Culture- and non-culture-based approaches reveal unique features of the ocular microbiome in dry eye patients.

PURPOSE: The purpose of this study was to investigate the ocular microbiome in individuals with dry eye disease and to identify features of their ocular microbiome of possible health and diagnostic significance. METHODS: Conjunctival samples were collected from both eyes in duplicate from 91 individuals (61 dry eye, 30 healthy) and used for both culture-dependent and culture-independent analyses. Samples were either analysed using next generation sequencing (V3-V4 16S rDNA) or inoculated on a wide range of agar types and grown under a broad range of conditions to maximize recovery. Isolates were identified by partial sequencing of the 16S rDNA and rpoB genes and tested for antibiotic susceptibility. We applied a L2-regularized logistic regression model on the next generation sequencing data to investigate any potential association between severe dry eye disease and the ocular microbiome. RESULTS: Culture-dependent analysis showed the highest number of colony forming units in healthy individuals. The majority of isolates recovered from the samples were Corynebacterium, Micrococcus sp., Staphylococcus epidermidis, and Cutibacterium acnes. Culture independent analysis revealed 24 phyla, of which Actinobacteria, Firmicutes and Proteobacteria were the most abundant. Over 405 genera were detected of which Corynebacterium was the most dominant, followed by Staphylococcus and Cutibacterium. The L2-regularized logistic regression model indicated that Blautia and Corynebacterium sp. may be associated with severe DED. CONCLUSIONS: Our study indicates that the ocular microbiome has characteristic features in severe DED patients. Certain Corynebacterium species and Blautia are of particular interest for future studies.

The Conjunctival Microbiome and Dry Eye: What We Know and Controversies.

ABSTRACT: Dry eye disease is a common multifactorial condition that may be idiopathic or associated with autoimmune conditions, such as Sjogren syndrome. Commensal microorganisms modify immune responses, so it is relevant to understand how they modify such immune-mediated diseases. Microbiota in the gut regulate inflammation in the eye, and conversely, severe inflammation of the ocular surface results in alteration of gut microbiome. The conjunctiva microbiome can be analyzed using 16S or shotgun metagenomics. The amount of microbial DNA in ocular surface mucosa relative to human DNA is limited compared with the case of the intestinal microbiome. There are challenges in defining, harvesting, processing, and analyzing the microbiome in the ocular surface mucosa. Recent studies have shown that the conjunctiva microbiome depends on age, presence of local and systemic inflammation, and environmental factors. Microbiome-based therapy, such as the use of oral probiotics to manage dry eye disease, has initial promising results. Further longitudinal studies are required to investigate the alteration of the conjunctival microbiome after local therapy and surgery.

Can Gut Microbiota Affect Dry Eye Syndrome?

Using metagenomics, continuing evidence has elicited how intestinal microbiota trigger distant autoimmunity. Sjögren's syndrome (SS) is an autoimmune disease that affects the ocular surface, with frequently unmet therapeutic needs requiring new interventions for dry eye management. Current studies also suggest the possible relation of autoimmune dry eye with gut microbiota. Herein, we review the current knowledge of how the gut microbiota interact with the immune system in homeostasis as well as its influence on rheumatic and ocular autoimmune diseases, and compare their characteristics with SS. Both rodent and human studies regarding gut microbiota in SS and environmental dry eye are explored, and the effects of prebiotics and probiotics on dry eye are discussed. Recent clinical studies have commonly observed a correlation between gut dysbiosis and clinical manifestations of SS, while environmental dry eye portrays characteristics in between normal and autoimmune. Moreover, a decrease in both the Firmicutes/Bacteroidetes ratio and genus Faecalibacterium have most commonly been observed in SS subjects. The presumable pathways forming the "gut dysbiosis-ocular surface-lacrimal gland axis" are introduced. This review may provide perspectives into the link between the gut microbiome and dry eye, enhance our understanding of the pathogenesis in autoimmune dry eye, and be useful in the development of future interventions.

Next-Generation Sequencing of the Ocular Surface Microbiome: In Health, Contact Lens Wear, Diabetes, Trachoma, and Dry Eye.

