How the microbiome powerfully modulates our ocular health Harvey A. - - PowerPoint PPT Presentation

How the microbiome powerfully modulates our ocular health

Harvey A. Fishman, MD, PhD Comprehensive Ophthalmology 706 Webster St. Palo Alto, CA

Financial Disclosures

• Co-Founder of EyeCareLive
• Co-Founder of TearBio
• Digital Health Patents
• Speaking Consultant with Lumenis
• Consultant with 23andMe
• Past Research Consulting or Speaker for AMO, Allergan,

Zeiss, OptoVue, Bio-Tissue

Overview

I. Definitions: “High-Yield Biology Primer” II. Human Microbiome Project (HMP)

• III. Genetic techniques for microbiome analysis
• IV. Non-ocular microbiome and Ocular health

V. Ocular Microbiome and Ocular health

• VI. Future directions

DNA Chip Technology

“High-Yield Biology Primer”

What is a microbiome?

• Nobel Prize Laureate, Dr. Joshua Lederberg,

coined “microbiome” in 2001 to signify the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease.

“Ome sweet 'omics: -- A genealogical treasury of words. The Scientist. 2001; 15:8.

• Phylogenetics - the study of the evolutionary history and relationships among

individuals or groups of organisms (e.g. species, or populations)

• Taxonomy - the identification, naming and classification of organisms- Taxon

(plural taxa)- is a group of one or more populations of an organism or

• rganisms to form a unit)
• “Microbiota” - the microbial taxa associated with humans
• “Microbiome” - the catalog of these microbes and their genes
• “Metagenomics” - originally referred to shotgun characterization of total DNA,

although now it is being applied to studies of marker genes such as the 16S rRNA gene

• Ribosome - complex molecular machine, found within all living cells, that

serves as the site of biological protein synthesis (translation)

• “OTUs” (Operational Taxonomic Units) - clusters of (uncultivated or unknown)
• rganisms, grouped by DNA sequence similarity of a specific taxonomic

marker gene- pragmatic proxies for microbial "species" at different taxonomic levels, in the absence of traditional systems of biological classification as are available for macroscopic organisms. Typically, OTUs are based on similar 16S rRNA sequences

“High-Yield Biology Primer”

NIH Human Microbiome Project (HMP)

The overall mission of the HMP is to generate resources to facilitate characterization of the human microbiota to further our understanding of how the microbiome impacts human health and disease a. Pregnancy & Preterm Birth: How microbiome and host profiles change throughout pregnancy and influence the establishment of the nascent microbiome in neonates b. Onset of Inflammatory Bowel Disease (IBD): How the human gut microbiome changes

• ver time in adults and children with IBD

c. Onset of Type 2 Diabetes Type 2 diabetes mellitus (T2D): Performing a detailed analysis

• f the biological processes that occur in the

microbiome and human host by longitudinal profiling of patients at risk for T2D.

Genetic Techniques for Microbiome Analysis

16S ribosomal RNA (or 16S rRNA) sequencing has played a pivotal role in the accurate identification of bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories a. The 16S rRNA gene is used for phylogenetic studies as it is highly conserved between different species of bacteria and archaea b. Archaea constitute a domain of single-celled

• microorganisms. These microbes are

prokaryotes, meaning they have no cell nucleus c. 16S rRNA gene sequences contain hypervariable regions that can provide species-specific signature sequences useful for identification of bacteria.

Pearson Education, Inc.

Genetic Techniques for Microbiome Analysis

16S ribosomal RNA (or 16S rRNA) sequencing has played a pivotal role in the accurate identification of bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories

• d. 16S rRNA is the component of the 30S small

subunit of a prokaryotic ribosome that binds to the Shine-Dalgarno sequence. e .The genes coding for it are referred to as 16S rRNA gene and are used in reconstructing phylogenies, due to the slow rates of evolution of this region of the gene.

• f. 16S rDNA sequencing is particularly important for

identification of bacterial with unusual phenotypic profiles, rare bacteria, slow-growing bacteria, uncultivable bacteria and culture-negative infections.

Genetic Techniques for Microbiome Analysis

Ilumina Chip Technology: Oligonucleotides, cDNA or small fragments

• f PCR products corresponding to specific

genes are spotted on the chip.

Non-ocular microbiome and ocular health

• Human microbiota consists of the 10-100 trillion symbiotic microbial

cells harbored by each person, primarily bacteria in the gut; the human microbiome consists of the genes these cells harbor

• Meta- HIT consortium reported a gene catalog of 3.3 million non-

redundant genes in the human gut microbiome alone as compared to the ∼22,000 genes present in the entire human genome

• Decreased Bacteriodetes and increased Firmicutes have been found

in genetically obese mice (ob/ob) when compared to their lean counterparts, and the obesity phenotype can even be transferred to a germ- free but genetically wild-type mouse by way of the microbiota

• Most of the 10–100 trillion microorganisms in the human

gastrointestinal tract live in the colon.

