suspension was homogenized for 2 min at 1500 rpm on a mechanical shaker and adjusted to 109 CFU per mL using standard plate count methods (data not shown).
Six different types of catheters were used, as shown in Table 1. These different catheters were selected to cover a broad range of commercially available products and observe the performance of the treatments in all of them.
Table 1. Characteristics of the catheters used in this study
Reference for experiments
|
Specifications
|
Catheter A
|
2-way Foley catheter, silicone, Balloon 30cc, 18Ch
|
Catheter B
|
Straight whistle tip catheter, silicone, 40cm, 18Ch
|
Catheter C
|
Bladder catheter without balloon, PVC, 37cm, 18Ch
|
Catheter D
|
Transurethral Foley catheter, 2-way, silicone, 41cm, 18Ch
|
Catheter E
|
Suprapubic catheter, polyurethane, 65cm, 10Ch
|
Catheter F
|
Transurethral Foley Nelaton balloon catheter, silicone, 41cm, 18Ch
|
Thirty (30) catheters of each type were used for the decolonization test. The catheters were incubated with 5 ml of the mixed bacterial suspension for 4 h at 37°C, after which the catheters were irrigated 2 x 400 ml of an organic load suspension (0.3 % bovine albumin + 3.0 % urea, reagents from Carl Roth Germany) per day to simulate the process of contamination with urine and organic materials. After 72 h, ten of the catheters were irrigated with 100 mL Uro-Tainer 0.02 % PHMB (B. Braun Medical, Switzerland ) with 5 minutes exposure time, ten catheters were irrigated with 100 mL Uro-Tainer 0.9% NaCl (B. Braun Medical, Switzerland) with an exposure time of 5 minutes and 10 catheters were not treated (controls).
After treatment, the microbial count was determined by irrigation of the catheters with 100 ml of a TLH-SDS neutralizer solution (0.1 % polysorbat 80, 0.1 % g/L lecithin, 0.1 % histidine, 0.2 % SDS, all reagents from Carl Roth Germany) and membrane filtration (0.45 µm pore size, MF-Millipore USA) of 50 mL via serial dilution method on trypticase soy bean agar (TSA, Oxoid Germany). No measurements of pH were made for the rinsed filtrates as the slightly acidic pH of the Uro-Tainer 0.02% PHMB (pH at 20 °C of 5.5) was neutralized with the use of the TLH-SDS solution. We anticipated that the pH of the filtrates did not modify the pH of the culture media used in this study and therefore did not affect the growth of the surviving bacteria.
In addition, all catheters were cut and the material in the lumen was extracted with a sterile cotton swab. The swab was suspended in 0.9% NaCl solution (Carl Roth Germany) and the microbial count was determined via serial dilution method on TSA plates.
All nutrient media were incubated at 37 °C for 2 days. Mean values of microbial count (log10 CFU) after the different treatments were calculated as well as the reduction factors for the Uro-Tainer 0.02% PHMB solution compared to Uro-Tainer 0.9% NaCl and no treatment. Statistical analyses were done using the two-tailed Student’s ‘t’ test and p values of (* ≤ 0.05) were considered as significant.
After negative reduction factors were observed for the type E catheters, an estimation of the biofilm mass was performed on them. Thirty new E catheters were artificially colonized as described before. For the decolonization, two solutions were used: 100 mL Uro-Tainer® with 0.02% PHMB and 100 mL Uro-Tainer 0.9% NaCl. Each of the solutions was connected to ten catheters and closed with a clamp after the catheter was filled with liquid with ten catheters left untreated as controls. After an exposure time of 5 min, the clamp was opened, and the remaining liquid was flushed through the catheter. All catheters were then thoroughly rinsed with 10 ml of sterile, bi-distilled water to remove planktonic or detached cells and filled with 1% crystal violet (Merck, Germany). After an incubation period of 15 min at room temperature, the catheters were again thoroughly rinsed with 10 mL sterile, bi-distilled water. Afterwards, 2 mL of 70% ethanol were carefully poured through the catheter into an acrylic cuvette (Sarstedt, Germany) in order to remove the crystal violet from the biofilms still present. The absorbance at 595 nm of the obtained solutions was measured in comparison with a blank cuvette with 2 mL of 70% ethanol using a SPECTRAmax PLUS384 Microplate Spectrophotometer (Molecular Devices, United States). By subtracting the absorbance of the blank value from the measured absorbance of the samples, it was possible to make indirect comparative statements on the layer thickness of the biofilm in the type E catheters.