OBJECTIVES: To assess publications examining the occurrence, composition, and clinical significance of a microbiome at the ocular surface. METHODS: MEDLINE, EMBASE, and Google Scholar were searched. Reference lists of included articles were also searched for relevant citations. All publications up to June 1, 2019, were analyzed. RESULTS: Eleven articles and 1 abstract were included, analyzing 661 patients. Articles generally report bacteria to the genus level. The presence of DNA associated with diverse bacterial species was reported including pathogenic species, such as Pseudomonas and Neisseria. Bacterial DNA that makes up the microbiome, such as Acinetobacter, Actinomyces, Aquabacterium, Bradyrhizobium, Corynebacterium, Sphingomonas, Staphylococcus, and Streptococcus, in other parts of the body was found. The putative ocular microbiome is consistent between right and left eyes and is affected by contact lens use (higher Pseudomonas levels) and blepharitis (higher Staphylococcus levels). CONCLUSIONS: There is a significant likelihood that there is at least a transitory ocular surface microbiome, with Acinetobacter, Corynebacterium, Propionibacterium, Staphylococcus, and Streptococcus detected in at least 7 of 11 studies. However, further investigation attempting to control for environmental and methodological contaminants (Aquabacterium and Bradyrhizobium are commonly identified as contaminants in DNA extraction kits) is required. Bacteria, such as Propionibacterium, Staphylococcus, and Streptococcus, capable of causing sight-threatening infections may reside on a healthy ocular surface. With greater understanding, we can establish whether elements of the ocular surface microbiome are harmful or protective (despite their small quantities); furthermore, new therapeutic agents can be identified to treat and prevent ocular surface infection and inflammation.

Resistance of the murine cornea to bacterial colonization during experimental dry eye.

The healthy cornea is remarkably resistant to infection, quickly clearing deliberately inoculated bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus. Contrasting with the adjacent conjunctiva and other body surfaces, it also lacks a resident viable bacterial microbiome. Corneal resistance to microbes depends on intrinsic defenses involving tear fluid and the corneal epithelium. Dry eye, an ocular surface disease associated with discomfort and inflammation, can alter tear fluid composition and volume, and impact epithelial integrity. We previously showed that experimentally-induced dry eye (EDE) in mice does not increase corneal susceptibility to P. aeruginosa infection. Here, we explored if EDE alters corneal resistance to bacterial colonization. EDE was established in mice using scopolamine injections and dehumidified air-flow, and verified by phenol-red thread testing after 5 and 10 days. As expected, EDE corneas showed increased fluorescein staining versus controls consistent with compromised epithelial barrier function. Confocal imaging using mT/mG knock-in mice with red-fluorescent membranes revealed no other obvious morphological differences between EDE corneas and controls for epithelium, stroma, and endothelium. EDE corneas were imaged ex vivo and compared to controls after alkyne-functionalized D-alanine labeling of metabolically-active colonizing bacteria, or by FISH using a universal 16S rRNA gene probe. Both methods revealed very few viable bacteria on EDE corneas after 5 or 10 days (median of 0, upper quartile of ≤ 1 bacteria per field of view for each group [9-12 eyes per group]) similar to control corneas. Furthermore, there was no obvious difference in abundance of conjunctival bacteria, which included previously reported filamentous forms. Thus, despite reduced tear flow and apparent compromise to corneal barrier function (fluorescein staining), EDE murine corneas continue to resist bacterial colonization and maintain the absence of a resident viable bacterial microbiome.

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.

Interferons and Dry Eye in Sjögren's Syndrome.

Various cytokines, including interferon (IFN)-γ and IL-17, are augmented, and autoreactive T cells and B cells are activated in the immune pathogenesis of Sjögren's syndrome (SS). In particular, IFNs are involved in both the early stages of innate immunity by high level of type I IFN in glandular tissue and sera and the later stages of disease progression by type I and type II IFN producing T cells and B cells through B cell activating factor in SS. Genetically modified mouse models for some of these molecules have been reported and will be discussed in this review. New findings from human SS and animal models of SS have elucidated some of the mechanisms underlying SS-related dry eye. We will discuss IFN-γ and several other molecules that represent candidate targets for treating inflammation in SS-related dry eye.

Aging Eye Microbiota in Dry Eye Syndrome in Patients Treated with Enterococcus faecium and Saccharomyces boulardii.