• More than 90% of all phylogenetic types (phylotypes) of colonic

bacteria belong to just 2 of the 70 known divisions (phyla) in the domain Bacteria: the Firmicutes and the Bacteroidetes.

19th January 2018 by Laura Berry

Non-ocular microbiome and ocular health

Microbiome dysbiosis and ocular disease

• Macular degeneration
• Uveitis
• Non-specific autoimmune ocular disease
• Dry eye disease
• Glaucoma

AMD- EFEI Uveitis - Medicine.net

The severity of SS ocular and systemic disease was inversely correlated with microbial diversity. These findings suggest that SS is marked by a dysbiotic intestinal microbiome driven by low relative abundance of commensal bacteria and high relative abundance of potentially pathogenic genera that is associated with worse

• cular mucosal disease in a mouse model of SS and in SS

patients.

Highlights

• T cell activation in the gut correlates with

spontaneous uveitis in R161H mice

• Elimination of gut microbiota attenuates

disease and reduces T cell activation in the gut

• Presence of endogenous antigen is not

required for intestinal T cell activation

• Extracts of intestinal contents signal via

retina-specific TCR and trigger uveitis

Chronic inflammation and tissue damage may be the result of an exaggerated or dysregulated host response to the infection and the microbiome might be a significant source of infectious antigen and antigen-specific T cells. Thus, chronic or recurrent uveitic disease may be caused by local reactivations of persistent microbial agents or inadequately cleared antigen, including retroviral antigen, which intermittently disrupt the Treg/ T-effector cell ratio. In addition, a dysregulated microbiome may predispose to, or even be the source of, uveitogenic pathogens or adjuvants.

The role of IM in ocular Inflammatory disease

Certain bacterial strains, segmented filamentous bacteria found in rodents and analogous bacterial strains found in human can promote differentiation of T helper 17 cells (Th17) in the gut.

Five main therapeutic strategies for targeting the intestinal microbiome for treatment of ocular inflammatory disease

• 1. Targeting specific causative bacteria
• 2. Target the intestinal microbiome could be through the

administration of oral live bacterial strains that are known to promote immune homeostasis by enhancing, for instance, regulatory T cell differentiation.

• 3. Chemical drugs- antibiotics that are not broad-spectrum

but instead are chemicals designed to target the metabolic pathways of only a specific community of bacteria.

• 4. Dietary modifications, for example exposing individuals to

a diet high in nondigestible fibres to enhance the produc- tion of endogenous short chain fatty acids by the intestinal microbiota

• 5. Supplant an entire community of intestinal bacteria with a

normal community using a faecal microbial transplant

Controversy surrounding the existence of the ocular microbiome

Gut- Skin Conection

“These studies, and many similar, have given rise to 2 views. In the first, microbial DNA and organisms may be isolated sporadically from the

• cular surface, but without the implication that they are stable colonizers.

Rather, their fate is to be killed or removed from the eye” “By contrast, others suggest that, similar to other mucosal sites, there is a normal consortium of microbes that colonize the ocular surface”

CONCLUSIONS: Relative to adjacent skin or other mucosa, healthy ocular surface microbiome is paucibacterial. Its flora are distinct from adjacent skin. Torque teno virus is a frequent constituent of the ocular surface

• microbiome. (ClinicalTrials.gov number, NCT02298881.)

Using culture dependent and independent methods, the ocular surface does not appear to support a substantial core microbiome. However, consistently present taxa could be observed within individuals suggesting the possibility of individual-specific core microbiomes.

Certain bacteria species were found in samples from dry eye subjects only, including Bacillus sp. and Klebsiella oxytoca, in addition to an association between elevated bacterial count (CFU/swab) and the incidence of blepharitis Increasing bacterial count was correlated with a decrease in goblet cells among the subgroup of 27 subjects, consistent with previous studies that have demonstrated a depletion of goblet cells in other areas of the body after colonization by bacteria

Oral antibiotic treatment increases susceptibility to

• P. aeruginosa–induced ocular infection

The existence of the ocular microbiota has been reported but functional analyses to evaluate its significance in regulating ocular immunity are currently lacking.

SPF SW mice were treated orally with an antibiotic cocktail before the infections experiments, which resulted in a significant decrease in numbers of gut microbiota while preserving the ocular commensals in the conjunctiva ABX mice showed increased susceptibility to ocular P. aeruginosa challenge, which was exemplified by elevated bacterial burden in the corneas and increased corneal pathology.

Curr Opin Ophthalmol. 2016 Jan;27(1):9-23. Grzybowski A1, Kuklo P, Pieczynski J, Beiko G PURPOSE OF REVIEW: Postoperative endophthalmitis is an ophthalmic surgeon's most feared complication, as it portends a high probability of visual loss. The purpose of this article is to review the literature on the subject of preoperative measures used for prevention of postoperative endophthalmitis and to determine the effectiveness of these measures. RECENT FINDINGS: The present reviewed literature regarding topical prophylaxis of endophthalmitis in cataract surgery. SUMMARY: There is strong evidence for the use of povidone-iodine preoperatively for prophylaxis of postoperative endophthalmitis in ocular surgery; the evidence for topical antibiotics is not as compelling.

