Lid Margin Microbiome in Stevens-Johnson Syndrome Patients With Lid Margin Keratinization and Severe Dry Eye Disease.

Purpose: The current study evaluated the lid margin microbiome of keratinized lid margins of patients with chronic Stevens-Johnson syndrome (SJS) and compared it with healthy controls and historically reported lid margin microbiome of patients with meibomian gland dysfunction (MGD). Methods: Eyelid margin swabs of 20 asymptomatic adults (mean age = 29 ± 12 years) and 10 patients with chronic SJS (mean age = 31.2 ± 14 years) with lid margin keratinization were sequenced using next generation of 16S rDNA V3 to V4 variable region. Within SJS, the keratinized lid margin microbiome was compared with adjacent eyelid skin. Results: All patients had obstructive MGD, and mean Schirmer I value was 2.8 ± 1.9 mm. The phyla were similar in two groups, whereas at the genera level, an increase in the relative abundance of Corynebacterium, Haemophilus, Azotobacter, and Afipia and a decrease of Acinetobacter was noted in SJS compared to healthy lid margins. SJS-associated microbiota displayed lesser diversity and more heterogeneity than healthy controls. The Principal Components Analysis (PCA) plot revealed wide separation in the SJS and the control groups. Correlational network analysis revealed Corynebacterium and Sphingomonas forming a major hub of negative interactions with other bacterial genera in the SJS group. Significant differences exist in the prevalent genera between keratinized lid margins and historically reported meibum microbiome of patients with MGD. In addition, the eyelid skin of patients with SJS had predominant Staphylococcus, whereas Corynebacterium and Pseudomonas were more in the keratinized lid margins compared to the eyelid skin microbiome. Conclusions: Lid margin microbiome is significantly altered in the keratinized lid margins of patients with SJS compared to the eyelid skin of patients with SJS, normal lid margins, and patients with MGD.

Hyperkeratinization and Proinflammatory Cytokine Expression in Meibomian Glands Induced by Staphylococcus aureus.

Purpose: This exploratory study aimed to investigate the morphological and pathological alterations of the meibomian gland (MG) with the Staphylococcus aureus crude extracts (SACEs) treatment. Methods: Mouse MG explants were cultured and differentiated with or without SACEs for 48 hours. Explant's viability and cell death were determined by thiazolyl blue tetrazolium bromide (MTT) assay and TUNEL assay. MG morphology was observed by Hematoxylin and Eosin staining. Lipid droplet production was detected by Nile Red staining and LipidTox immunostaining. The pro-inflammatory cytokines were detected by ELISA. The relative gene and protein expression in MG explants was determined via quantitative RT-PCR, immunostaining, and immunoblotting. The components of the SACEs were analyzed by immunoblotting and silver staining. Results: Our findings demonstrated that the SACEs treatment induced overexpression of keratin 1 (Krt1) in the ducts and acini of MG explants, accompanied by a decrease in viability and an increase in cell death in explants. Furthermore, the SACEs treatment dose-dependently increased the levels of TNF-α, IL-1ß, and IL-6 in MG explants. The SACEs treatment induced activation of the nuclear factor kappa B (NF-κB) and AIM2 (absent in melanoma 2)/ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) inflammasome signaling pathway in explants. Further investigation showed expression of the key adipogenesis-related molecule peroxisome proliferator-activated receptor γ was decreased after SACEs treatment. However, no change was found in the lipid synthesis of MG explants after treatment with the SACEs. Staphylococcal enterotoxins B (SEB) was detected in the SACEs. SEB induced the overexpression of Krt1 and IL-1ß in ducts and acini of MG explants. Conclusions: Our findings confirm that Staphylococcus aureus induced hyperkeratinization and pro-inflammatory cytokines expression in MG explants ducts and acini. These effects might be mediated by SEB. Activation of the NF-κB and AIM2/ASC signaling pathway is involved in this process.

Metagenomic Profiling of Ocular Surface Microbiome Changes in Meibomian Gland Dysfunction.

Purpose: Ocular surface microbiome changes can affect meibomian gland dysfunction (MGD) development. This study aimed to delineate differences among the microbiome of eyelid skin, conjunctiva, and meibum in healthy controls (HCs) and patients afflicted with MGD. Methods: Shotgun metagenomic analysis was used to determine if there are differences between the microbial communities in ocular sites surrounding the meibomian gland in healthy individuals and patients afflicted with MGD. Results: The meibum bacterial content of these microbiomes was dissimilar in these two different types of individuals. Almost all of the most significant taxonomic changes in the meibum microbiome of individuals with MGD were also present in their eyelid skin, but not in the conjunctiva. Such site-specific microbe pattern changes accompany increases in the gene expression levels controlling carbohydrate and lipid metabolism. Most of the microbiomes in patients with MGD possess a microbe population capable of metabolizing benzoate. Pathogens known to underlie ocular infection were evident in these individuals. MGD meibum contained an abundance of Campylobacter coli, Campylobacter jejuni, and Enterococcus faecium pathogens, which were almost absent from HCs. Functional annotation indicated that in the microbiomes of MGD meibum their capability to undergo chemotaxis, display immune evasive virulence, and mediate type IV secretion was different than that in the microbiomes of meibum isolated from HCs. Conclusions: MGD meibum contains distinct microbiota whose immune evasive virulence is much stronger than that in the HCs. Profiling differences between the meibum microbiome makeup in HCs and patients with MGD characterizes changes of microbial communities associated with the disease status.

