Summary of Enterococcus faecalis Modulates Immune Activation and Slows Healing During Wound Infection

• Enterococcus faecalis (E. faecalis) is frequently found in wounds, but its pathogenic mechanisms are not well understood.
• A mouse wound model was used to study E. faecalis infection dynamics.
• Initial inoculum dose determines the infection state:
  • Low dose: short-term, low-titer colonization.
  • High dose: acute replication and long-term persistence, immune cell infiltration, suppressed inflammatory cytokines, and delayed healing.

Key Findings

• Minimum Colonization Dose:
  • The colonization dose (CD50) for E. faecalis in wounds is $5.8 \times 10^1$ CFU.
  • The CD90 was $6.2 \times 10^2$ CFU.
  • $10^6$ CFU defined as infectious dose (ID90).
  • At $10^6$ CFU, macroscopic inflammation and wound exudates are observed.
• High-titer E. faecalis infections lead to bacterial persistence in wounds.
  • With $10^6$ CFU, bacteria rapidly increase to $10^8$ CFU by 8 hours post-inoculation (hpi), then decrease to $10^5$ CFU by 3 days post-inoculation (dpi), maintaining this level throughout the experiment.
• Multiple peptide resistance factor (MprF) contributes to E. faecalis fitness during wound infection.
  • A ΔmprF1/2 mutant is less fit during co-infection at 3 dpi.
• E. faecalis forms microcolonies on the wound surface.
  • Scanning electron microscopy (SEM) shows microcolonies encased in a matrix at 8 hpi.
  • Fluorescence in situ hybridization (FISH) reveals microcolonies at the wound edge and bed at 3 dpi.
• High-titer E. faecalis infection delays wound closure.
  • Histology shows hyperthickened epidermis, many polymorphonuclear leukocytes, and impaired granulation tissue formation at 7 dpi.
• E. faecalis can persist within wounds, escaping immune clearance.
  • Wounds infected with $10^6$ CFU show higher levels of inflammatory cytokines (IL-1b) and factors/chemokines (CSF3, CXCL1, CCL2, CCL3, CCL4) at 8 hpi.
  • At 3 dpi, reduced levels of IL-2, IL-5, IL-10, IL12-p70, CCL11, IFN-γ, and CSF2 are observed.
  • Principal component analysis (PCA) highlights differences in IL-1β, IL-2, IL-12p70, and CCL11.
  • Flow cytometry indicates increased immune cell types in infected wounds. Neutrophil infiltration correlates with neutrophil-related chemokine expression.

Methods

• Genetic manipulation involved creating deletion mutants (ΔmprF1, ΔmprF2, ΔmprF1/2).
• Mouse wound excisional model used male C57BL/6 mice.
• Competitive index (CI) calculated as: $CI = \frac{OG1RF<em>{output} / OG1X</em>{output}}{OG1RF<em>{input} / OG1X</em>{input}}$
• E. faecalis detected with oligonucleotide probe: 5′-GGTGTTGTTAGCATTTCG/Cy3/-3′

Discussion

• Acute high-titer E. faecalis wound infection is associated with a host immune response and inflammation, delaying wound healing.
• An MprF null strain is attenuated at 3 dpi, suggesting its role in immune defense.
• E. faecalis wound infection results in immunomodulation, with decreased cytokine levels at 3 dpi.
• Further studies needed to understand E. faecalis pathogenesis and its impact on wound healing.