OCULAR SURFACE MICROBIOTA IN NAÏVE KERATOCONUS: A MULTICENTER VALIDATION STUDY
Published 2025 - 29th ESCRS Winter Meeting
Reference: PO164 | Type: Free Paper | DOI: 10.82333/t92j-9j52
Authors: Carlos Rocha Rocha-De-Lossada* 1 , Cosimo Mazzotta 2 , Federico Gabrielli 3 , Filomena Tiziana Papa 3 , Carmen Gómez-Huertas 4 , José-María Sánchez-González 5 , Miguel Rechichi 6 , Giovanni Rubegni 7 , Davide Borroni 8
1Hospital Regional Universitario de Malaga,Malaga,Spain, 2Siena Crosslinking Center,Siena,Italy, 3Biolab SRL, Laboratorio di Genetica e Genomica Molecolare,Ascoli,Italy, 4Hospital Virgen de las Nieves,Granada,Spain, 5Department of Physics of Condensed Matter, Optics Area,Universidad de Sevilla,Sevilla,Spain, 6Centro Polispecialistico Mediterraneo,Sellia Marina,Italy, 7Postgraduate Ophthalmology School, University of Siena,Siena,Italy, 8Eyemetagenomics Ltd., 71–75, Shelton Street, Covent Garden, London,Milano,Italy
Purpose
This study aims to characterize the ocular microbiota of patients diagnosed with keratoconus who have not undergone any prior surgical treatment using the mNGS 16S rRNA sequencing method.
Setting
Patients were recruited in the Opthalmology department of Virgen de las Nieves University Hospital, Siena Crosslinking Center, Siena, Italy, and Centro Oculistico Borroni (Gallarate, Italy) from January–October 2022
Methods
Samples of naïve keratoconus patients were collected with an eNAT with 1 mL of Liquid Amies Medium. The eNAT was applied on the inferior surface of the eye and moved two times, “limbus to fornix to limbus”. Microbial DNA from keratoconus and healthy control samples were standardly isolated using QIAamp DNA Microbiome Kit.
The Microbial DNA was checked and quantified using Genomic DNA ScreenTape at Agilent TapeStation 4150. DNA libraries were set with Ion 16S Metagenomics Kit. Amplified products were purified using AMPure XP beads and end-repaired for barcode ligation. Later, libraries were verified for quality and quantified using Agilent High Sensitivity D1000 ScreenTape (Agilent Tech- nologies, USA). Equimolar libraries were pooled together at the final concentration of 5 pM. Template preparations were performed with Ion Chef according to the Ion 540 Kit-Chef protocol (Thermo Fisher, Waltham, MA, USA). Raw reads were analyzed with GAIA
Results
The microbiota analysis showed a relative abundance of microorganisms at the phylum level in each sample collected from 38 patients with KC and 167 healthy controls. A comparison between healthy control and keratoconus samples identified two genera unique to keratoconus, Pelomonas and Ralstonia.
The Chao1 index, a measure of species richness, revealed that the KC group has a lower microbial richness. Conversely, the control group demonstrated a broader distribution of Chao1 values, indicating a higher microbial richness and suggesting a more diverse and potentially more resilient microbiota. Similarly, the Shannon diversity index, revealed a moderate level of diversity in the KC group, while the control group displayed a higher level of diversity.
Conclusion
Our findings suggest that alterations in the microbiota may play a role in the complex scenario of KC development.