Microbial Dynamics in Ophthalmic Health: Exploring the Interplay between Human Microbiota and Glaucoma Pathogenesis.

The human microbiome has a crucial role in the homeostasis and health of the host. These microorganisms along with their genes are involved in various processes, among these are neurological signaling, the maturation of the immune system, and the inhibition of opportunistic pathogens. In this sense, it has been shown that a healthy ocular microbiota acts as a barrier against the entry of pathogens, contributing to the prevention of infections. In recent years, a relationship has been suggested between microbiota dysbiosis and the development of neurodegenerative diseases. In patients with glaucoma, it has been observed that the microbiota of the ocular surface, intraocular cavity, oral cavity, stomach, and gut differ from those observed in healthy patients, which may suggest a role in pathology development, although the evidence remains limited. The mechanisms involved in the relationship of the human microbiome and this neurodegenerative disease remain largely unknown. For this reason, the present review aims to show a broad overview of the influence of the structure and composition of the human oral and gut microbiota and relate its dysbiosis to neurodegenerative diseases, especially glaucoma.

Disturbances in the ocular surface microbiome by perioperative antimicrobial eye drops.

We aimed to elucidate the effects of antimicrobial eye drops used in the perioperative period of ophthalmic surgery on the ocular surface microbiome by metagenomic analysis. Twenty-eight eyes from 15 patients (mean age 74.1 years) with no history of eye drop use within 3 months before cataract surgery were included in this study. Gatifloxacin eye drops were used in all patients in the perioperative period. The antimicrobial eye drops were started 3 days before surgery. They were discontinued after conjunctival sac specimen collection for 2 weeks after the surgery. Conjunctival sac specimens were collected to investigate the alterations in the ocular surface microbiome by meta-16S analysis targeting the V3-V4 region of the bacterial 16S rRNA gene. Principal coordinate analysis showed that the bacterial composition tended to be different before and 2 and 4 weeks after surgery. Individual observations on six eyes showed that the bacterial composition at 12 weeks after surgery was closer to that before surgery than to that at 4 weeks after surgery in two eyes, while the bacterial composition in the remaining four eyes was different at various time points. Before surgery, Firmicutes, Proteobacteria, and Bacteroidetes were predominant; however, 2 weeks after surgery, the proportion of Proteobacteria increased and that of Firmicutes decreased. A similar trend was noticed 4 weeks after surgery, although antibacterial eye drops had been discontinued 2 weeks after surgery. The Shannon-Weaver coefficient showed a decreasing trend at 2-, 4-, and 12-weeks post operation compared to that before operation. The diversity of the microbiome decreased significantly at 2- and 4-weeks after surgery when compared to that before surgery (p < 0.05). The ocular surface microbiome is easily disrupted by antimicrobial eye drops, and it needs recovery time. In such cases, the ocular surface microbiome is presumed to contain many antimicrobial-resistant bacteria. In some cases, it may not recover, and a new microbiome is formed.

Genetic Manipulation of Corynebacterium mastitidis to Better Understand the Ocular Microbiome.

Purpose: Corynebacterium spp. are Gram-positive bacteria commonly associated with the ocular surface. Corynebacterium mastitidis was isolated from mouse eyes and was demonstrated to induce a beneficial immune response that can protect the eye from pathogenic infection. Because eye-relevant Corynebacterium spp. are not well described, we generated a C. mast transposon (Tn) mutant library to gain a better understanding of the nature of eye-colonizing bacteria. Methods: Tn mutagenesis was performed with a custom Tn5-based transposon that incorporated a promoterless gene for the fluorescent protein mCherry. We screened our library using flow cytometry and enzymatic assays to identify useful mutants that demonstrate the utility of our approach. Results: Fluorescence-activated cell sorting (FACS) of mCherry+ bacteria allowed us to identify a highly fluorescent mutant that was detectable on the murine ocular surface using microscopy. We also identified a functional knockout that was unable to hydrolyze urea, UreaseKO. Although uric acid is an antimicrobial factor produced in tears, UreaseKO bacterium maintained an ability to colonize the eye, suggesting that urea hydrolysis is not required for colonization. In vitro and in vivo, both mutants maintained the potential to stimulate protective immunity as compared to wild-type C. mast. Conclusions: In sum, we describe a method to genetically modify an eye-colonizing microbe, C. mast. Furthermore, the procedures outlined here will allow for the continued development of genetic tools for modifying ocular Corynebacterium spp., which will lead to a more complete understanding of the interactions between the microbiome and host immunity at the ocular surface.

