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in 2004 that before using protocols based on enumerating bacterial survivors, the absence of cytotoxicity of the tested detergent must be confirmed. It is simply wrong to equate the killing of bacteria to the removal of the biofilm from the surface.
The minimum bactericidal concentration (MBC) of benzalkonium chloride (BAC) on Escherichia coli is approximately 45 ppm.
were assaying biofilms by enumerating survivors after exposure of the biofilm to 1,000 ppm of BAC (>20 times greater than the MBC). To confirm, we performed a quick test on the MBC of BAC against E coli (as per the U.S. Environmental Protection Agency [EPA] methodology
) and confirmed that there were no survivors when approximately 200 organisms of E coli (ACCC 8196) were exposed to 100 ppm of BAC (B6295 Sigma) for 2 minutes. It is of no surprise that both groups of researchers have not recovered any survivors after exposing biofilms to the interfering substance at concentrations of 10-20 times the MBC. All AOAC, European Standard, and U.S. EPA test protocols emphasize the need for cytotoxicity validation, and it is rather surprising that the authors overlooked this textbook validation step.
Strong cationic detergents such as BAC (used in both Matrix and Intercept formulations; Sigma Aldrich, St. Louis, MO) result in dense clusters of dead cells as can be seen in the Scanning Electron Micrographs images from Ren et al
(Fig 1A and Fig 1B). These clusters form pockets of protein and carbohydrate-rich bioburden, which potentially interferes with the subsequent disinfection-sterilization step. The thicker the bioburden, the greater the probability of failure. If one evaluate the distribution of bioburden using the U.S. EPA criteria for biofilm cleaners—“prepares the surface for application of a registered disinfectant intended to kill biofilm,”
with respect to replacing the nutrient-poor conditions for nutrient-rich conditions and replacing the validated recommended test organism (biofilm forming strain of Pseudomonas aeruginosa) for E coli. Both deviations appear rather unwarranted: it is widely accepted that the biofilm in endoscope lumens is most likely to occur during storage when airborne P aeruginosa is contaminating washed and disinfected (ie, nutrient poor) lumen. The scenario envisaged by the authors—biofilm resulting from E coli from patient feces—is extremely unlikely.
The biofilm removal and accessibility of bacteria to disinfectant action should be regarded as a major risk when assessing the feasibility of replacing manual brushing with automated reprocessing in automatic endoscope reprocessors. The research on removal of biofilms is expensive, time consuming, and limited to specialized laboratories only. This is why the wider infection control community heavily relies on the published results—the conclusions of the articles similar to Ren et al
are copied-and-pasted into process risk assessment reports and product marketing sheets. The erroneous test methodologies result in erroneous conclusions that in turn lead to underestimating the risks and might cause major outbreaks, similar to the recent incident at University of California, Los Angeles.