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Biofilm removal: Erroneous methodologies cause even more confusion?

Published:October 22, 2015DOI:https://doi.org/10.1016/j.ajic.2015.04.211
      To the Editor:
      We would like to bring the readers' attention to the gross experimental error in the article “Evaluation of detergents and contact time on biofilm removal from flexible endoscopes” by Ren et al
      • Ren W.
      • Sheng X.
      • Huang X.
      • Zhi F.
      • Cai W.
      Evaluation of detergents and contact time on biofilm removal from flexible endoscopes.
      that completely invalidates the results and conclusions of the article.
      It appears the authors overlooked our warning
      • Sava A.
      Biofilm digestion: more confusion than answers.
      on a similar results-invalidating error made by Vickery et al
      • Vickery K.
      • Pajkos A.
      • Cossart Y.
      Removal of biofilm from endoscopes: evaluation of detergent efficiency.
      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.
      • Fazlara A.
      • Ekhtelat M.
      The disinfectant effects of benzalkonium chloride on some important foodborne pathogens.
      The Intercept detergent (tested by Ren et al
      • Ren W.
      • Sheng X.
      • Huang X.
      • Zhi F.
      • Cai W.
      Evaluation of detergents and contact time on biofilm removal from flexible endoscopes.
      ) contains 4.8% BAC,

      INTERCEPT® Detergent Safety Data Sheet. (2014). Available from: http://www.medivators.com/customer-support/material-safety-data-sheets/endoscopy-msds Accessed October 13, 2015.

      or 480 ppm, at use dilution of 1:100. In other words, the authors disregarded the presence of interfering substance at levels of approximately 10 times greater than the MBC. Our previous letter
      • Sava A.
      Biofilm digestion: more confusion than answers.
      was pointing at the same experimental error when Vickery et al
      • Vickery K.
      • Pajkos A.
      • Cossart Y.
      Removal of biofilm from endoscopes: evaluation of detergent efficiency.
      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

      U.S. Environmental Protection Agency. Standard operating procedure for single tube method for measuring disinfectant efficacy against biofilm grown in the CDC biofilm reactor. (2013). Available from: http://www2.epa.gov/sites/production/files/2014-11/documents/mb-20-01.pdf. Accessed October 13, 2015.

      ) 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
      • Ren W.
      • Sheng X.
      • Huang X.
      • Zhi F.
      • Cai W.
      Evaluation of detergents and contact time on biofilm removal from flexible endoscopes.
      (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,”

      U.S. Environmental Protection Agency. Determining if a cleaning product is a pesticide under FIFRA. (2015). Available from: http://www2.epa.gov/pesticide-registration/determining-if-cleaning-product-pesticide-under-fifra. Accessed October 13, 2015.

      —the surface covered by easily accessible single cells as in Fig 1B
      • Ren W.
      • Sheng X.
      • Huang X.
      • Zhi F.
      • Cai W.
      Evaluation of detergents and contact time on biofilm removal from flexible endoscopes.
      is far more desirable than the dense clustered nutrient-rich conglomerates of Fig 1A.
      • Ren W.
      • Sheng X.
      • Huang X.
      • Zhi F.
      • Cai W.
      Evaluation of detergents and contact time on biofilm removal from flexible endoscopes.
      In other words, when using the U.S. EPA criteria, the rating of the cleaners and conclusion of the study should be exactly opposite to the one made by the authors.
      Figure thumbnail gr1
      Fig 1Scanning Electron Micrograph micrographs taken from Ren et al
      • Ren W.
      • Sheng X.
      • Huang X.
      • Zhi F.
      • Cai W.
      Evaluation of detergents and contact time on biofilm removal from flexible endoscopes.
      indicating (A) disperse and hydrated cells and (B) clustered and dehydrated dead cell bodies.
      The authors
      • Ren W.
      • Sheng X.
      • Huang X.
      • Zhi F.
      • Cai W.
      Evaluation of detergents and contact time on biofilm removal from flexible endoscopes.
      have also chosen to deviate significantly from standard methods,

      ISO. ISO 15883–5F: Test soils and methods for demonstrating cleaning efficiency. Annex F - Test soil and method for flexible endoscopes. 21–24 (2005). Available from: http://www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.htm?csnumber=41175. Accessed October 13, 2015.

      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
      • Ren W.
      • Sheng X.
      • Huang X.
      • Zhi F.
      • Cai W.
      Evaluation of detergents and contact time on biofilm removal from flexible endoscopes.
      and Vickery et al
      • Vickery K.
      • Pajkos A.
      • Cossart Y.
      Removal of biofilm from endoscopes: evaluation of detergent efficiency.
      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.
      • Terhune C.
      • Petersen M.
      Scope maker Olympus faces scrutiny over patient deaths, infections.

      References

        • Ren W.
        • Sheng X.
        • Huang X.
        • Zhi F.
        • Cai W.
        Evaluation of detergents and contact time on biofilm removal from flexible endoscopes.
        Am J Infect Control. 2013; 41: 89-92
        • Sava A.
        Biofilm digestion: more confusion than answers.
        Am J Infect Control. 2005; 33: 614
        • Vickery K.
        • Pajkos A.
        • Cossart Y.
        Removal of biofilm from endoscopes: evaluation of detergent efficiency.
        Am J Infect Control. 2004; 32: 170-176
        • Fazlara A.
        • Ekhtelat M.
        The disinfectant effects of benzalkonium chloride on some important foodborne pathogens.
        Am J Agric Environ Sci. 2012; 12: 23-29
      1. INTERCEPT® Detergent Safety Data Sheet. (2014). Available from: http://www.medivators.com/customer-support/material-safety-data-sheets/endoscopy-msds Accessed October 13, 2015.

      2. U.S. Environmental Protection Agency. Standard operating procedure for single tube method for measuring disinfectant efficacy against biofilm grown in the CDC biofilm reactor. (2013). Available from: http://www2.epa.gov/sites/production/files/2014-11/documents/mb-20-01.pdf. Accessed October 13, 2015.

      3. U.S. Environmental Protection Agency. Determining if a cleaning product is a pesticide under FIFRA. (2015). Available from: http://www2.epa.gov/pesticide-registration/determining-if-cleaning-product-pesticide-under-fifra. Accessed October 13, 2015.

      4. ISO. ISO 15883–5F: Test soils and methods for demonstrating cleaning efficiency. Annex F - Test soil and method for flexible endoscopes. 21–24 (2005). Available from: http://www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.htm?csnumber=41175. Accessed October 13, 2015.

        • Terhune C.
        • Petersen M.
        Scope maker Olympus faces scrutiny over patient deaths, infections.
        Los Angeles Times, 2015 (Available from: http://www.latimes.com/business/la-fi-olympus-outbreak-20150302-story.html#page=1. Accessed October 13, 2015.)