Doctor of Philosophy (PhD)
Biomedical and Veterinary Medical Sciences - Veterinary Clinical Sciences
Escherichia coli biofilm formation is believed to be a significant cause of chronic equine endometritis, but limited studies exist to support this theory. In total 130 equine reproductive tract E. coli were collected, with 35 isolates showing strong in vitro biofilm formation (OD570 > 0.4). Changing incubation conditions did not significantly alter the distribution of E. coli between biofilm-forming groups, and strong biofilm formation began as early as 4 hours after initiation of incubation. The collected uterine isolates could be screened for biofilm formation using an adapted crystal violet (CV) assay consisting of chromogenic agar incubation and polyvinyl chloride conical tubes, with results demonstrating an in-house technique for screening isolates for biofilm formation. The strong biofilm-forming uterine E. coli showed a 7- to 11-fold increase over corresponding planktonic state in antibiotic resistance to ampicillin, ticarcillin, ceftiofur, gentamicin, amikacin, tobramycin, polymixin B and enrofloxacin. The exposure of certain biofilm-forming uterine E. coli isolates to 12.5% and 25% sub-MIC ceftiofur concentrations resulted in a significant dose-dependent increase in biofilm biomass after 24 hours incubation. All E. coli isolates evaluated (total = 101) were shown to carry the genes for Type-1 fimbriae, S-fimbriae, curli and the PGA polysaccharide, all of which are associated with biofilm formation. When exposed to endometrial explants E. coli adherence occurred rapidly and early stages of biofilm formation were visible on endometrial surfaces using SEM. Attempts to demonstrate uropathogenic E.coli associated cellular internalization were unsuccessful, but instead showed that adhered E. coli acquire biofilm associated antibiotic resistance rapidly after adhering to endometrial surfaces. In conclusion these experiments have shown that approximately 30% of equine reproductive tract E. coli can form strong in vitro biofilm, and show significant resistance to commonly used equine reproductive antibiotics. Also there is potential for increased biofilm formation to occur after exposure to low-level antibiotic exposure. The CV assay is an important screening assay for biofilm formation and can be adapted for use by clinicians in equine practice. The results of these experiments have shown the need to include biofilm-screening assays in equine practice for effective treatment of chronic endometritis.
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Beehan, David Paul, "The Investigation of Biofilm Formation by Equine Reproductive Tract Escherichia Coli" (2016). LSU Doctoral Dissertations. 4444.