BACKGROUND: Aging and oxidative stress seem to play a key role in the onset and progression of ocular surface diseases. Dry Eye Syndrome (DES) is a multifactorial disease of the tears and ocular surface in which symptoms may interfere with the ability to work and carry out daily functions. METHODS: This clinical trial was a pilot study to evaluate the effects of supplementation with mixture (Saccharomyces boulardii MUCL 53837 and Enterococcus faecium LMG S-28935) on the tear film. Following the run-in period subjects were randomized in two groups: group A (n.30 subjects) and group B (n.30 subjects). Group A (control) treated only with substitute tear and group B treated with substitute tear + mixture (probiotic). RESULTS: The data obtained in the two study groups A and B were, respectively the following: Schirmer I: 9.2±0.2 vs. 12.8±0.4 (p< 0.001); Schirmer II: 3.6±0.1 vs. 4.6±0.2 (p<0.001); BUT 3.8±0.3 vs. 6.2±0.2 (p<0.001). Culture test showed initial bacterial growth in group "A" (placebo) 27 out of 60 samples tested, corresponding to 45.0% and "B" after treatment (probiotic) was found positive culture whit growth of bacteria in 18 tests equal to 30.0%. The total numbers of isolations of aerobic and anaerobic bacteria found group A and B after treatment. A reduction of 23 to 16 strains of aerobic and anaerobic isolates from 13 to 7 has been found. CONCLUSION: The administration of probiotics strains was effective in reducing DES. In light of these results, we have identified our probiotic (Saccharomyces boulardii MUCL 53837 and Enterococcus faecium LMG S-28935) activity integration with the action of tear substitutes, along with standardization of clinical parameters of the tear film and microbiological activity in restoring of the microbiota ocular surface subject with DES.

Conjunctival Microbial Flora in Ocular Stevens-Johnson Syndrome Sequelae Patients at a Tertiary Eye Care Center.

PURPOSE: To evaluate the conjunctival microbial flora in cases of ocular Stevens-Johnson Syndrome (SJS) in a tertiary eye care center. METHODS: This prospective study comprised 176 eyes of 88 patients with ocular SJS compared with 124 eyes of normal subjects. The conjunctival swabs were collected and sent for microbiological analysis for bacterial isolation and antibiotic sensitivity examination. The type of bacteria isolated and its antibiotic sensitivity pattern were studied. RESULTS: Of 176 eyes, 104 (59%) had positive cultures for bacteria in cases of SJS and 16 (12.9%) had positive culture in the control group, the difference being statistically significant (P = 0.001). In the SJS group, 14 different types of bacterial isolates were identified. The most common isolate was coagulase-negative staphylococci (CNS) (30/104, 28.8%) followed by Corynebacteria species (35/104, 33.6%) and Staphylococcus aureus (19/104, 18.2%). More than 1 bacteria were isolated in 7 eyes (6.7%). Most of the isolates showed resistance to ciprofloxacin with no resistance to gatifloxacin and moxifloxacin. In the control group, only 2 bacteria were isolated, which included CNS (14/16, 87.5%) and Streptococcus pneumoniae (2/16, 12.5%). CNS showed resistance to ciprofloxacin, and S. pneumoniae was resistant to tobramycin and gentamycin. CONCLUSIONS: Ocular SJS is associated with alteration of the normal microbial flora residing in the conjunctival sac. The study of which is vital in cases of infection in these eyes with compromised ocular surface. Mixed flora are seen more often in cases of ocular SJS as compared with controls.

Altered Mucosal Microbiome Diversity and Disease Severity in Sjögren Syndrome.

There is mounting evidence that the microbiome has potent immunoregulatory functions. We assessed the effects of intestinal dysbiosis in a model of Sjögren syndrome (SS) by subjecting mice to desiccating stress (DS) and antibiotics (ABX). We characterized the conjunctival, tongue and fecal microbiome profiles of patients with SS. Severity of ocular surface and systemic disease was graded. 16S ribosomal RNA gene sequencing characterized the microbiota. ABX + DS mice had a significantly worse dry eye phenotype compared to controls, a decrease in Clostridium and an increase in Enterobacter, Escherichia/Shigella, and Pseudomonas in stool after ABX + DS for 10 days. Goblet cell density was significantly lower in ABX treated groups compared to controls. Stool from SS subjects had greater relative abundances of Pseudobutyrivibrio, Escherichia/Shigella, Blautia, and Streptococcus, while relative abundance of Bacteroides, Parabacteroides, Faecalibacterium, and Prevotella was reduced compared to controls. 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 ocular mucosal disease in a mouse model of SS and in SS patients.

Comparative role of 20% cord blood serum and 20% autologous serum in dry eye associated with Hansen's disease: a tear proteomic study.

