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

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.

Alterations in the conjunctival surface bacterial microbiome in bacterial keratitis patients.

Microbial keratitis is an infectious disease of the eye, in which the cornea is inflamed. Under severe conditions, keratitis can lead to significant loss of vision and enucleation of the eye. Ocular trauma is the major risk factor causing keratitis and microorganisms viz., bacteria, fungi, viruses are the causative agents. The current study characterized the conjunctival bacterial microbiomes of healthy individuals and individuals with bacterial keratitis (BK) and assessed whether ocular microbiome dysbiosis is prevalent in BK patients. Ocular bacterial microbiomes were generated from the conjunctival swabs of healthy controls (HC-SW) and conjunctival swabs (BK-SW) and corneal scrapings (BK-CR) of BK patients using V3-V4 amplicon sequencing and data analysed using QIIME and R software. The Alpha diversity indices, diversity and abundance of different phyla and genera, heat map analysis, NMDS plots and inferred functional pathway analysis clearly discriminated the bacterial microbiomes of conjunctival swabs of healthy controls from that of BK patients. Preponderance of negative interactions in the hub genera were observed in BK-CR and BK-SW compared to HC-SW. In addition, a consistent increase in the abundance of pathogenic bacteria, as inferred from published literature, was observed in the conjunctiva of BK patients compared to HC and this may be related to causing or exacerbating ocular surface inflammation. This is the first study demonstrating dysbiosis in the ocular bacterial microbiome of conjunctiva of bacterial keratitis patients compared to the eye of healthy controls. The bacterial microbiome associated with the corneal scrapings of keratitis individuals is also described for the first time.

Phylogenetic Grouping of Human Ocular Escherichia coli Based on Whole-Genome Sequence Analysis.

Escherichia coli is a predominant bacterium in the intestinal tracts of animals. Phylogenetically, strains have been classified into seven phylogroups, A, B1, B2, C, D, E, and F. Pathogenic strains have been categorized into several pathotypes such as Enteropathogenic (EPEC), Enterotoxigenic (ETEC), Enteroinvasive (EIEC), Enteroaggregative (EAEC), Diffusely adherent (DAEC), Uropathogenic (UPEC), Shiga-toxin producing (STEC) or Enterohemorrhagic (EHEC) and Extra-intestinal pathogenic E. coli (ExPEC). E. coli also survives as a commensal on the ocular surface. However, under conditions of trauma and immune-compromised states, E. coli causes conjunctivitis, keratitis, endopthalmitis, dacyrocystitis, etc. The phylogenetic affiliation and the pathotype status of these ocular E. coli strains is not known. For this purpose, the whole-genome sequencing of the 10 ocular E. coli strains was accomplished. Based on whole-genome SNP variation, the ocular E. coli strains were assigned to phylogenetic groups A (two isolates), B2 (seven isolates), and C (one isolate). Furthermore, results indicated that ocular E. coli originated either from feces (enteropathogenic and enterotoxigenic), urine (uropathogenic), or from extra-intestinal sources (extra-intestinal pathogenic). A high concordance was observed between the presence of AMR (Antimicrobial Resistance) genes and antibiotic resistance in the ocular E. coli strains. Furthermore, several virulent genes (fimB to fimI, papB to papX, etc.) and prophages (Enterobacteria phage HK97, Enterobacteria phage P1, Escherichia phage D108 etc.) were unique to ocular E. coli. This is the first report on a whole-genome analysis of ocular E. coli strains.

Alterations in the Ocular Surface Fungal Microbiome in Fungal Keratitis Patients.

Keratitis, an inflammatory disease of the eye, when neglected could lead to sight-threatening complications and ultimately blindness. Globally, over a million people are affected by keratitis annually. Keratitis has a microbial etiology and is caused by bacteria, fungi, viruses, etc. The present study compared the ocular surface fungal microbiome of healthy individuals and individuals with fungal keratitis. Fungal microbiomes from the conjunctival swabs of healthy individuals and from conjunctival swabs and corneal scrapings of individuals with fungal keratitis were generated using ITS2 region amplicons. Microbiomes were sequenced using Illumina MiSeq 2 × 250 base pair chemistry with a paired-end protocol. Based on Alpha diversity indices, phylum and genera level diversity, abundance differences, and heat map analysis, the fungal microbiomes of conjunctival swabs and corneal scrapings of individuals with fungal keratitis exhibited dysbiosis (alterations in the diversity and abundance) compared to the ocular surface microbiome of the healthy control individuals. This is the first report indicating dysbiosis in the fungal microbiome of conjunctival swabs and corneal scrapings in individuals with fungal keratitis. A total of 11 genera present in the majority of the eyes constituted the variable core ocular microbiome.