Validation of 16S rRNA Gene Sequencing of the Periocular Microbiome and Lack of Alteration by Topical Eyedrops.

Purpose: Genomic techniques for characterizing the ocular microbiome require further validation. We compared the microbiome of patients' eyelids through both conventional culture and 16S rRNA analysis and analyzed the impact of eyedrop use on microbiome diversity. Methods: Ninety-eight patients followed for management of glaucoma or suspicion of glaucoma had eyelid swabs performed with Isohelix MS Mini DNA Swabs (98 participants) and ESwabs (49 participants) for 16S rRNA analysis and conventional culture, respectively. The effect of preservative-containing eyedrops on the microbiomes detected using these two techniques were analyzed and compared across techniques. Results: Forty-five of the 50 (non-unique) genera (90%) identified by conventional culture were also identified by each individual's 16S rRNA analysis within the top 14 most abundant organisms present based on operational taxonomic unit. All conventional cultures performed had at least one or more genera also identified by each participant's 16S rRNA analysis. There was no difference in the conventional culture positivity rate or proportion of participants with a particular genus present on conventional culture based on whether preservative-containing eyedrops were regularly used. Similarly, in eyes using versus not using eyedrops, no differences were observed in the proportions of participants with a particular genus present or the Shannon index as determined by 16S rRNA analysis. Conclusions: 16S rRNA analysis correlates well with conventional culture results for the eyelid microbiome, with results from neither technique demonstrating an association of microbiome composition and eyedrop use. The clinical relevance of the large numbers of microbes detected via 16S rRNA analysis requires further study. Translational Relevance: 16S rRNA analysis of the periocular microbiome is consistent with conventional culture and enables further study of physiologic and pathologic ocular processes possibly related to microbiome diversity.

An insight on the eye bacterial microbiota and its role on dry eye disease.

The human ocular surface hosts a bacterial assemblage that integrates a diverse and complex microbiome. This bacterial microbiota is part of a healthy eye and plays a protective role in it. However, this ocular bacterial assemblage may alter the ocular surface inflammation response and can influence the development and progression of dry eye disease. For this reason, the present review describes the changes generated on the ocular surface by bacterial assemblages during the development of dry eye disease. Likewise, the interaction of this microbiota with the other inflammatory factors that influence the development of this disease is analyzed, as well as the use of treatments focused on modifying the bacteria on the ocular surface.

Nocardia infection following ocular surface surgery.

OBJECTIVE: To investigate the clinical characteristics and treatment outcomes of Nocardia infection after ocular surface surgery. METHODS: This is a retrospective study. Eight cases of culture-proven Nocardia infection, which developed within 1 month after ocular surface surgery were included. Demographics and clinical history of patients were investigated. RESULTS: There were 8 eyes (2 left and 6 right) of 8 patients (5 males and 3 females), aged 27-65, with a median age of 52.9 years. Three cases underwent pterygium excision, three were subjected to conjunctival flap covering, and two were treated with lamellar corneal transplantation. The time interval between previous surgery and the onset of symptoms varied from 7 to 28 days (mean = 20.5 ± 7.13 days). All the cases presented grey-white infiltrates at the surgical incision site while appearing with six corneal ulcers and two conjunctival ulcers. Filaments of Nocardia were founded by confocal microscopy in two of the five cases. All responded poorly to medical therapy. Seven of the eight cases were treated with reoperation. Nocardia infection recurred in three cases after reoperation, and one was eviscerated. CONCLUSIONS: Surgical trauma is a risk factor for ocular Nocardia infection. Nocardia infection should be suspected when secondary infection occurs in a surgical incision with an atypical clinical presentation. The use of corticosteroids may influence the efficacy of drugs. Complete removal of lesions may lower the recurrence of Nocardia infection with poor drug treatment effects.