BACKGROUND: To compare the role of topically applied serum therapy with preservative-free artificial tear (AT) drops in patients with moderate to severe dry eye in Hansen's disease along with change in tear protein profile. METHODS: 144 consecutive patients were randomly divided into three groups. After a baseline examination of clinical parameters, each of the patients received designated modality of topical therapy six times a day for 6 weeks. Post-treatment documentation of clinical parameters was done at 6 weeks, and then at 12 weeks after discontinuation of topical therapy. Analysis of three tear proteins using gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was done at baseline, at the first and second post-treatment visits. RESULTS: In the cord blood serum (CBS) group, except for McMonnies score and staining score, all other clinical parameters showed continued improvement in the first and second post-treatment analyses. In the autologous serum (ALS) group, all the clinical parameters except Schirmer's I showed significant improvement in the first post-treatment analysis .This was sustained at a significant level in the second analysis except for tear film break-up time (TBUT) and conjunctival impression cytology grading. In the AT group, all the parameters improved at a non-significant level except for TBUT in the first analysis. In the next analysis, apart from McMonnies score and TBUT, other clinical parameters did not improve. In the ALS and CBS groups, tear lysozyme, lactoferrin levels improved in both post-treatment measurements (statistically insignificant).Total tear protein continued to increase at statistically significant levels in the first and second post-treatment analyses in the CBS group and at a statistically insignificant level in the ALS group. In the AT group, the three tear proteins continued to decrease in both the analyses. CONCLUSIONS: In moderate to severe dry eye in Hansen's disease, serum therapy in comparison with AT drops, improves clinical parameters and causes betterment in tear protein profile. TRIAL REGISTRATION NUMBER: CTRI/2013/07/003802.

[Bacterial profiles in conjunctival sac of dry eyes and normal eyes in middle- and old-aged Yi populations].

OBJECTIVE: To compare the bacterial profiles in conjunctival sac of dry eyes and normal eyes in Yi people aged 40 years or old. METHODS: A cross-sectional survey was conducted with standardized training and protocol. A total of 140 dry eyes of 70 individuals from Yi people in Jiulong county underwent ophthalmological examinations. The secretions of the inferior palpebral conjunctival sac were embrocated and inoculated on blood plates for 48 hours. The bacteria were separated and identified. Another 132 normal eyes from 66 Yi individuals were examined as controls. RESULTS: Bacterial positive cultivations were found in 72.1% (101/140) of dry eyes and 67.4% (89/132) of normal eyes, respectively. The difference was not statistically significant (P = 0.397). No gender difference in the bacterial positive rates was found either. Gram-positive bacteria were the main bacteria in both dry eyes (95.2%, 98/103) and normal eyes (91.1%, 82/90), predominantly staphylococcus epidemids and corynebacterium. There were no statistical differences in the constituents of bacteria in Gram classification and numbers of bacteria between dry eyes and normal eyes (P > 0.05). CONCLUSION: The bacterial profile in conjunctival sac of dry eyes is similar to that of normal eyes in Yi people aged 40 years or over. Gram-positive bacteria are the main bacteria.

[Bacterial profiles in conjunctival sac of middle-aged and elderly Qiang with dry eyes and normal eyes].

OBJECTIVE: To compare the bacteria profiles in conjunctival sac of Qiang aged 40 years and over between those with dry eyes and those with normal eyes. METHODS: This survey was undertaken with a standardized protocol in Beichuan county. Ophthalmological examinations were performed on 54 individuals with dry eyes (108 eyes) and 52 individuals with normal eyes (104 eyes). The secretion of the inferior palpebral conjunctival sac was embrocated and inoculated on blood plate for 48-72 hours. The bacteria was separated and identified. RESULTS: Positive cultural results were found in 57.4% (62/108) of dry eyes and 61.5% (64/104) of normal eyes. The difference was not statistically significant (P=0.540). No gender and occupational differences in positive cultural results were found (P>0.05). Both the dry eyes and normal eyes were dominated by Gram-positive bacteria, with corynebacterium, staphylococcus epidemids and sphingomonas paucimobilis as main bacteria. Samples taken from the dry eyes produced 73 strains bacteria, 52 (83.8%) with a single strain of bacteria and 10 (16.2%) with co-existed strains of bacteria. The samples taken from the normal eyes were more likely to have co-existed strains of bacteria (P=0.000). Of the 87 strains of bacteria, 64.0%, were single strain and 36.0% were coexisted strains. Fifteen species of bacteria were cultivated from the dry eyes, significantly less than the number of species (24) cultivated from the normal eyes (P=0.027). CONCLUSION: There is no difference in bacterial positive rate in the conjunctival sac between dry eyes and normal eyes of Qiang aged 40 years and over. Gram-positive bacteria is the dominant bacteria. Normal eyes are more likely to have multiple coexisted bacteria compared with dry eyes.