Effect of Eyelid Treatments on Bacterial Load and Lipase Activity in Relation to Contact Lens Discomfort.

OBJECTIVES: To evaluate the effect of microblepharon exfoliation on the number of eyelid bacteria and their lipase activity and the relationship of these to contact lens discomfort. METHODS: Thirty experienced contact lens wearers had their eyelid margin physiology, tear properties, and comfort scores assessed. The number, type, and frequency of lower eyelid margin bacteria, and their lipase activity, were measured. Eyelids were treated with a foam cleanser or microblepharon exfoliation. Clinical and microbiological tests were repeated at each visit. Changes and correlations were examined. RESULTS: Symptomatic lens wearers had a higher ratio for the number and frequency of gram-positive rods and cocci. Microblepharon exfoliation reduced the number and ratio of gram-positive rods to cocci from baseline for symptomatic wearers that lasted 7 to 10 days after treatment (P<0.05). Numbers of bacteria, the ratio of rods to cocci, and lipase activity correlated with lash contamination (r≥0.385; P≤0.046) and anterior blepharitis (r≥0.359; P≤0.048). Bacterial lipase correlated with meibomian gland secretions (r=0.422; P=0.038) and the tear evaporation rate (r=0.479; P=0.022). Microblepharon exfoliation produced a significant reduction in CLDEQ-8 scores and converted 10 symptomatic into asymptomatic lens wearers. CONCLUSIONS: There was dysbiosis in the lid microbiome of symptomatic lens wearers. Microblepharon exfoliation reduced the number, frequency of isolation, and ratio of gram-positive rods and cocci. Bacterial numbers and their lipase production correlated with changes to clinical signs and symptoms. Symptomatic lens wearers could be converted to asymptomatic lens wearers after microblepharon exfoliation.

Composition and Diversity of Bacterial Community on the Ocular Surface of Patients With Meibomian Gland Dysfunction.

Purpose: To investigate the composition and diversity of bacterial community on the ocular surface of patients with meibomian gland dysfunction (MGD) via 16S rDNA sequencing. Methods: Forty-seven patients with MGD, who were divided into groups of mild, moderate, and severe MGD, and 42 sex- and age-matched participants without MGD (control group) were enrolled. Samples were collected from the upper and lower conjunctival sac of one randomly chosen eye of each participant. Through sequencing the hypervariable region of 16S rDNA gene obtained from samples, differences in the taxonomy and diversity between groups were compared. Results: Principle coordinate analysis showed significantly distinct clustering of the conjunctival sac bacterial community between the severe MGD group and the other groups. At the phylum level, the relative abundances of Firmicutes (31.70% vs. 19.67%) and Proteobacteria (27.46% vs. 14.66%) were significantly higher (P < 0.05, Mann-Whitney U), and the abundance of Actinobacteria (34.17% vs. 56.98%) was lower in MGD than controls (P < 0.05, Mann-Whitney U). At the genus level, the abundances of Staphylococcus (20.71% vs. 7.88%) and Sphingomonas (5.73% vs. 0.79%) in patients with MGD were significantly higher than the controls (P < 0.05, Mann-Whitney U), while the abundance of Corynebacterium (20.22% vs. 46.43%) was significantly lower (P < 0.05, Mann-Whitney U). The abundance of Staphylococcus was positively correlated with the meiboscores in patients with MGD (r = 0.650, P < 0.001, Spearman). Conclusions: Patients with MGD can have various degrees of bacterial microbiota imbalance in the conjunctival sac. Staphylococcus, Corynebacterium, and Sphingomonas may play roles in the pathophysiology of MGD.

Comparative portrayal of ocular surface microbe with and without dry eye.

rRNA gene high-throughput sequencing was performed in the conjunctival swab samples to investigate the composition of the OS bacterial community in DE (n=35) and NDE (n=54) and compared the composition of MGD (n=25) and NMGD (n=10) among DE subjects. Deep sequencing of OS 16S rDNA from DE (n=35) and NDE (n=54) demonstrated great a difference in alpha and beta diversity between the OS bacterial flora (P < 0.05). The similar OS microbial structures were shown at the phylum and genus levels by bioinformatics analysis between them, and in LEfSe (linear discriminant analysis effect size) analysis, Bacteroidia and Bacteroidetes were enriched in DE, while Pseudomonas was plentiful in NDE (linear discriminant analysis [LDA] > 4.0). Among the DE group, there was no significant difference in α and ß diversity between MGD and NMGD (P > 0.05). Surprisingly, Bacilli was the dominant microbe in MGD, and Bacteroidetes was the superior bacteria in NMGD among DE subjects (LDA > 4.0). Different diversity of OS bacteria composition between DE and NDE and the altered diversity of OS bacteria may play an important role in DE. Moreover, the lower dominance of OS bacteria in DE may be associated with the occurrence and development of DE. Although there was no significant difference in alpha and beta analysis, the OS dominant microbe between MGD and NMGD among DE was different.