Molecular typing, biofilm characteristics and biofilm-targeted inhibition strategies of Streptococcus equi subsp. zooepidemicus isolated from donkey endometritis.

Overview

• This study investigated the biofilm-forming behavior of Streptococcus equi subsp. zooepidemicus (SEZ) strains isolated from donkeys with endometritis and evaluated various antibiotic and non-antibiotic combinations for inhibiting these biofilms.
• Results revealed strain-specific differences in biofilm inhibition and showed that combinations of antibiotics with agents like Tris-EDTA or DMSO could effectively disrupt SEZ biofilms, suggesting new treatment strategies for managing endometritis in donkeys.

Background and Purpose

• Endometritis, an inflammation of the uterus lining, is a significant cause of infertility in intensively farmed donkeys.
• SEZ is a predominant pathogen in equine endometritis and is known for forming biofilms—complex bacterial communities adhering to surfaces—which contribute to treatment resistance.
• The goal was to characterize the biofilm patterns of SEZ isolated from donkeys and to assess potential anti-biofilm strategies using various drug combinations.

Methods

• Sample Collection: Uterine lavage fluids were collected from 30 Dezhou Black donkeys diagnosed with clinical endometritis.
• Bacterial Isolation and Identification: Samples were cultured on blood agar and MacConkey agar, followed by 16S rDNA PCR sequencing to confirm SEZ isolates.
• Antimicrobial Sensitivity Testing: Broth microdilution methods were used to determine the susceptibility profiles of the SEZ isolates against antibiotics.
• Strain Typing: Multilocus sequence typing (MLST) classified the isolates into different genetic types to understand strain diversity.
• Biofilm Assessment: In vitro biofilm formation was studied over time using crystal violet staining to quantify biomass.
• Biofilm Inhibition Testing: Six agents—Tris-EDTA, hydrogen peroxide, DMSO, penicillin potassium, ceftiofur, and ciprofloxacin—were tested alone and in combinations for their ability to inhibit biofilm formation or disrupt established biofilms.

Key Findings

• Prevalence and Diversity: Out of 31 total bacterial isolates, 13 (41.9%) were identified as SEZ.
• Strain Types: Four novel MLST sequence types were identified—named STnovel, ST554, ST555, and ST556—indicating genetic diversity.
• Biofilm Formation: SEZ strains formed biofilms rapidly within 12 to 24 hours, highlighting the importance of timely intervention.
• Strain-Dependent Inhibition:
  • Hydrogen peroxide showed the strongest inhibition against biofilms formed by the STnovel strain (about 74% inhibition).
  • Combination therapies were more effective on other strains:
    • Ceftiofur combined with Tris-EDTA inhibited ST554 biofilms by approximately 67%.
    • Ciprofloxacin combined with DMSO inhibited ST555 biofilms by about 83%.
    • Penicillin combined with DMSO inhibited ST556 biofilms by approximately 83%.

Conclusions and Implications

• The study demonstrated that SEZ biofilm inhibition varies significantly depending on the strain, emphasizing the need for tailored therapeutic approaches.
• Combining antibiotics with non-antibiotic agents like Tris-EDTA or DMSO can enhance anti-biofilm effects, offering promising new strategies for treating SEZ-related endometritis in donkeys.
• These findings could lead to improved clinical management by overcoming biofilm-associated antibiotic resistance and reducing infertility caused by persistent uterine infections.