Characterization of fusarium keratitis outbreak isolates: contribution of biofilms to antimicrobial resistance and pathogenesis

Abstract

Purpose: Fusarium is a major cause of microbial keratitis, and its ability to form biofilms was suggested as a contributing factor in recent outbreaks. We investigated the ability of outbreak Fusarium isolates (F. solani species complex [FSSC] and F. oxysporum species complex [FOSC]) to form biofilms in vitro and in vivo, and evaluated their antifungal susceptibilities.

Methods: Biofilm formation was assessed using our in vitro contact lens model and in vivo murine model. Biofilm architecture was assessed using confocal laser scanning microscopy (CLSM). Susceptibility against amphotericin B (AmB), voriconazole (VCZ), and natamycin (NAT) was determined using the CLSI-M38-A2 method and XTT metabolic assay.

Results: FSSC strains formed more biofilms than FOSC, in a strain- and clade-dependent manner. CLSM analyses revealed that "high biofilm forming" (HBF) strains had denser and thicker biofilms than "low biofilm forming" (LBF) strains of both species (thickness 51 vs. 41 μm for FSSC and 61 vs. 45 μm for FOSC strains, P < 0.05 for both comparisons). Fusarium biofilms exhibited species-dependent antifungal susceptibilities (e.g., FSSC biofilms AmB minimal inhibitory concentrations [MIC] ≥16 μg/mL, while NAT or VCZ MICs were 2-8 μg/mL). FSSC-infected mice had severe corneal opacification independent of biofilm thickness, while FOSC infection resulted in moderate corneal opacification. Corneal fungal burden of mice infected with HBF strains was higher than those of the LBF strains. In contrast, the reference ATCC isolate was unable to cause infection.

Conclusions: The ability to form biofilms is a key pathogenicity determinant of Fusarium, irrespective of the thickness of these biofilms. Further studies are warranted to explore this association in greater detail.

Figure 1.

Comparison of the ability of Fusarium isolates obtained from keratitis outbreak to form biofilms. Metabolic activity (XTT) assay was used to evaluate biofilm formation by isolates belonging to FSCC (A, C) or FOSC (B, D). Panels (A) and (B) show variation of biofilm forming ability by strain type, while panels (C) and (D) present results showing variation in biofilm formation by clade type. Horizontal bars: significantly different comparisons (P ≤ 0.05). MRL8609 and MRL8996 were non-outbreak isolates obtained from keratitis patients.

Figure 2.

Confocal microscopy analysis of biofilms formed by FSSC (A, B) and FOSC (C, D) isolates. Formed biofilms were stained with fluorescent stains FUN-1 (red) and concanavalin A-Alexa Fluor 488 conjugate (green), and examined using a Zeiss LSM510 confocal scanning laser microscope equipped with argon and HeNe lasers, and mounted on a Zeiss Axiovert100 M microscope. All observations were conducted with a water immersion C-apochromat objective. Representative images are shown for (A) FSSC 6914, (B) FSSC 6970, (C) FOSC 6908, and (D) FOSC 6936. Magnification 20×.

Figure 3.

Evaluation of in vivo virulence of Fusarium keratitis outbreak isolates using contact lens-associated biofilms or intrasomal injection. Murine corneas were infected with F. solani or F. oxysporum strains either by (A, B) adding a biofilm-contaminated contact lens to abraded cornea, or (C, D) intrastromal injection with conidia. Corneas of C57BL/6 mice were infected with isolates belonging to FSSC (B6914, B6970), FOSC (B6908, B6936), or ATCC reference strains by abrading the cornea and adding a contact lens containing biofilms or intrasomally. (A, C) Corneal opacification in infected mice, showing representative eyes from two repeat experiments with 5 mice per group. (B, D) Corneal fungal burden of infected mice, expressed as CFUs/eye. Data points represent individual corneas.