Does corneal tattooing affect the conjunctival microbiota?

PURPOSE: This study aimed to investigate the effects of commercial tattoo inks used in corneal tattooing on conjunctival microbiota. METHOD: This prospective case control study consisted of 125 participants divided in the following three groups: 35 patients with corneal tattoos, 40 patients with corneal leukoma, and 50 healthy subjects. Corneal tattooing was performed in all the cases in this study using a tattoo pen machine and commercial tattoo ink. A total of 500 cultures were taken from 250 eyes of 125 individuals on chocolate and sheep blood agar. Bacteriological samples were taken from the inferior eyelid conjunctiva using a sterile cotton swab. Without any contact elsewhere, the swabs were smeared on bedside chocolate agars and 5% sheep blood agar. RESULTS: In tattooed eyes, bacterial growth was detected in 42.9% of the chocolate and sheep blood agar samples. In other healthy eyes of patients with corneal tattoos, 54.5% bacterial growth on chocolate agar and 57.1% on sheep blood agar were detected. No statistical difference was detected in the conjunctival microbiota of chocolate and sheep blood agar (p = 0.254, p = 0.134, respectively) in the tattooed eyes compared to the other eye of the individual. No statistically significant difference was found in terms of bacterial growth in tattooed, leukoma, or healthy eyes on chocolate and sheep blood agar (p = 0.408, p = 0.349). The growth rate of Staphylococcus epidermidis decreased by 33.3% (from 12 to 8) on chocolate agar in 35 tattooed eyes, and it decreased by 28.5% (from 14 to 10) on sheep blood agar, while gram-negative bacteria Brevundimonas diminuta, Acinetobacter lwoffii, and Psychrobacter faecalis were detected in three patients. CONCLUSION: Corneal tattooing using commercial dye does not affect conjunctival microbiota. In the past 3 years, 120 patients have been tattooed with commercial tattoo ink in Istanbul Medeniyet University Göztepe Training and Research Hospital. No complications related to infection were found in the 3-year follow-up. The gram-negative bacteria detected in the healthy control group and tattooed eyes were bacteria found on normal skin or in the respiratory tract. Although some gram-negative bacteria do not cause infection, careful eye examination, follow-up, and culture are required in suspicious cases.

Initial description of the core ocular surface microbiome in dogs: Bacterial community diversity and composition in a defined canine population.

OBJECTIVE: To characterize the bacterial community residing on the conjunctiva of clinically healthy dogs. METHODS: Bacterial DNA from conjunctival swabs of 10 dogs with normal ocular examinations (both OD and OS, n = 20) was extracted, and 16S rRNA amplicons were sequenced using Illumina MiSeq 600. Resulting data were subjected to quality control steps, and analyzed for bacterial community richness and diversity, within- and between-group dissimilarity, and relative taxonomic composition. RESULTS: High-quality reads (2.22 million bp) resulted in a mean of 159 068 sequences per sample. Bacterial community evenness and diversity was high when compared to other species, and did not significantly differ when samples were grouped by dogs or eyes. As expected, within-dog samples were more similar than between-dog samples. Taxonomic classification revealed that >95% of the community consisted of Firmicutes (34.9 ± 8.8%), Actinobacteria (26.3 ± 7.1%), Proteobacteria (26.2 ± 6.6%), and Bacteroidetes (9.4 ± 2.4%). Key members of the dog ocular surface microbiome, found in all dogs and corresponding to >25% of all identified OTUs (operational taxonomic units), were part of the Bifidobacteriaceae, Lachnospiraceae, Moraxellaceae, Corynebacteriaceae families. Genera previously thought to account for the majority of the core ocular surface microbiome in the dog (Staphylococcus sp., Streptococcus sp., and Bacillus sp.) were associated with only 2.63% of overall reads. CONCLUSIONS: This study shows the feasibility of conjunctival swabs and high-throughput sequencing to profile the bacterial community structure of the canine ocular surface. A core ocular surface microbiome was identified for this canine population.