Avaliação da microbiota ocular em pacientes com disfunção do filme lacrimal / Evaluation of conjunctival flora in patients with tear film dysfunction

OBJETIVO: Avaliar a microbiota conjuntival em olhos com disfunção do filme lacrimal, e a modificação desta microbiota após a colocação de plug de silicone no canalículo inferior. MÉTODOS: Série de casos intervencionais não comparativos para avaliar 68 olhos de 41 pacientes com disfunção do filme lacrimal, durante o período de 2002 a 2007, na Universidade Federal de São Paulo. Todos os pacientes foram submetidos à colheita de amostras de raspado conjuntival de fundo-de-saco inferior para cultivo em Brain heart infusion broth. Os vinte e dois pacientes submetidos à colocação de plug de silicone repetiram a colheita de raspado conjuntival um mês após o procedimento. RESULTADOS: Dos 68 olhos avaliados, 47 apresentaram crescimento bacteriano nas amostras colhidas. Nove diferentes espécies de bactérias foram identificadas: Staphylococcus coagulase negativa em 66,66 por cento, Staphylococcus aureus em 13,72 por cento, Corynebacterium sp em 5,86 por cento, Enterobacter aerogenes em 3,92 por cento, Streptococcus hemolítico do grupo viridans em 1,96 por cento, Serratia sp em 1,96 por cento, Alcaligenes xylosoxidans spp em 1,96 por cento, Corynebacterium xerosis em 1,96 por cento, e Proteus mirabilis em 1,96 por cento. Staphylococcus coagulase negativa (SCN) foi o microrganismo mais frequentemente isolado tanto antes quanto após o plug de silicone. A sensibilidade do SCN à Oxacilina antes da colocação do plug era de 87,50 por cento, e, após, de 73,68 por cento. CONCLUSÃO: A microbiota em olhos com disfunção do filme lacrimal é bastante semelhante à encontrada em olhos normais. A resistência de SCN à Oxacilina foi um pouco maior após o implante do plug de silicone.

PURPOSE: To evaluate conjunctival microbiota in eyes with tear film dysfunction and its modification after punctal occlusion with silicone plug. METHODS: Non comparative interventional case series study to evaluate 68 eyes of 41 patients with tear film dysfunction, from 2002 to 2007, followed in Federal University of Sao Paulo. Samples for culture were all obtained from conjunctival swabs and inoculated in Brain heart infusion broth (BHI broth). Twenty two patients that undergone punctal plug occlusion repeated culture procedure one month after plug insertion. RESULTS: 47 of the 68 eyes evaluated had positive culture in their samples. Nine different types of bacteria were identified: Coagulase negative Staphylococcus in 66,66 percent, Staphylococcus aureus, in 13,72 percent, Corynebacterium sp, in 5,86 percent, Enterobacter aerogenes, in 3,92 percent, Streptococcus hemolítico do grupo viridans, in 1,96 percent, Serratia sp, in 1,96 percent, Alcaligenes xylosoxidans spp, in 1,96 percent, Corynebacterium xerosis, in 1,96 percent, and Proteus mirabilis in 1,96 percent. Coagulase negative Staphylococcus (CNS) was the most frequently isolated microorganism before and after punctal occlusion. CNS sensibility to Oxacilin before plug insertion was 87,50 percent, and after, 73,68 percent. CONCLUSION: Microbiota found in eyes with tear film dysfunction seems to be similar to that found in normal eyes. CNS resistance to oxacilin was slightly higher after silicone plug insertion.

The role of microbial flora on the ocular surface.

