Activity of Hospital Disinfectants against Vegetative Cells and Spores of Clostridioides difficile Embedded in Biofilms

Abstract

<i>Clostridioides difficile</i> spores can survive in the environment in either mono- or mixed-species biofilms. However, no previous studies have investigated chemical disinfection of <i>C. difficile</i> spores embedded in biofilms. Thus, the purpose of this study was to assess the <i>in vitro</i> effectiveness of hospital disinfectants against <i>C. difficile</i> spores embedded within biofilms. Five unique <i>C. difficile</i> strains embedded in three different biofilm types grown for 72 or 120 h were exposed to seven different hospital disinfectants. <i>C. difficile</i> abundance [as log(number of CFU/milliliter)] was calculated after manufacturer-determined contact times along with biofilm biomass and microscopy. The primary analysis compared differences between <i>C. difficile</i> vegetative cell and spore counts as well as amounts of biomass after exposure to disinfectants. <i>C. difficile</i> vegetative cells and spores were recovered from biofilms regardless of the type of biofilm growth or biofilm growth time. No disinfectant was able to completely eliminate <i>C. difficile</i> from the biofilms. Overall, Clorox, <i>ortho</i>-phthalaldehyde (OPA), and Virex were most effective at killing <i>C. difficile</i> spores regardless of biofilm age, ribotype, or wash conditions (whether biofilms are washed or unwashed) (<i>P</i> = 0.001, each). Clorox and OPA were also effective at killing total vegetative cell growth (<i>P</i> = 0.001, each), but Virex was found to be ineffective against vegetative cell growth in biofilms (<i>P</i> = 0.77). Clorox and Virex were most effective in reducing biomass, followed by Nixall, OPA, and Vital Oxide. No disinfectant was able to completely eliminate <i>C. difficile</i> embedded within biofilms although differences among disinfectants were noted. Future research will be required to determine methods to eradicate this persister reservoir.