Conjunctival Microbiota in Patients With Type 2 Diabetes Mellitus and Influences of Perioperative Use of Topical Levofloxacin in Ocular Surgery.

Background: Patients with type 2 diabetes mellitus (T2DM) are prone to ocular surface infections. We therefore characterized the conjunctival microbiome of T2DM patients and the influence of topical levofloxacin to investigate whether a dysbiosis is associated with this phenomenon. Methods: Conjunctival microbiome of 79 T2DM patients and 113 non-diabetic controls was profiled using the 16S rDNA sequencing approach. Furthermore, 21 T2DM and 14 non-diabetic patients who underwent cataract surgeries were followed up perioperatively and the influence of pre- and post-operative levofloxacin on the conjunctival microbiome was further investigated prospectively and compared longitudinally. Results: The α-diversity of the conjunctival microbiota was significantly higher in T2DM patients than in controls (P < 0.05). Significant differences in both composition and function of the conjunctival microbiome were identified on the ocular surface of T2DM patients as compared to non-diabetic controls. Particularly, phylum Bacteroidetes and Fusobacteria, genus Pseudomonas, Haemophilus, and Empedobacter were enriched, while genus Streptococcus was reduced on the T2DM ocular surface. Microbial genes functioning of bacterial chemotaxis was elevated in the conjunctival microbiome of T2DM patients. Furthermore, compared to the initial status, several genera including Staphylococcus were more abundant in the conjunctival microbiome of T2DM patients after 3-days use of preoperative levofloxacin topically, while no genus was more abundant in the non-diabetic follow-up group. No difference was observed between initial status and 7 days after ceasing all postoperative medications in both diabetic and non-diabetic follow-up groups. Conclusions: The conjunctival microbiome of T2DM patients was more complex and may respond differently to topical antibiotics.

Characterization of Ocular Surface Microbial Profiles Revealed Discrepancies between Conjunctival and Corneal Microbiota.

The ocular microbiome composition has only been partially characterized. Here, we used RNA-sequencing (RNA-Seq) data to assess microbial diversity in human corneal tissue. Additionally, conjunctival swab samples were examined to characterize ocular surface microbiota. Short RNA-Seq reads, obtained from a previous transcriptome study of 50 corneal tissues, were mapped to the human reference genome GRCh38 to remove sequences of human origin. The unmapped reads were then used for taxonomic classification by comparing them with known bacterial, archaeal, and viral sequences from public databases. The components of microbial communities were identified and characterized using both conventional microbiology and polymerase chain reaction (PCR) techniques in 36 conjunctival swabs. The majority of ocular samples examined by conventional and molecular techniques showed very similar microbial taxonomic profiles, with most of the microorganisms being classified into Proteobacteria, Firmicutes, and Actinobacteria phyla. Only 50% of conjunctival samples exhibited bacterial growth. The PCR detection provided a broader overview of positive results for conjunctival materials. The RNA-Seq assessment revealed significant variability of the corneal microbial communities, including fastidious bacteria and viruses. The use of the combined techniques allowed for a comprehensive characterization of the eye microbiome's elements, especially in aspects of microbiota diversity.

Metagenomic Analysis Reveals the Heterogeneity of Conjunctival Microbiota Dysbiosis in Dry Eye Disease.