Microbiological Characteristics of Ocular Surface Associated With Dry Eye in Children and Adolescents With Diabetes Mellitus.

Purpose: To analyze the characteristics of ocular surface microbial composition in children and adolescents with diabetes mellitus and dry eye (DE) by tear analysis. Methods: We selected 65 children and adolescents aged 8 to 16 years with DE and non-DE diabetes mellitus and 33 healthy children in the same age group from the Shanghai Children and Adolescent Diabetes Eye Study. Tears were collected for high-throughput sequencing of the V3 and V4 region of 16S rRNA. The ocular surface microbiota in diabetic DE (DM-DE; n = 31), diabetic with non-DE (DM-NDE; n = 34), and healthy (NDM; n = 33) groups were studied. QIIME2 software was used to analyze the microbiota of each group. Results: The DM-DE group had the highest amplicon sequence variants, and the differences in α-diversity and ß-diversity of micro-organisms in the ocular surfaces of DM-DE, diabetic with non-DE, and healthy eyes were statistically significant (P < 0.05). Bacteroidetes (15.6%), Tenericutes (9.3%), Firmicutes (21.8%), and Lactococcus (7.9%), Bacteroides (7.8%), Acinetobacter (3.9%), Clostridium (0.8%), Lactobacillus (0.8%) and Streptococcus (0.2%) were the specific phyla and genera, respectively, in the DM-DE group. Conclusions: Compared with the patients with non-DE and healthy children, the microbial diversity of the ocular surface in children and adolescents with diabetes mellitus and DE was higher with unique bacterial phyla and genera composition.

Microbial Signatures in The Rodent Eyes With Retinal Dysfunction and Diabetic Retinopathy.

Purpose: The gut microbiome has been linked to disease pathogenesis through their interaction in metabolic, endocrine, and immune functions. The goal of this study was to determine whether the gut and plasma microbiota could transfer microbes to the retina in type 1 diabetic mice with retinopathy. Methods: We analyzed the fecal, plasma, whole globe, and retina microbiome in Akita mice and compared with age-matched wild-type (WT) mice using 16S rRNA sequencing and metatranscriptomic analysis. To eliminate the contribution of the ocular surface and plasma microbiome, mice were perfused with sterile saline solution, the whole globes were extracted, and the neural retina was removed under sterile conditions for retinal microbiome. Results: Our microbiome analysis revealed that Akita mice demonstrated a distinct pattern of microbes within each source: feces, plasma, whole globes, and retina. WT mice and Akita mice experienced transient bacteremia in the plasma and retina. Bacteria were identified in the retina of the Akita mice, specifically Corynebacterium, Pseudomonas, Lactobacillus, Staphylococcus, Enterococcus, and Bacillus. Significantly increased levels of peptidoglycan (0.036 ± 0.001 vs. 0.023 ± 0.002; P < 0.002) and TLR2 (3.47 ± 0.15 vs. 1.99 ± 0.07; P < 0.0001) were observed in the retina of Akita mice compared to WT. Increased IBA+ cells in the retina, reduced a- and b-waves on electroretinography, and increased acellular capillary formation demonstrated the presence of retinopathy in the Akita cohort compared to WT mice. Conclusions: Together, our findings suggest that transient bacteremia exists in the plasma and retina of both cohorts. The bacteria found in Akita mice are distinct from WT mice and may contribute to development of retinal inflammation and barrier dysfunction in retinopathy.