PURPOSE OF REVIEW: Presence and interplay of microbial flora at the ocular surface reveal dynamic and evolving interactions with implications for both ocular surface health and disease. Data in this area are scarce or non-existent. The purpose of this review is to provide a snapshot of new and emerging developments in this area over the last 12 months. RECENT FINDINGS: Recent findings signal potential roles for ocular surface microbial flora in both the preservation and extension of ocular surface health and in the initiation of new or escalation of common surface disorders. Contributions range from priming surface epithelial immune cells to regulating mucin composition and production. Other findings explore the emergent role of ocular microbial flora cross talk with pattern recognition receptors to protect and strengthen local and adaptive mucosal immunity while preserving vision. SUMMARY: Deciphering the functional role of microbial communities at the ocular surface could bring new insights into and clarify the epidemiology and pathology of ocular surface dynamics in health and disease.

Bacteriologic profile of the conjunctiva in the patients with dry eye.

PURPOSE: To assess the conjunctival bacterial profiles in dry eye and their fluoroquinolone susceptibility patterns. DESIGN: Prospective, observational study. METHODS: Sixty-seven female patients with dry eye (29 with Sjögren syndrome and 38 without Sjögren syndrome) who received artificial tears were enrolled at Osaka University Hospital in Japan. Twenty-three patients received additional topical steroids. Twenty-six puncta were occluded with plugs. Cultures were obtained with conjunctival swabs at the right eye of the subjects. The minimum inhibitory concentrations of isolated strains were determined for the fluoroquinolones (levofloxacin and gatifloxacin). The profiles of conjunctival bacteria of patients with dry eye were compared with those obtained before surgery from 56 female control patients. RESULTS: Eighty-eight strains were isolated (48 strains of Propionibacterium acnes, 26 coagulase-negative Staphylococcus [CNS] species, six Staphylococcus aureus strains, and eight others). Of the 26 CNS strains, 17 (65.4%) were fluoroquinolone resistant, including four (33.3%) of 12 methicillin-sensitive CNS and 13 (92.9%) of 14 methicillin-resistant CNS. All methicillin-sensitive S. aureus strains and P. acnes strains were sensitive to fluoroquinolones; one methicillin-resistant S. aureus strain was resistant. There was no significant difference in the conjunctival isolation rates between patients with dry eye and controls. However, the dry eye group had a significantly higher incidence of fluoroquinolone-resistant methicillin-sensitive CNS and of fluoroquinolone-resistant methicillin-resistant CNS than controls (P = .018 and P = .024, respectively). There were no significant differences in bacteria isolated between subgroups with or without punctal plugs and with or without topical steroids. CONCLUSIONS: Patients with dry eye are more likely to have fluoroquinolone-resistant conjunctival bacteria than controls. These results may help prevent infectious keratoconjunctivitis in patients with dry eye.

Ocular pathogen or commensal: a PCR-based study of surface bacterial flora in normal and dry eyes.

PURPOSE: To compare the bacterial population of the ocular surface of normal and dry eye subjects using conventional culture and 16S rDNA PCR. METHODS: Ninety-one subjects were classified as normal (n = 57) or dry eye (n = 34) by using tear break-up time, McMonnies survey, goblet cell density, and meibomian gland assessment. Conventional bacterial culture and broad-range 16S rDNA PCR, cloning, and DNA sequencing were used for bacterial identification. Repeated sampling was performed in a subset of subjects over a 3-month period. The association between goblet cell loss and bacterial counts in a subgroup of subjects was assessed. RESULTS: Most of the bacteria identified by culture were coagulase negative staphylococci, whereas molecular methods demonstrated a considerable number of additional bacteria. Atypical ocular surface bacteria including Rhodococcus erythropolis, Klebsiella oxytoca, and Erwinia sp., were identified in cases of overt inflammation and, surprisingly, on the normal ocular surface. The same bacteria remained on the ocular surface after repeated sampling. Increased bacterial flora was associated with reduced goblet cell density. CONCLUSIONS: Molecular analysis revealed a diverse ocular surface bacterial population. In addition to the normal flora, various potentially pathogenic bacteria were identified. The detection of known pathogens in both normal and dry eyes, with minimal signs of infection, presents a diagnostic dilemma. It remains unknown whether their presence is associated with inflammation and reduced goblet cell density or whether they adversely affect the ocular surface predisposing it to abnormal microbial colonization. In the absence of overt clinical infection, it is unknown whether such results should prompt intervention with therapy.

Effect of antibacterial honey on the ocular flora in tear deficiency and meibomian gland disease.

