In this study, we evaluated the ability of the equine clinical treatments N-acetylcysteine, EDTA, and hydrogen peroxide to disrupt in vitro biofilms and kill equine reproductive pathogens (Escherichia coli, Pseudomonas aeruginosa, or Klebsiella pneumoniae) isolated from clinical cases. N-acetylcysteine (3.3%) decreased biofilm biomass and killed bacteria within the biofilms of E. coli isolates. The CFU of recoverable P. aeruginosa and K. pneumoniae isolates were decreased, but the biofilm biomass was unchanged. Exposure to hydrogen peroxide (1%) decreased the biofilm biomass and reduced the CFU of E. coli isolates, K. pneumoniae isolates were observed to have a reduction in CFU, and minimal effects were observed for P. aeruginosa isolates. Chelating agents (EDTA formulations) reduced E. coli CFU but were ineffective at disrupting preformed biofilms or decreasing the CFU of P. aeruginosa and K. 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. An in vivo equine model of infectious endometritis was also developed to monitor biofilm formation, utilizing bioluminescence imaging with equine P. 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 biofilm in vitro, and P. aeruginosa is capable of producing biofilm-like material in vivo. Bacterial endometritis is an important cause of subfertility in mares (1). Endometrial infections are reported in 25 to 60% of mares who fail to become pregnant following breeding (1), contributing to a major economic loss for the equine industry (1, 2). The most common species of bacteria identified during the clinical diagnosis of bacterial endometritis include Streptococcus equi subsp. zooepidemicus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa (3, 4). The isolation of one of these species of bacteria is considered to be clinically relevant due to observed reductions in pregnancy rates after identification (2). The detection of these bacteria in the uterus would result in treatment with uterine lavage and broad-spectrum antibiotics to reduce bacterial load and eradicate the remaining bacteria (5, 6).Bacterial endometritis that is refractory to traditional antimicrobial treatment is a significant challenge in the equine breeding industry (4). One possible explanation often cited for the failure of antibiotic treatment is the growth of bacterial pathogens in a biofilm (1, 5). Uterine isolates of E. coli, P. aeruginosa, and K. pneumoniae have been proposed to most likely be associated with a biofilm, due to the observation of repeated antibiotic treatment failures in equine reproducti...

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