This study investigates the survival of microorganisms in buildup biofilm (BBF) formed in flexible endoscopes, which undergo repeated cycles of patient-use and reprocessing. The authors developed a novel modeling approach using the MBEC (Minimum Biofilm Eradication Concentration) system to evaluate microbial survival in BBF. The model mimics the cumulative effect of reprocessing protocols, including drying, disinfectant exposure, and re-exposure to the test organism. Glutaraldehyde (GLUT) and accelerated hydrogen peroxide (AHP) were evaluated for their effectiveness in killing microbes in traditional biofilm (TBF) and BBF. Key findings include: - The combination of an organic matrix and aldehyde disinfection (GLUT) quickly produced a protective BBF that facilitated high levels of microbial survival. - In cross-linked BBF formed under high nutrient conditions, maximum colony-forming units (CFU) reached ~6 Log10 CFU/peg. - Organism survival was significantly higher in BBF compared to TBF, especially when glutaraldehyde was used. - The rate of biofilm survival post high-level disinfection (HLD) for E. faecalis and P. aeruginosa in BBF was 10 and 8.6, respectively, compared to a survival rate in TBF of ~2 for each organism. - Indirect outgrowth testing demonstrated the presence of surviving organisms in both TBF and BBF when glutaraldehyde was used. - AHP was less effective in eliminating microbial survival in BBF compared to glutaraldehyde. The study concludes that the BBF model demonstrates for the first time that survival of a wide range of microorganisms occurs in BBF, with significantly more rapid outgrowth compared to TBF. This highlights the need for meticulous cleaning and reprocessing of flexible endoscopes to prevent the accumulation of organic material and microorganisms, which can hinder effective disinfection. The data suggests that the assurance of effective high-level disinfection may decrease if BBF develops within the channels of flexible endoscopes.This study investigates the survival of microorganisms in buildup biofilm (BBF) formed in flexible endoscopes, which undergo repeated cycles of patient-use and reprocessing. The authors developed a novel modeling approach using the MBEC (Minimum Biofilm Eradication Concentration) system to evaluate microbial survival in BBF. The model mimics the cumulative effect of reprocessing protocols, including drying, disinfectant exposure, and re-exposure to the test organism. Glutaraldehyde (GLUT) and accelerated hydrogen peroxide (AHP) were evaluated for their effectiveness in killing microbes in traditional biofilm (TBF) and BBF. Key findings include: - The combination of an organic matrix and aldehyde disinfection (GLUT) quickly produced a protective BBF that facilitated high levels of microbial survival. - In cross-linked BBF formed under high nutrient conditions, maximum colony-forming units (CFU) reached ~6 Log10 CFU/peg. - Organism survival was significantly higher in BBF compared to TBF, especially when glutaraldehyde was used. - The rate of biofilm survival post high-level disinfection (HLD) for E. faecalis and P. aeruginosa in BBF was 10 and 8.6, respectively, compared to a survival rate in TBF of ~2 for each organism. - Indirect outgrowth testing demonstrated the presence of surviving organisms in both TBF and BBF when glutaraldehyde was used. - AHP was less effective in eliminating microbial survival in BBF compared to glutaraldehyde. The study concludes that the BBF model demonstrates for the first time that survival of a wide range of microorganisms occurs in BBF, with significantly more rapid outgrowth compared to TBF. This highlights the need for meticulous cleaning and reprocessing of flexible endoscopes to prevent the accumulation of organic material and microorganisms, which can hinder effective disinfection. The data suggests that the assurance of effective high-level disinfection may decrease if BBF develops within the channels of flexible endoscopes.