PURPOSE: To assess for differences in the ocular flora of patients with dry eye caused by tear deficiency and/or meibomian gland disease and to assess the effect of antibacterial honey on the ocular flora in these forms of dry eye. METHODS: In this prospective, open-label pilot study, bacteria isolated from the eyelid margin and conjunctiva were identified and quantified before and at 1 and 3 months after initiation of treatment with topical application of antibacterial honey 3 times daily. Subjects had non-Sjogren tear deficiency (n = 20), Sjogren syndrome tear deficiency (n = 11), meibomian gland disease (n = 15), and non-Sjogren tear deficiency with meibomian gland disease (n = 20), and there were 18 non-dry eye subjects. RESULTS: The total colony-forming units (CFUs) isolated from each of the dry eye subgroups before antibacterial honey use was significantly greater than the total CFU isolated from the non-dry eye group. Antibacterial honey use significantly reduced total CFUs for the eyelids and the conjunctiva of dry eye subjects from baseline at month 1 (eyelids: P = 0.0177, conjunctiva: P = 0.0022) and month 3 (eyelids: P < 0.0001, conjunctiva: P < 0.0001). At month 3, there were reductions in total CFUs for all dry eye subgroups such that the CFUs were not significantly different from those of the non-dry eye group. CONCLUSION: From these results, there is sufficient preliminary data to warrant further study of the effects of antibacterial honey in chronic ocular surface diseases.

Short term oral minocycline treatment of meibomianitis.

AIM: To evaluate the clinical impact, aqueous tear parameters, and meibomian gland morphology in patients with primary meibomianitis before, during, and 3 months after a course of oral minocycline. METHODS: 16 patients were prospectively enrolled, 11 male and five female (mean age 69 years old). Each patient received routine clinical evaluations before, after 3 months therapy, and at 6 month study follow up visit. The clinical appearance, tear volume, flow and turnover, evaporation, Schirmer I test, meibomian gland dropout, lissamine green staining, and bacteriology wer evaluated. RESULTS: Improvement was observed in clinical signs of meibomianitis at the second and third visits. Microbial culture findings improved. Decreased aqueous tear volume and flow, and increased evaporation rate range at 35-45% relative humidity (RH) (p < 0.05) were also detected. Other related tear parameters did not change. Meibomian gland dropout showed no improvement. CONCLUSIONS: 3 months of oral minocycline resulted in clinical improvements in all meibomianitis signs that persisted for at least 3 months after discontinuation despite decreased aqueous tear volume and flow with increased evaporation (35-45% RH). However, there was improvement in the turbidity of secretions. Short term minocycline therapy probably has efficacy in the management of meibomianitis that extends beyond eradication of bacteria.

The treatment of severe trachomatous dry eye with canalicular silicone plugs.

PURPOSE: To evaluate the effects of temporary canalicular occlusion with silicone plugs on trachomatous dry eye patients who were on maximal tolerable medical therapy. METHODS: Forty-four trachomatous dry eye patients who had Schirmer testing with topical anaesthetic measuring 5 mm or less and a tear film break-up time of 5 s or less were included. After the lacrimal efficiency test with dissolvable collagen punctal plugs, silicone canalicular plugs were placed in 22 trachomatous dry eye patients. The other 22 patients in the untreated control group were allowed to continue their medical therapy. Pretreatment and post-treatment evaluations included subjective patient assessment, rose Bengal and fluorescein staining, tear film break-up time, Schirmer testing, conjunctival impression cytology and goblet cell counting. RESULTS: Six months after plug placement, 18 eyes (82%) of 22 patients had subjective improvement and all these patients successfully wore plugs for at least 6 months. There were statistically significant differences between the pretreatment and post-treatment test results including rose Bengal and fluorescein staining scores, tear film break-up times and Schirmer testing measurements. Impression cytology showed improvement of squamous metaplasia in 17 eyes (77%). Eight of the patients (36%) were able to decrease dependency on topical therapy. Ten of the patients (45%) completely stopped using artificial tears. There were statistically significant differences between the two groups in the total symptom scores, staining scores, tear film break-up time, Schirmer testing, impression cytology scores and goblet cell counts. CONCLUSION: In cases where topical tear supplementation is insufficient to relieve the signs and symptoms of severe dry eye and the lacrimal puncta have not already been closed by the trachomatous cicatrising process, occlusion of the canaliculi may be useful to prevent drainage of both natural and artificial tears. Canalicular occlusion improves the objective signs and subjective symptoms and may significantly decrease dependency on tear supplements in selected patients.