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L. Wiley, S. Jamerson, L. A. Wiley, T. Elliott, J. Olson; Analysis of Contact Lens Case Bacterial Microbiota by 16S Ribosomal DNA Sequencing: Predominance of Achromobacter in Patients With Contact Lens Related Keratitis. Invest. Ophthalmol. Vis. Sci. 2009;50(13):6333.
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© ARVO (1962-2015); The Authors (2016-present)
Biofilm formation in contact lens cases may serve as a source of microbial contamination and toxin production, which can contribute to contact lens related ocular disease. This study used 16S ribosomal DNA (rDNA) sequencing to analyze bacterial populations in contact lens cases of patients with contact-lens-related keratitis and corneal ulcers.
Biofilms were removed from lens cases by scraping and sonication, and combined with lens case solutions for DNA extraction, using the Mo Bio Microbial DNA Isolation Kit. Universal bacterial 16S rRNA primers (forward: GAGTTTGATYMTGGCTCAG; reverse: CAGGAAACAGCTATGAC) were used to amplify the 16S rRNA gene, PCR products were purified, cloned into the pCR 4-TOPO vector (Invitrogen), then re-amplified and sequenced. Sequences were classified by BLAST analysis against Genbank. Each sequence was matched with at least one database entry at the species level (identity >97%); results were then clustered at the genus level (identity >95%, or slightly higher for diverse genera).
In analysis of cases from 5 patients with contact-lens-related keratitis, 70 of 125 of the 16S rDNA clones were identified as Achromobacter. Bacterial diversity from keratitis patients was low, averaging 3 different genera per patient, and 2 of 9 species were identified as Gram-positive. In analysis of cases from 3 patients with contact-lens-related ulcers, 1 of 80 of the 16S rDNA clones was Achromobacter. Species diversity from corneal ulcer patients was much greater than that of keratitis patients, averaging 10 different genera per patient and 15 of 24 of the species were Gram-positive.
Bacterial species in lens cases from patients with keratitis had limited diversity and were dominated by Gram-negative, Achromobacter. Bacterial species in lens cases of patients with corneal ulcers were more diverse and included multiple Gram-positive species. Differences in bacterial microbiota in lens cases of keratitis and ulcer patients may reflect a succession of biofilm species over time, which can, in turn, contribute to disease outcomes.
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