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

Ranjith Konduri,Chinthala Reddy Saiabhilash,Sisinthy Shivaji

doi:10.3390/microorganisms9061124

Ranjith Konduri, Chinthala Reddy Saiabhilash + Show 1 more

Open Access

https://doi.org/10.3390/microorganisms9061124

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Journal: Microorganisms	Publication Date: May 22, 2021
Citations: 18	License type: CC BY 4.0

Affiliation: L V Prasad Eye Institute

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.

Highlights

Ocular fluid S. aureus and S. epidermidis grew as black colonies on CRA plates indicative of biofilm formation (Figure 1A,B)
When biofilm formation was monitored by the XTT method it was observed that both S. aureus and S. epidermidis were probably more efficient in biofilm formation potential and by 48–72 h reached peak biofilm formation which was sustained till 120 h (Figure 1D)
This study confirms earlier observations demonstrating that ocular Staphylococcus aureus and S. epidermidis collected from the cornea, conjunctiva, eyelid margin, intraorbital foreign body, intraocular lenses, vitreous and aqueous humors of corneal ulcer patients and from patients with other ocular diseases including endophthalmitis exhibit biofilmforming capacity as determined by CRA [54], Crystal violet (CV) [54,76], and scanning electron microscopy (SEM) methods [55,77,78]

Summary

Introduction

The cornea has an immune surveillance system [5,6] comprised of innate defenses contributed by numerous cellular (corneal epithelial cells, corneal nerves, keratocytes, polymorphonuclear cells, neutrophils, eosinophils, macrophages, NK cells, Langerhans cells, etc.) and molecular elements (components of complement, interferons, interleukins, etc.) to eliminate pathogens [5,6,7] Despite these defense mechanisms, many microorganisms do survive on the ocular surface and recent studies either based on 16S ribosomal RNA (rRNA) gene amplification, cloning and sequencing or by using NGS (next-generation sequencing) based on 16S rRNA gene amplification and analysis (16S rRNA meta-barcoding) revealed a greater degree of diversity and abundance in the bacterial microbiome of the ocular surface [8,9,10,11,12,13]. Keilty [14] was the first to cultivate hemolytic Staphylococcus from the conjunctival swabs of normal subjects

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Conclusion