A review of preoperative manoeuvres for prophylaxis of endophthalmitis in intraocular surgery: topical application of antibiotics, disinfectants, or both?

Eur J Ophthalmol. 2009 Sep-Oct;19(5):717-22 Miño De Kaspar H1, Ta CN, Froehlich SJ, Schaller UC, Engelbert M, Klauss V, Kampik A. To determine whether a patient's age, gender, local or systemic risk factors affect the rate of preoperative bacterial contamination. METHODS: Consecutive 1,474 patients . The patients were divided into four groups: a control group (without local or systemic risk factors), those with local risk factors (chronic use of topical medications, contact lens wear, blepharitis, chronic eyelid or conjunctival inflammation), those with systemic risk factors (immunosuppression, diabetes, autoimmune conditions, and asthma), and those with both. Conjunctival cultures were obtained before surgery. RESULTS: Advanced age was associated with a higher rate of positive conjunctival cultures (p<0.005). No statistical difference was found with regard to gender (p=0.7173). Among the 282 patients in the control group, 14 (5%) had a positive conjunctival culture. Compared to the control group, positive conjunctival cultures were found in 118 out of 503 patients (23.5%) with local risk factors (p<0.0001), 65 out of 545 patients (11.9%) with systemic risk factors (p=0.0019), and 22 out of 144 (15.3%) with both (p=0.0006). Two patients developed postoperative endophthalmitis (0.14%), one with both local risk and systemic factors and the other with a systemic risk factor. CONCLUSIONS: Patients with local or systemic risk factors or advanced age were found to have a higher rate of bacterial conjunctival contamination before intraocular surgery.

Prospective study of risk factors for conjunctival bacterial contamination in patients undergoing intraocular surgery

Preliminary evidence of the efficacy of probiotic eye-drop treatment in patients with vernal keratoconjunctivitis. Graefes Arch Clin Exp Ophthalmol 2008;246:3:435-41. Lovieno A, Lambiase A, Sacchetti M, et al.

Seven patients (mean age 11.8 ‰± ‰4.3; five M, two F) with mild to moderate VKC were included in the study. Lactobacillus Acidophilus diluted in saline solution (2 ‰× ‰108 CFU/ml) was administrated as eye-drops four times daily for 4 weeks in both eyes. Clinical signs (conjunctival hyperemia, chemosis, secretion, Trantas dots, superficial punctuate keratitis) and symptoms (itching, photophobia, burning, tearing) were evaluated and scored from 0 to 3 at baseline, after 2 and 4 weeks of treatment. Total sign (TSS) and symptom (TSyS) scores were calculated. Conjunctival impression cytology was performed in three patients at baseline and after 4 weeks of treatment, in order to evaluate the expression of ICAM-1 and TLR-4. Results In the six out of seven patients who completed the study, symptoms were significantly improved after both 2 weeks (TSyS: baseline 6.7 ‰± ‰0.9 vs 4.1 ‰± ‰1.2; p ‰= ‰0.017) and 4 weeks (TSyS: baseline 6.7 ‰± ‰0.9 vs 3.6 ‰± ‰1.2, p ‰= ‰0.011) of

• treatment. A significant improvement of clinical signs was observed after 4 weeks of treatment (TSS: baseline 7.5 ‰± ‰

1.6 vs 3.9 ‰± ‰1.7, p ‰= ‰0.034) but not after 2 weeks of treatment (TSS: baseline 7.5 ‰± ‰1.6 vs 5.3 ‰± ‰1.5; NS). In particular, photophobia was significantly reduced (2 ‰± ‰0.6 vs 1 ‰± ‰0.3; p ‰= ‰0.023) at 2 weeks, while at 4 weeks the scores for itching (1.8 ‰± ‰0.3 vs 1 ‰± ‰0.3), tearing (1.6 ‰± ‰0.4 vs 0.8 ‰± ‰0.2), conjunctival hyperemia (2.3 ‰± ‰0.2 vs 1.4 ‰± ‰0.5) and chemosis (1.2 ‰± ‰0.4 vs 0.4 ‰± ‰0.4) were significantly lower compared to baseline. A down-regulation of ICAM-1 and TLR-4 was observed in two patients showing clinical improvement after 4 weeks of treatment. Conclusion Our open pilot study showed that 1-month treatment with probiotic eye-drops improves signs and symptoms in patients with VKC. Additional double-blind controlled clinical trials with a larger sample of patients are needed to confirm the effects of topical Lactobacilli on VKC patients.

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“A human gut microbial gene catalog established by metagenomic sequencing.” Junjie Qin et al. Nature. 2010 March 4; 464(7285): 59– 65. Open source Wikipedia for various definitions above