Enzymes Enhance Biofilm Removal Efficiency of Cleaners

Efficient removal of biofilms from medical devices is a big challenge in health care to avoid hospital-acquired infections, especially from delicate devices like flexible endoscopes, which cannot be reprocessed using harsh chemicals or high temperatures. Therefore, milder solutions such as enzymatic cleaners have to be used, which need to be carefully developed to ensure efficacious performance. In vitro biofilm in a 96-well-plate system was used to select and optimize the formulation of novel enzymatic cleaners. Removal of the biofilm was quantified by crystal violet staining, while the disinfecting properties were evaluated by a BacTiter-Glo assay. The biofilm removal efficacy of the selected cleaner was further tested by using European standard (EN) for endoscope cleaning EN ISO 15883, and removal of artificial blood soil was investigated by treating TOSI (Test Object Surgical Instrument) cleaning indicators. Using the process described here, a novel enzymatic endoscope cleaner was developed, which removed 95% of Staphylococcus aureus and 90% of Pseudomonas aeruginosa biofilms in the 96-well plate system. With a >99% reduction of CFU and a >90% reduction of extracellular polymeric substances, this cleaner enabled subsequent complete disinfection and fulfilled acceptance criteria of EN ISO 15883. Furthermore, it efficiently removed blood soil and significantly outperformed comparable commercial products. The cleaning performance was stable even after storage of the cleaner for 6 months. It was demonstrated that incorporation of appropriate enzymes into the cleaner enhanced performance significantly.


Cleaning performance using EN ISO 15883
Biofilm was formed in a system according to ISO/TS 15883-5:2005(E) Annex F with slight adaptions. Polytetrafluoroethylene (Teflon) tube (KARL STORZ, Germany) with a diameter of 4 mm was used, and flow rates of 1 ml/min inlet and 40 ml/min circling were applied. The whole system was placed in an oven at 30°C instead of using a water bath described in EN ISO 15883. A 2.5 m long Teflon tube was used per experiment. P. aeruginosa DSM No. 1117 was used instead of the P. aeruginosa strains described in the norm. Biofilm was grown for 72 hours.
Tubes were cut into 30 mm parts and rinsed with 0.9% NaCl solution for 1 minute with peristaltic pump (~20 ml/min). Treatment with cleaner or WSH (negative control) was done with ~200 ml/min flow for 15 minutes at 25°C. Disinfection (only done if indicated) after cleaning was done with deconex ® HLD PA / PA20 (Borer Chemie AG) flowing through the 30 mm tube parts for 15 min using a peristaltic pump (~5 ml/min). The tube parts were rinsed with 0.9% NaCl solution for 1 minute with peristaltic pump (~20 ml/min). The tubes parts were cleaned outside with a 70% EtOH containing paper towel and cut longitudinally into half and into 5 mm pieces. The small pieces were added to a Falcon tube containing 20 ml 0.9% NaCl solution and vortexed for 5 minutes to detach the bacterial cells.
The following quantifications were done: 1) Total biomass cells were measured by optical density of the suspension at 600 nm 2) Viable cells were quantified by plating dilution series A 1:5 dilution series (30 μl in 120 μl 0.9% NaCl solution) was prepared in 96-well plates down to a dilution of 5 -11 . 5 μl of each sample and dilution were pipetted onto TSA square plates.
Colonies were counted after 1 day incubation at 25°C.
3) Proteins were quantified by Lowry assay (1) Complex-forming reagent was prepared immediately before use by mixing the following three stock solutions A, B, and C in the proportion 100:1:1 (v:v:v), respectively: Solution A: 2% (w/v) Na2CO3 in distilled water; Solution B: 1% (w/v) CuSO4·5H2O in distilled water; Solution C: 2% (w/v) sodium potassium tartrate in distilled water. 1 ml sample was mixed with 1 ml of complex-forming reagent and incubated for 10 min. 0.5 ml Folin reagent was added and mixed by vortexing. The mixture was incubated for 30 min before measuring the absorbance at 650 nm. 4) Polysaccharides were quantified by the phenol sulfuric acid method (2) 1 ml of 5% phenol solution was added to a 2 ml sample, afterward immediately 5 ml of 95% sulfuric acid was added. The mixture was incubated for 10 min, carefully shaken and incubated for another 20 min. Absorbance was measure at 490 nm.