Population-Based Prevalence of Ocular Chlamydia trachomatis Infection among Infants in the Trachoma Endemic Amhara Region, Ethiopia.

Infants ages < 6 months do not receive azithromycin as part of trachoma control and thus may serve as an infection reservoir in persistently endemic districts. The aim of this study was to determine the population-based Chlamydia trachomatis infection prevalence and infectious load among infants ages 1-12 months in persistently trachoma endemic districts in Amhara, Ethiopia. Across six districts, 475 infants were enumerated, and of these 464 (97.7%) were swabbed for infection testing. The C. trachomatis infection prevalence in the study area among infants was 0.2% (95% CI: 0.0-1.5). Among children ages 0-5 years positive for C. trachomatis, the median load was 31 elementary bodies (EB) (Inter quartile range: 7-244 EB), and the infection-positive infant had a load of 7,755 EB. While it is worth reconsidering azithromycin treatment recommendations for the potential mortality benefits, these results do not support lowering the treatment age for trachoma control.

The crested caracara (Caracara plancus) eye: Morphologic observations and results of selected diagnostic tests.

PURPOSE: To provide a descriptive investigation about relevant features of the crested caracara's eye (Caracara plancus) and bony orbit, as well as provide data for ophthalmic tests. METHODS: Morphological observations and the following diagnostic tests were performed: Schirmer tear test (STT), conjunctival flora evaluation, corneal touch threshold (CTT), intraocular pressure (IOP), central corneal thickness (CCT), B-mode ocular biometry, palpebral fissure length (PFL), and corneal diameter (CD) in 19 healthy birds, plus two macerated skulls. Not all birds were used for each test. RESULTS: STT: 7.84 ± 3.05 mm/min; CTT: 2.46 ± 1.10 cm; IOP: 19.18 ± 3.07 mmHg; CCT: 0.31 ± 0.02 mm; PFL: 13.32 ± 1.06 mm; CD: 10.26 ± 2.43 mm; Axial globe length: 1.89 ± 0.06 cm; Anterior chamber depth: 0.27 ± 0.06 cm; Lens axial length: 4.55 ± 0.06 cm; Vitreous chamber depth: 1.2 ± 0.07 cm. The most frequent conjunctival bacterial isolates were Corynebacterium sp. (10/23.8%), Staphylococcus sp. (9/21.42%), Streptococcus sp. (7/16.6%), and E. coli (7/16.66%). The large lateral part of the palatine bone likely plays a role in the ventral protection of the globe against the impact of prey. Observed results are generally reflective of increased body mass compared to other Falconiformes, with values approaching those of similar sized Accipitriformes. CONCLUSIONS: These data may help veterinarians recognize peculiar morphologic features and perform a more accurate diagnosis of eye diseases of this avian species.

Genomics of Staphylococcus aureus ocular isolates.

Staphylococcus aureus is a major cause of ocular infections, often resulting in devastating vision loss. Despite the significant morbidity associated with these infections, little is yet known regarding the specific strain types that may have a predilection for ocular tissues nor the set of virulence factors that drive its pathogenicity in this specific biological niche. Whole genome sequencing (WGS) can provide valuable insight in this regard by providing a prospective, comprehensive assessment of the strain types and virulence factors driving disease among specific subsets of clinical isolates. As such, a set of 163-member S. aureus ocular clinical strains were sequenced and assessed for both common strain types (multilocus sequence type (MLST), spa, agr) associated with ocular infections as well as the presence/absence of 235 known virulence factors in a high throughput manner. This ocular strain set was then directly compared to a fully sequenced 116-member non-ocular S. aureus strain set curated from NCBI in order to identify key differences between ocular and non-ocular S. aureus isolates. The most common sequence types found among ocular S. aureus isolates were ST5, ST8 and ST30, generally reflecting circulating non-ocular pathogenic S. aureus strains. However, importantly, ocular isolates were found to be significantly enriched for a set of enterotoxins, suggesting a potential role for this class of virulence factors in promoting ocular disease. Further genomic analysis revealed that these enterotoxins are located on mobile pathogenicity islands, thus horizontal gene transfer may promote the acquisition of enterotoxins, potentially amplifying S. aureus virulence in ocular tissues.

