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

Abstract

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.

FIG 1

Bioluminescent imaging of the equine uterus at 5 days postinoculation with lux-labeled P. aeruginosa. Luminescence was detected upon opening the uterine body, indicating the abundant presence of lux-labeled P. aeruginosa on the endometrial surface and in the intraluminal fluid pooling at the bottom left of the image. Min, minimum; Max, maximum.

FIG 2

Bioluminescent imaging of the equine uterus at 5 days postinoculation with lux-labeled P. aeruginosa. Luminescence was detected following repeated washing of the endometrial surface of the uterine body, indicating that the bioluminescent biofilm-like material produced by lux-labeled P. aeruginosa was strongly adherent and difficult to remove from the endometrium.