The Removal Effect Of Different Detergents At Different Treatment Tiem Points On Endoscopic Biofilm

Endoscopic biofilm is one of the important reasons resulting in nosocomial infection. Biofilms are a structured community of bacterial cells enclosed in a self-produced extracellular polymeric substance (EPS) adherent to an inert or living surface. It protects the bacteria from the environmental influence and increases resistance to biocide. Bacteria within biofilms are up to 1000 times more resistant to antimicrobials than the same bacteria in suspension. Study found that formation of biofilm could significantly reduce efficacy of disinfectant. Technolgic advances in recent years have resulted in the development of complex, expensive, and heat sensitive medical instrumentation, including flexible gastrointestinal endoscopes. Because of the design complexity and the region of use which could contribute to formation of biofilm, gastrointestinal endoscopes present special challenges to cleaning. Biofilm which plays an important role in the transmission of infectious agents via contaminated endoscopes was present in endoscopic channels after disinfecting. The presence of bacterial biofilm may account for decontamination failures of adequate disinfection processes and results in nosocomial infection and increase in patient morbidity and health care costs. It is worthy of discussion to understand influence factor of endoscopic biofilm formation and to find effective method of controlling and removing the endoscopic biofilm. In this study, we established a closed continuous circulatory incubation system for generating biofilm on the inner surface of Teflon tube commonly used in endoscope. We used this apparatus to establish endoscopic biofilm model for investigating the effect of different incubation time on formation of biofilm and testing the removal efficacy of different detergents on biofilm recommended for use in endoscope reprocessing for guiding the right choice and use of detergents and provides the reliable theoretic basis for removal of biofilm from endoscopes in endoscope reprocessing. Only by doing so can we better control cross-infection and ensure medical safety and it has important clinical significance.ObjectiveTo established a closed continuous circulatory incubation system for generating biofilm on the inner surface of Teflon tube commonly used for endoscope channels. We used this apparatus to establish endoscopic biofilm model for investigating the effect of different incubation time on formation of biofilm and evaluating the removal efficacy of enzymatic cleaners and cleaner containing no enzymes on biofilm recommended for use in endoscope reprocessing for guiding the right choice and use of detergents and providing the reliable theoretic basis for removal of biofilm in endoscope reprocessing and revision of "the Guideline of Cleaning and Disinfection Techniques of Endoscopes (2004 Edition)".MethodsEstablishing a closed continuous circulatory incubation systemWe performed this model design based on endoscopic biofilm model established by Vickery. A closed continuous circulatory incubation system was consisted of a 500 ml reservoir containing the inoculated test media, a 100 cm long test tubing marked at 2 cm intervals, and another piece of tubing that led back into the reservoir. These three parts were connected to set-up the biofilm apparatusEstablishing endoscopic biofilm modeThe closed continuous circulatory incubation system was placed into an Excella E24 Incubator shaker series, reservoir inoculated with midlgarithmic phase E coli, and the circuit was filled with bacterial suspension containing E coli 1.5×10 cfu/mL. The reservoir and the tubing were maintained at 37℃in the Excella E24 Incubator shaker series. The pump was adjusted to a speed of 80-90 ml per hour and the media re-circulated for 10 days. Media and bacterial suspension were changed daily. The tubing was coiled in the same manner each experiment. At termination of the experiment the test tubing was disconnected from the apparatus and drained. The external surface of the tubing was first wiped with sterile gauze moistened with alcohol and then with sterile gauze soaked in Phosphate Buffered Saline (PBS). Approximately 10 cm of tubing at each end was discarded, and the tubing was then cut into 2 cm lengths with a sterile sharp scalpel.Every sections were placed into 25 ml PBS contained in a V shaped sterile glass container for washing. Each washing step was carried out by gently inverting the container five times, decanting the PBS and removing any left over solution with sterile gauze and refilling the container with 25 ml PBS and washed 3 times by gently submerging the tubes in 25 mL of the PBS to remove any loosely attached bacteria. After a total of three washes each section was removed excess fluid with sterile gauze.Viable counts of within biofilmTo recover bacteria from the internal surfaces of Teflon tube each section was scraped with a 2 mm wide sterile wooden stick. The tubing was then cut in half and the two segments and the scraper were placed into a 5 ml test tube containing exactly five milliliters of PBS, and at a time they were placed in the test tube rack and immersed in an ultrasonic bath for 10 minutes with a sweeping frequency of 42-47 