Phylogenetic diversity, functional pathways, and network interactions of ocular chlamydia-like organisms (CLOs) in...

Trachoma is the leading infectious cause of blindness worldwide and classically attributed to Chlamydia trachomatis (Ct). However, other members of the phylum Chlamydiae, particularly environmental chlamydia-like organisms (CLOs), may modulate ocular ecology and influence disease outcomes. Here, we investigated CLO distribution, phylogeny, and microbiome associations among 1,059 individuals from trachoma-endemic communities in Ethiopia using targeted 16S rRNA sequencing and metagenomic shotgun sequencing. CLOs were detected in 249 (23.3%) participants of all ages and sexes and were significantly less likely to be associated with Ct or trachomatous scarring (TS) and trichiasis (TT). Phylogenetic analyses revealed extensive CLO diversity with six novel phylotypes, the most abundant of which was ancestral to Sorochlamydiaceae-a family linking pathogenic Chlamydiaceae, which includes the genus Chlamydia, and symbionts of protists. CLO-positive microbiomes exhibited significantly greater species richness and evenness with distinct differences in community composition relative to CLO-negative microbiomes. These effects were most pronounced among males and older adults. Functional profiling revealed widespread depletion of biosynthetic and metabolic pathways in CLO-positive microbiomes, particularly in participants with TS/TT, suggesting reduced community biosynthetic capacity and niche modification. Species interaction network analyses demonstrated substantial reorganization of microbial associations in the presence of CLOs with increased connectivity and centrality compared to CLO-negative networks. These findings identify CLOs as prevalent, phylogenetically diverse, and ecologically influential members of the microbiome. Their inverse association with Ct and TS/TT underscores the importance of considering intracellular symbionts beyond Ct in understanding conjunctival microbial ecology, resilience, and trachoma pathogenesis and for designing novel control strategies.IMPORTANCETrachoma caused by Chlamydia trachomatis (Ct) remains the leading infectious cause of blindness globally. While control efforts focus exclusively on Ct, other members of the phylum Chlamydiae, such as chlamydia-like organisms (CLOs), inhabit mucosal surfaces but remain understudied in the eye. Using targeted 16S rRNA and metagenomic shotgun sequencing of conjunctival samples from villagers in trachoma-endemic Ethiopia, CLOs were prevalent (23.3%; 249/1,059), phylogenetically diverse, including novel Chlamydiae phylotypes, and inversely associated with both Ct infection and severe scarring disease. CLO microbiomes had increased microbial diversity, altered community composition, depleted metabolic pathway abundance, and reorganized species interaction networks compared to CLO-negative microbiomes. These findings challenge the singular focus on Ct in trachoma control and research and suggest that CLOs represent ecologically significant members of the conjunctival microbiome. Further research on their interactions with ocular microbial communities could reveal new insights into trachoma pathogenesis and inform more holistic approaches to disease control.