A novel application of bubble-eye strain of Carassius auratus for ex vivo fish immunological studies.

In this study, we investigated a new application of bubble-eye goldfish (commercially available strain with large bubble-shaped eye sacs) for immunological studies in fishes utilizing the technical advantage of examining immune cells in the eye sac fluid ex vivo without sacrificing animals. As known in many aquatic species, the common goldfish strain showed an increased infection sensitivity at elevated temperature, which we demonstrate may be due to an immune impairment using the bubble-eye goldfish model. Injection of heat-killed bacterial cells into the eye sac resulted in an inflammatory symptom (surface reddening) and increased gene expression of pro-inflammatory cytokines observed in vivo, and elevated rearing temperature suppressed the induction of pro-inflammatory gene expressions. We further conducted ex vivo experiments using the immune cells harvested from the eye sac and found that the induced expression of pro-inflammatory cytokines was suppressed when we increased the temperature of ex vivo culture, suggesting that the temperature response of the eye-sac immune cells is a cell autonomous function. These results indicate that the bubble-eye goldfish is a suitable model for ex vivo investigation of fish immune cells and that the temperature-induced infection susceptibility in the goldfish may be due to functional impairments of immune cells.

Clinical and mycological characteristics of keratitis caused by Colletotrichum gloeosporioides: A case report and review of literature.

INTRODUCTION: Colletotrichum species are well-known plant pathogens, which have been increasingly reported as the cause of keratitis or subcutaneous lesions in humans. In this study we reported a rare case of fungal keratitis from Iran and reviewed the literature. CASE PRESENTATION: A 69-year-old man whose right eye was injured by herbal material was examined by slit-lamp biomicroscopy and mycology investigation of corneal scrapings was done. The grown filamentous fungal was identified as Colletotrichum gloeosporioides based on morphological characteristics and DNA sequence of the internal transcribed spacer region. The isolated strain was sensitive to amphotericin B, caspofungin, anidolafungin, micafungin, voriconazole, and relatively resistant to fluconazole, and itraconazole. Patient was successfully treated with voriconazole. CONCLUSIONS: This report highlights that the early and accurate identification and therapy can helpful to management keratitis caused by C. gloeosporioides.

Intravitreal Injection and Quantitation of Infection Parameters in a Mouse Model of Bacterial Endophthalmitis.

Intraocular bacterial infections are a danger to the vision. Researchers use animal models to investigate the host and bacterial factors and immune response pathways associated with infection to identify viable therapeutic targets and to test drugs to prevent blindness. The intravitreal injection technique is used to inject organisms, drugs, or other substances directly into the vitreous cavity in the posterior segment of the eye. Here, we demonstrated this injection technique to initiate infection in the mouse eye and the technique of quantifying intraocular bacteria. Bacillus cereus was grown in brain heart infusion liquid media for 18 hours and resuspended to a concentration 100 colony forming units (CFU)/0.5 µL. A C57BL/6J mouse was anesthetized using a combination of ketamine and xylazine. Using a picoliter microinjector and glass capillary needles, 0.5 µL of the Bacillus suspension was injected into the mid vitreous of the mouse eye. The contralateral control eye was either injected with sterile media (surgical control) or was not injected (absolute control). At 10 hours post infection, mice were euthanized, and eyes were harvested using sterile surgical tweezers and placed into a tube containing 400 µL sterile PBS and 1 mm sterile glass beads. For ELISAs or myeloperoxidase assays, proteinase inhibitor was added to the tubes. For RNA extraction, the appropriate lysis buffer was added. Eyes were homogenized in a tissue homogenizer for 1-2 minutes. Homogenates were serially diluted 10-fold in PBS and track diluted onto agar plates. The remainder of the homogenates were stored at -80 °C for additional assays. Plates were incubated for 24 hours and CFU per eye was quantified. These techniques result in reproducible infections in mouse eyes and facilitate quantitation of viable bacteria, the host immune response, and omics of host and bacterial gene expression.

