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      The conjunctival microbiome in health and trachomatous disease: a case control study


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          Trachoma, caused by Chlamydia trachomatis, remains the world’s leading infectious cause of blindness. Repeated ocular infection during childhood leads to scarring of the conjunctiva, in-turning of the eyelashes (trichiasis) and corneal opacity in later life. There is a growing body of evidence to suggest non-chlamydial bacteria are associated with clinical signs of trachoma, independent of C. trachomatis infection.


          We used deep sequencing of the V1-V3 region of the bacterial 16S rRNA gene to characterize the microbiome of the conjunctiva of 220 residents of The Gambia, 105 with healthy conjunctivae and 115 with clinical signs of trachoma in the absence of detectable C. trachomatis infection. Deep sequencing was carried out using the Roche-454 platform. Sequence data were processed and analyzed through a pipeline developed by the Human Microbiome Project.


          The microbiome of healthy participants was influenced by age and season of sample collection with increased richness and diversity seen in younger participants and in samples collected during the dry season. Decreased diversity and an increased abundance of Corynebacterium and Streptococcus were seen in participants with conjunctival scarring compared to normal controls. Abundance of Corynebacterium was higher still in adults with scarring and trichiasis compared to adults with scarring only.


          Our results indicate that changes in the conjunctival microbiome occur in trachomatous disease; whether these are a cause or a consequence is yet unknown.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13073-014-0099-x) contains supplementary material, which is available to authorized users.

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          Bacterial diversity in the oral cavity of 10 healthy individuals.

          The composition of the oral microbiota from 10 individuals with healthy oral tissues was determined using culture-independent techniques. From each individual, 26 specimens, each from different oral sites at a single point in time, were collected and pooled. An 11th pool was constructed using portions of the subgingival specimens from all 10 individuals. The 16S ribosomal RNA gene was amplified using broad-range bacterial primers, and clone libraries from the individual and subgingival pools were constructed. From a total of 11,368 high-quality, nonchimeric, near full-length sequences, 247 species-level phylotypes (using a 99% sequence identity threshold) and 9 bacterial phyla were identified. At least 15 bacterial genera were conserved among all 10 individuals, with significant interindividual differences at the species and strain level. Comparisons of these oral bacterial sequences with near full-length sequences found previously in the large intestines and feces of other healthy individuals suggest that the mouth and intestinal tract harbor distinct sets of bacteria. Co-occurrence analysis showed significant segregation of taxa when community membership was examined at the level of genus, but not at the level of species, suggesting that ecologically significant, competitive interactions are more apparent at a broader taxonomic level than species. This study is one of the more comprehensive, high-resolution analyses of bacterial diversity within the healthy human mouth to date, and highlights the value of tools from macroecology for enhancing our understanding of bacterial ecology in human health.
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            Diversity of bacteria at healthy human conjunctiva.

            Ocular surface (OS) microbiota contributes to infectious and autoimmune diseases of the eye. Comprehensive analysis of microbial diversity at the OS has been impossible because of the limitations of conventional cultivation techniques. This pilot study aimed to explore true diversity of human OS microbiota using DNA sequencing-based detection and identification of bacteria. Composition of the bacterial community was characterized using deep sequencing of the 16S rRNA gene amplicon libraries generated from total conjunctival swab DNA. The DNA sequences were classified and the diversity parameters measured using bioinformatics software ESPRIT and MOTHUR and tools available through the Ribosomal Database Project-II (RDP-II). Deep sequencing of conjunctival rDNA from four subjects yielded a total of 115,003 quality DNA reads, corresponding to 221 species-level phylotypes per subject. The combined bacterial community classified into 5 phyla and 59 distinct genera. However, 31% of all DNA reads belonged to unclassified or novel bacteria. The intersubject variability of individual OS microbiomes was very significant. Regardless, 12 genera-Pseudomonas, Propionibacterium, Bradyrhizobium, Corynebacterium, Acinetobacter, Brevundimonas, Staphylococci, Aquabacterium, Sphingomonas, Streptococcus, Streptophyta, and Methylobacterium-were ubiquitous among the analyzed cohort and represented the putative "core" of conjunctival microbiota. The other 47 genera accounted for <4% of the classified portion of this microbiome. Unexpectedly, healthy conjunctiva contained many genera that are commonly identified as ocular surface pathogens. The first DNA sequencing-based survey of bacterial population at the conjunctiva have revealed an unexpectedly diverse microbial community. All analyzed samples contained ubiquitous (core) genera that included commensal, environmental, and opportunistic pathogenic bacteria.
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              Characterization of the normal microbiota of the ocular surface.

              The ocular surface is continually exposed to the environment and as a consequence to different types of microbes, but whether there is a normal microbiota of the ocular surface remains unresolved. Using traditional microbial culture techniques has shown that <80% of swabs of the conjunctiva yield cultivable microbes. These usually belong to the bacterial types of the coagulase-negative staphylococci, Propionibacterium sp., with low frequency of isolation of bacteria such as Staphylococcus aureus, Micrococcus sp., Gram-negative bacteria or fungi. Even when these are grown, the numbers of colony forming units (cfu) per swab of the conjunctiva is usually much less than 100 cfu. Swabs of the lid more commonly result in microbial growth, of the same species as from the conjunctiva and slightly higher cfu. Contact lenses have also been cultured, and they yield similar microbial types. Microbes can be isolated from the ocular surface almost immediately after birth. The advent of molecular techniques for microbial identification based on 16S rRNA sequencing has opened up the possibility of determining whether there are non-cultivable microbes that can colonise the ocular surface. Additionally, use of these techniques with cross-sectional and longitudinal studies may help to understand whether the ocular surface harbours its own unique microbiota, or whether the microbiota are only transiently present. Copyright © 2013 Elsevier Ltd. All rights reserved.

                Author and article information

                Genome Med
                Genome Med
                Genome Medicine
                BioMed Central (London )
                15 November 2014
                15 November 2014
                : 6
                : 11
                : 99
                [ ]The Genome Institute, Washington University, St Louis, MO 63108 USA
                [ ]Department of Pediatrics, Washington University School of Medicine, St Louis, MO 63130 USA
                [ ]Department of Clinical Research, London School of Hygiene and Tropical Medicine, London, WC1E 7HT UK
                [ ]Disease Control and Elimination Theme, Medical Research Council Unit, Fajara, POB273 The Gambia
                [ ]The Jackson Laboratory for Genomic Medicine, Farmington, CT 06030 USA
                © Zhou et al.; licensee BioMed Central Ltd. 2014

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                : 27 May 2014
                : 28 October 2014
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                © The Author(s) 2014

                Molecular medicine
                Molecular medicine


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