KH. The temperature of the water was maintained at 20℃. The samples were vigorously shaken for 2 minutes post-ultrasonication. For each 2cm section, serial 10-fold dilutions were made in PBS and lml from each dilution was seeded onto a Nutrient Agar plate and spread over the surface with a sterile glass spreader. Plates were incubated at 37℃. Colonies were counted the next day on all plates with colony number. The results were expressed as the number of log Colony Forming Units (CFUs) per cm2. Per cm2 were based on calculation of the internal surface area of the Teflon tube. The results from each control or test replica were averaged.Scanning Electron Microscopic examination of biofilmTow-centimeter sections of the tubing from each experimental condition were fixed in Phosphate buffer solution containing 2.5% buffered glutaraldehyde at 4℃. After 2 hours they were washed three times in Phosphate buffer solution. Before critical point drying the bisected segments were dehydrated through a series of acetone and replaced with isopentane acetate.Critical point drying was carried out in a commercial Critical Point Drier. The specimens were then cut longitudinally and mounted on stubs and coated with a 20 nm gold/platinum film in a Sputter Coater. The entire surface was systematically examined in a Scanning Electron Microscope.Intervention of different detergent for endoscopic biofilmBy the method used above to establish endoscopic biofilm model, two types of detergents recommended for use in endoscope cleaning were evaluated:(1) detergents containing no enzymes (Rapidmulti-enzyme cleaner 1:400, Scopezime 1:270) and (2) detergents containing enzymes (Intercept 1:385). Concentration of detergents recommended by Manufacturer for use in endoscope cleaning.One biofilm-coated segment was placed into a sterile vial containing test detergent and allowed to react for 3, 5 and 7 min at room temperature. This condition was repeated 3 times. The tubing was removed from the detergent. Residual detergent was removed by submerging the tubes in 25 ml PBS, and then the tubing was wiped with sterile gauze. Control tubing was not reacted by detergents. Residual biofilm was assessed by counting the number of bacteria remaining adherent to the surface of the tubing after washing and by scanning electron microscopy (SEM)ResultsOn the fifth, seventh, tenth day, the average standard colony counts (1gCFU/ cm2) within biofilm adherent to the internal surface of Teflon tube were 4.98±0.47, 4.82±0.46,4.43±0.52 respectively. The viable bacterial counts were significantly different among the three groups (P<0.05). Bacterial colony count of the tenth days within biofilm was significantly less than that of the fifth, seventh day respectively (P <0.05). There was no difference between that of the fifth and seventh day(P>0.05). By scanning electron microscope, a large number of bacteria closely adhered to the Teflon tube'internal surface whose extracellular polymeric substance (EPS) crossed network-like shape after continuous perfusion for 5 days. EPS adhered to single cells forming microcolony after continuous perfusion for 7 days. The stable mature biofilm formed on the internal surface of Teflon tube after continuous perfusion for 10 days.After same detergents reacted for 3,5 and 7 min separately, the average standard colony count within biofilm (lgcfu/cm2) was 4.61±0.52 for Rapidmulti-enzyme cleaner, it was 4.67±0.49 for Scopezime, it was 1.29±0.13 for Intercept, and it was 5.44±0.19 for the blank control group. And they were 3.53±1.65,3.52±1.65,3.52±1.65 respectively after different detergents reacted for 3,5 and 7 min. The viable bacterial counts within biofilm were significantly different among three detergents groups (P <0.001). There was not significant difference among the three groups of different time points (P> 0.05). The removal efficacy of cleaner containing no enzymes on the bacteria within biofilm was better (P<0.001). There was no difference between that of two enzymatic clears (P>0.05). There was significant difference among the four groups including the three experimental groups which three detergents reacted for 3 min and the blank control group (P<0.001). By scanning electron microscope, after different detergents acted on biofilm for 3,5 and 7 min, residual biofilms of the groups which enzymatic cleaners treated was significantly more than the cleaner containing no enzymes group, while difference was not obvious among the groups which the same detergent treated at different time point.ConclusionsBiofilm formation of E coli on the internal surface of Teflon tube is closely related to incubation time. With the increase of incubation time, the bacteria within biofilm gradually decreased and a mature and stable biofilm formed. The viable bacterial counts within biofilm of the groups which two enzymatic cleaners treated was significantly more than the cleaner containing no enzymes group, while difference was not obvious among the groups which the same detergent treated at different time points. Two enzymatic clears respectively failed to reduce the viable bacterial numbers more than 1 log, but removed bacterial EPS, to a certain extent, while cleaner containing no enzymes reduce more than 4 logs and obviously reduced bacterial viability and bacterial EPS.