Evaluation of the ocular surface mycobiota in clinically normal horses.

The eye is host to myriad bacterial, fungal, and viral organisms that likely influence ocular surface physiology in normal and diseased states. The ocular surface mycobiota of horses has not yet been described using NGS techniques. This study aimed to characterize the ocular surface fungal microbiota (mycobiota) in healthy horses in 2 environmental conditions (stalled versus pasture). Conjunctival swabs of both eyes were obtained from 7 adult stallions stabled in an open-air pavilion and 5 adult mares living on pasture. Genomic DNA was extracted from ocular surface swabs and sequenced using primers that target the Internal Transcribed Spacer 1 (ITS1) region of the fungal genome on an Illumina platform. Sequences were processed using Quantitative Insights Into Molecular Ecology (QIIME 2.0) and taxonomy assigned with the Findley et al. 2013 ITS1 database. The most abundant genera identified were Leptosphaerulina (22.7%), unclassified Pleosporaceae (17.3%), Cladosporium (16.2%), Alternaria (9.8%), unclassified Pleosporales (4.4%), unclassified Montagnulaceae (2.9%), Fusarium (2.5%), and Pestalotiopsis (1.4%). Fungal community composition (Jaccard, R = 0.460, p = 0.001) and structure (Bray-Curtis, R = 0.811, p = 0.001) were significantly different between pastured mares and stabled stallions. The ocular surface of pastured mares had significantly increased fungal species richness and diversity compared to stabled stallions (Shannon p = 0.0224, Chao1 p = 0.0118, Observed OTUs p = 0.0241). Relative abundances of Aspergillus (p = 0.005) and Alternaria spp. (p = 0.002) were significantly increased in the mycobiota of pastured mares. This is the first report to describe the mycobiota of the equine ocular surface. Environmental factors such as housing influence the composition, structure, and richness of the equine ocular surface mycobiota.

Microbes of the human eye: Microbiome, antimicrobial resistance and biofilm formation.

BACKGROUND: The review focuses on the bacteria associated with the human eye using the dual approach of detecting cultivable bacteria and the total microbiome using next generation sequencing. The purpose of this review was to highlight the connection between antimicrobial resistance and biofilm formation in ocular bacteria. METHODS: Pubmed was used as the source to catalogue culturable bacteria and ocular microbiomes associated with the normal eyes and those with ocular diseases, to ascertain the emergence of anti-microbial resistance with special reference to biofilm formation. RESULTS: This review highlights the genetic strategies used by microorganisms to evade the lethal effects of anti-microbial agents by tracing the connections between candidate genes and biofilm formation. CONCLUSION: The eye has its own microbiome which needs to be extensively studied under different physiological conditions; data on eye microbiomes of people from different ethnicities, geographical regions etc. are also needed to understand how these microbiomes affect ocular health.

A closer look at pathogenesis of cerebral mucormycosis in diabetic condition: A mini review.

Patients with diabetes are considered a high-risk group involved with cerebral mucormycosis (CM). Due to the potential of Mucorales to invade sinuses and its rapid progression into orbit and retro-orbital areas and even brain, in most cases, CM is fatal in patients with diabetes. In the last few decades, mucormycosis and background conditions responsible for the development of its infections have received a great deal of attention. Dysfunction of innate and adaptive immune system, the increased amount of available nutrition, expression of host factors, and free iron level in plasma in diabetic ketoacidosis are among the topics that have been mostly taken into account so far. Therefore, it is important to clarify the molecular mechanisms that let the Mucorales to involve the patients with diabetes, which even at early stages of diagnosis and treatment, there is minimum chance to control the disease.