Shotgun metagenomic sequencing analysis of ocular surface microbiome in Singapore residents with mild dry eye.

The ocular surface microbiome has implications for ocular surface inflammation and immunology. Previous shotgun metagenomics analyses were performed in China, showing results that differed according to environment and age. Patients with Sjogren's syndrome were reported to have altered conjunctival microbiome, but such studies have not been done in milder dry eye. The aim of this study is to describe the conjunctival microbiome in people with mild dry eye in Singapore. Samples were collected from 14 participants with mild dry eye and 10 age-matched comparison participants recruited from Singapore National Eye Centre (SNEC) clinics. Shotgun metagenomic sequencing analysis was employed to evaluate the conjunctival microbiome composition. Proteobacteria formed the predominant phylum in the conjunctiva. As in a study from a coastal city in China, Achromobacter spp. was numerically most abundant. Compared to age-matched controls, the conjunctival microbial composition in mild dry eye was similar. Several microorganisms, including Streptococcus spp. increased in representation with age, and the abundance of Staphylococcus correlated with Schirmer readings. In addition, when cultured corneal epithelial cells were exposed to three strains of Achromobacter xylosoxidans, cytokines such as TNF-α and IL-6 were upregulated in the cell lysates and supernatants. Ourresults suggest that age is an important factor that affects composition of the conjunctival microbiome, and relative abundance of specific microorganism may vary according to the environment of the human host.

Elevated c-di-GMP Levels and Expression of the Type III Secretion System Promote Corneal Infection by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is generally believed to establish biofilm-associated infections under the regulation of the secondary messenger c-di-GMP. To evaluate P. aeruginosa biofilm physiology during ocular infections, comparative transcriptomic analysis was performed on wild-type P. aeruginosa PAO1, a ΔwspF mutant strain (high c-di-GMP levels), and a plac-yhjH-containing strain (low c-di-GMP levels) from mouse corneal infection, as well as in vitro biofilm and planktonic cultures. The c-di-GMP content in P. aeruginosa during corneal infection was monitored using a fluorescent c-di-GMP reporter strain. Biofilm-related genes were induced in in vivo PAO1 compared to in vitro planktonic bacteria. Several diguanylate cyclases and phosphodiesterases were commonly regulated in in vivo PAO1 and in vitro biofilm compared to in vitro planktonic bacteria. Several exopolysaccharide genes and motility genes were induced and downregulated, respectively, in in vivo PAO1 and the in vivo ΔwspF mutant compared to the in vivo plac-yhjH-containing strain. Elevation of c-di-GMP levels in P. aeruginosa began as early as 2 h postinfection. The ΔwspF mutant was less susceptible to host clearance than the plac-yhjH-containing strain and could suppress host immune responses. The type III secretion system (T3SS) was induced in in vivo PAO1 compared to in vitro biofilm bacteria. A ΔwspF mutant with a defective T3SS was more susceptible to host clearance than a ΔwspF mutant with a functional T3SS. Our study suggests that elevated intracellular c-di-GMP levels and T3SS activity in P. aeruginosa are necessary for establishment of infection and modulation of host immune responses in mouse cornea.

Reactive oxygen species drive evolution of pro-biofilm variants in pathogens by modulating cyclic-di-GMP levels.

The host immune system offers a hostile environment with antimicrobials and reactive oxygen species (ROS) that are detrimental to bacterial pathogens, forcing them to adapt and evolve for survival. However, the contribution of oxidative stress to pathogen evolution remains elusive. Using an experimental evolution strategy, we show that exposure of the opportunistic pathogen Pseudomonas aeruginosa to sub-lethal hydrogen peroxide (H2O2) levels over 120 generations led to the emergence of pro-biofilm rough small colony variants (RSCVs), which could be abrogated by l-glutathione antioxidants. Comparative genomic analysis of the RSCVs revealed that mutations in the wspF gene, which encodes for a repressor of WspR diguanylate cyclase (DGC), were responsible for increased intracellular cyclic-di-GMP content and production of Psl exopolysaccharide. Psl provides the first line of defence against ROS and macrophages, ensuring the survival fitness of RSCVs over wild-type P. aeruginosa Our study demonstrated that ROS is an essential driving force for the selection of pro-biofilm forming pathogenic variants. Understanding the fundamental mechanism of these genotypic and phenotypic adaptations will improve treatment strategies for combating chronic infections.