Aging of the cleaner concentrates
One liter cleaner concentrate samples of the same batch were placed in ovens at 25°C, 40°C and 50°C, respectively. Higher temperatures should on the one hand simulate accelerated aging conditions and on the other hand reveal if the cleaner endures heating at unfavorable transport and storage conditions. 10 ml samples were taken after different incubation times to determine their performance of biofilm removal in the 96-well plate system and efficiency of cleaning artificial blood contaminations on TOSI ® slides as described above.

Microscopy
Biofilm containing endoscope tubes prepared and treated as described above were cut longitudinally into half and into 5 mm pieces. The pieces were individually added to a 12-well plate containing 1 ml 2.5 µM SYTO9 (life technologies) in 0.9% NaCl solution per well. After 30 min of incubation the tube pieces were placed into a Petri dish filled with water.
Microscopy pictures were immediately taken using a 20x water objective and GFP filters with the Leica DM6000 B microscope, Leica DFC450 C camera and the Leica LAS AF software.
Surface coverage was determined by CellProfiler software identifying objects above an intensity threshold of 0.15.

Selection of conditions for the 96-well plate system
Biofilm formation Biofilm formation in polystyrene 96-well plates for 24 hours at 33°C and 40 rpm was found to suit best our purpose. Sufficient biofilm of both species was formed for testing the efficacy of cleaner formulations. Additionally, physiological sodium chloride solution (0.9% NaCl solution) and water of standardized hardness (WSH) did not remove the biofilm, whereas the positive control (1% SDS, 1% EDTA, 1% NaOH, 0.1% NaClO) removed a substantial amount of the biofilm. Different temperatures for biofilm formation were investigated. For example, after 24 h more P. aeruginosa biofilm was formed at 37°C than at 33°C, however this former biofilm was found to be more easily removed by cleaners. Also biofilm formed for 48 h was investigated which displayed similar biomass quantity and cleaning resistance compared to 24 h incubation. Therefore, longer incubation time was not necessary. Different media were compared and 30% TSB supplemented with additional glucose was selected, because it supported strong biofilm formation of both strains.

Cleaner treatment
It was found that the cleaner removed more biofilm if it was diluted in WSH than in deionized water, especially in combination with enzymes. Static conditions at 25°C for different incubation times of 5, 10, 20 and 40 min were tested. For deconex ® PROZYME ACTIVE 5 min were sufficient to display almost maximal performance. However, longer incubation times were selected as the results were more reproducible and most other cleaners were less effective with short incubation times. Different temperatures for the cleaner treatment were also investigated. The performance was clearly reduced at 6°C, while there was no significant difference between 25°C and 35°C. Finally, 25°C and 40 min treatment were selected to simulate the manual cleaning conditions of the endoscope in the cleaner bath at room temperature.

Biofilm quantification
Staining with Crystal Violet was used to investigate the total amount of biomass, while the BacTiter-Glo assay is a sensitive and precise method to quantify the amount of viable cells (3).         Remaining S. aureus (a) and P. aeruginosa (b) biofilm after treatment with cleaners incubated at different temperature for 24 weeks. deconex ® PROZYME ACTIVE concentrate was either stored at 25°C (light green), 40°C (green) or 50°C (dark green). Base formulation B3A without enzymes (yellow) and positive control (red) are also displayed. Y-axis represents the biofilm amount quantified by Crystal Violet staining relative to the negative control (blue). Error bars represent the values obtained from 6 individual wells. A t-test was applied to calculate if the differences are statistically not significant (n.s., p>0.05) significant (*, p<0.05) or highly significant (**, p<0.001).