Many Escherichia coli (E. coli) strains produce biofilm that confers antimicrobial resistance. However, studies of biofilm production by E. coli from canine pyometra are lacking. Objectives were to elucidate the role of biofilm production by E. coli in pyometra by: (1) assessing the ability of E. coli to produce biofilm in vitro, and (2) confirming biofilm in situ. Endometrial biopsies were obtained from bitches with pyometra and preserved for microscopic analysis (n = 25). An endometrial swab was submitted for aerobic culture. Samples with confirmed E. coli were evaluated further for biofilm production in vitro and in vivo. Seventy percent of cases (16/23) resulted in pure growth of 1 or 2 E. coli strains, totaling 20 isolates. Fifteen isolates (15/20, 75%) had higher optical densities then negative controls (P < .05). On histopathology, all tissues exhibited endometrial inflammation and mucus was located within endometrial glands and occasionally overlying epithelium on 14 slides (14/16, 88%). Bacteria was noted in 50% of slides (8/16). During FISH acellular debris within the uterine lumen consistent with biofilm was noted on 94% of samples (15/16) and E coli was positively identified on all samples (15/15). Areas suggestive of the presence of biofilm were observed on all samples on scanning electron microscopy; but, bacteria consistent with E. coli were only visualized in 9 samples (9/16, 56%). In conclusion, we demonstrated that relevant strains of E. coli produce biofilm in vitro and in vivo, which may be considered in the development of new pyometra treatments aimed at disrupting these E. coli biofilm.

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