Biofilm-Forming Potential of Ocular Fluid Staphylococcus aureus and Staphylococcus epidermidis on Ex Vivo Human Corneas from Attachment to Dispersal Phase

Abstract

The biofilm-forming potential of Staphylococcus aureus and Staphylococcus epidermidis, isolated from patients with Endophthalmitis, was monitored using glass cover slips and cadaveric corneas as substrata. Both the ocular fluid isolates exhibited biofilm-forming potential by the Congo red agar, Crystal violet and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-(phenylamino) carbonyl-2H-tetra-zolium hydroxide (XTT) methods. Confocal microscopy demonstrated that the thickness of the biofilm increased from 4-120 h of biofilm formation. Scanning electron microscopic studies indicated that the biofilms grown on cover slips and ex vivo corneas of both the isolates go through an adhesion phase at 4 h followed by multilayer clumping of cells with intercellular connections and copious amounts of extracellular polymeric substance. Clumps subsequently formed columns and eventually single cells were visible indicative of dispersal phase. Biofilm formation was more rapid when the cornea was used as a substratum. In the biofilms grown on corneas, clumping of cells, formation of 3D structures and final appearance of single cells indicative of dispersal phase occurred by 48 h compared to 96-120 h when biofilms were grown on cover slips. In the biofilm phase, both were several-fold more resistant to antibiotics compared to planktonic cells. This is the first study on biofilm forming potential of ocular fluid S. aureus and S. epidermidis on cadaveric cornea, from attachment to dispersal phase of biofilm formation.

Keywords: S. aureus; S. epidermidis; antimicrobial resistance; biofilm; eye; ocular fluid.

Figure 1

Biofilm formation in ocular fluid Staphylococcus aureus L-1054/2020(2) (A) and S. epidermidis L-1058/2020(2) (B) by the Congo red agar method (A,B), crystal violet method (C) and XTT method (D). E. coli ATCC 25922 was used as a negative control and S. aureus 25923 was used as a positive control. Each bar in (C,D) represent average value ± standard deviation. Experiments were performed in triplicates. Significance was calculated against 4 h biofilm using statistical analysis such as unpaired t-test and p value calculation. Experiments were performed in triplicates. * Indicates significant increase in biofilm formation compared to 4 h based on t-test and p value calculation. (p ≤ 0.05).

Figure 2

Temporal increase in thickness of biofilm in ocular Staphylococcus aureus L-1054/2020(2) (A) and S. epidermidis L-1058/2020(2) (B) by confocal scanning laser microscopy monitored between 4 to 120 h of biofilm growth on cover slips. The biofilm was stained with Syto9^® and Calcofluor white M2R. Viable cells appear green in color and EPS appears blue in color.

Figure 3

Scanning Electron Microscopy monitoring of biofilm formation in ocular surface Staphylococcus aureus L-1054/2020(2) between 4 to 120 h of biofilm growth on a cover slip. Single arrows represent intercellular connections (4–120 h), E represents EPS (72–120 h), C represents a column (24 and 48 h), M represents micro-colony (24 h) and W represents a water channel (72 h). Inset at 4 and 96 h represent intercellular connections and inset at 72 h represents EPS.

Figure 4

Scanning Electron Microscopy monitoring of biofilm formation in ocular Staphylococcus epidermidis L-1058/2020(2) between 4 to 120 h of biofilm growth on a cover slip. Single arrow represents intercellular connections (4–120 h), C represents a column (48–120 h), E represent EPS (48–120 h) and M represents microcolony (4–72 h).

Figure 5

Scanning Electron Microscopy monitoring of biofilm formation in ocular Staphylococcus aureus L-1054/2020(2) between 4 to 120 h of biofilm growth on cadaveric cornea. Single arrow represents intercellular connections (4–48 h), C represent a column (4, 24, 48 and 96 h), E represents EPS (24–120 h), M represents microcolony (4 h) and W represents a water channel (24 and 96 h).

Figure 6

Scanning Electron Microscopy monitoring of biofilm formation in ocular surface Staphylococcus epidermidis L-1058/2020(2) between 4 to 120 h of biofilm growth on cadaveric cornea. Single arrow represents intercellular connections (4–48 h) (see inset at 4 h), C represents a column (24–72 h) and E represents EPS (24–120 h).