In Vitro Efficacy of Nonantibiotic Treatments on Biofilm Disruption of Gram-Negative Pathogens and an In Vivo Model of Infectious Endometritis Utilizing Isolates from the Equine Uterus

In this study, we evaluated the ability of the equine clinical treatmentsN -acetylcysteine, EDTA, and hydrogen peroxide to disruptin vitro biofilms and kill equine reproductive pathogens (Escherichia coli ,Pseudomonas aeruginosa , orKlebsiella pneumoniae ) isolated from clinical cases.N -acetylcysteine (3.3%) decreased biofilm biomass and killed bacteria within the biofilms ofE. coli isolates. The CFU of recoverableP. aeruginosa andK. pneumoniae isolates were decreased, but the biofilm biomass was unchanged. Exposure to hydrogen peroxide (1%) decreased the biofilm biomass and reduced the CFU ofE. coli isolates,K. pneumoniae isolates were observed to have a reduction in CFU, and minimal effects were observed forP. aeruginosa isolates. Chelating agents (EDTA formulations) reducedE. coli CFU but were ineffective at disrupting preformed biofilms or decreasing the CFU ofP. aeruginosa andK. pneumoniae within a biofilm. No single nonantibiotic treatment commonly used in equine veterinary practice was able to reduce the CFU and biofilm biomass of all three Gram-negative species of bacteria evaluated. Anin vivo equine model of infectious endometritis was also developed to monitor biofilm formation, utilizing bioluminescence imaging with equineP. aeruginosa isolates from this study. Following infection, the endometrial surface contained focal areas of bacterial growth encased in a strongly adherent “biofilm-like” matrix, suggesting that biofilms are present during clinical cases of infectious equine endometritis. Our results indicate that Gram-negative bacteria isolated from the equine uterus are capable of producing a biofilmin vitro , andP. aeruginosa is capable of producing biofilm-like materialin vivo .