Background: Dry eye disease (DED) is a multifactorial inflammatory disease of the ocular surface. It is hypothesized that dysbiosis of the conjunctival microbiota contributes to the development of DED. However, species-level compositions of the conjunctival microbiota in DED and the potential dysbiosis involving microorganisms other than bacteria remain largely uncharacterized. Methods: We collected conjunctival impression samples from a cohort of 95 individuals, including 47 patients with DED and 48 healthy subjects. We examined the conjunctival microbiota of these samples using shotgun metagenomic sequencing and analyzed microbial dysbiosis in DED at the species level. Results: The conjunctival microbiota in DED exhibited a decreased α-diversity and an increased inter-individual variation. The α-diversity of female patients with DED was higher than that of male patients. Despite a decreased prevalence in DED, 23 microbial species were identified to show abnormally high abundance in DED samples positive for the species. Among these species, a fungal species Malassezia globosa was enriched female patients. In addition, distinct patterns of associations with disease status were observed for different species of the same genus. For DED subtypes, Staphylococcus aureus and S. capitis were associated with meibomian gland dysfunction (MGD), whereas S. hominis was enriched in patients solely with aqueous tear deficiency (ATD). The microbiota of patients with a mixed type of diagnosis was more similar to MGD patients than ATD patients. Conclusion: We demonstrated that the conjunctival microbiota dysbiosis in DED is characterized by significant heterogeneity. Microbial signatures may offer novel insights into the complicated etiology of DED and potentially promote the development of personalized treatment for DED in the future.

Ocular Surface Microbiota in Contact Lens Users and Contact-Lens-Associated Bacterial Keratitis.

Our objectives were to investigate whether the conjunctival microbiota is altered by contact lens wear and/or bacterial keratitis and to explore the hypothesis that commensals of conjunctival microbiota contribute to bacterial keratitis. Swab samples from both eyes were collected separately from the inferior fornix of the conjunctiva of non-contact-lens users (nparticipants = 28) and contact lens users (nparticipants = 26) and from patients with contact-lens-associated bacterial keratitis (nparticipants = 9). DNA from conjunctival swab samples was analyzed with 16S rRNA gene amplicon sequencing. Pathogens from the corneal infiltrates were identified by cultivation. In total, we identified 19 phyla and 283 genera; the four most abundant genera were Pseudomonas, Enhydrobacter, Staphylococcus, and Cutibacterium. Several pathogens related to bacterial keratitis were identified in the conjunctival microbiota of the whole study population, and the same bacteria were identified by both methods in the conjunctiva and cornea for four patients with contact-lens-associated bacterial keratitis. The overall conjunctival microbiota profile was not altered by contact lens wear or bacterial keratitis; thus, it does not appear to contribute to the development of bacterial keratitis in contact lens users. However, in some individuals, conjunctival microbiota may harbor opportunistic pathogens causing contact-lens-associated bacterial keratitis.

Effect of scleral lens use on conjunctival microbiota.

AIM: This study aimed to investigate the effect of scleral lens (SL) use on conjunctival microbiota. METHOD: A total of 26 eyes of 26 patients using an SL and 25 eyes of 25 healthy controls were included in the study. The samples were obtained from the lower fornices of the eyes using sterile swabs. For the bacteriological examination, a bacterial culture was obtained by inoculating the samples on chocolate agar, blood agar, MacConkey agar, and fluid thioglycollate medium. After 24-48 h of incubation at 37 0C, the growth of different colonies of bacteria was identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Bruker MALDI Biotyper). RESULTS: The mean age of the study group was 41.6 ± 19.1 years (18-65); the mean age of the control group was 40 ± 6 (21-62) (p = 0.69). There were 10 male patients and 16 female patients in the study group and 9 male patients and 16 female patients in the control group (p = 0.86). The mean duration of SL use was 13.7 ± 13.4 months (1-42 months). No bacterial growth was observed in 17 (65.4 %) of the 26 eyes in the SL group and 5 (20 %) of the 25 eyes in the control group (p = 0.001). The most commonly observed microorganisms were Staphylococcus epidermidis (S.epidermidis) and Staphylococcus aureus (S.aureus) in both groups. CONCLUSION: SL users were found to have a higher rate of culture negativity in comparison to the healthy controls, suggesting that SLs have a significant effect on conjunctival microbiota.

Microbiota conjuntival preoperatoria de pacientes diabéticos candidatos a cirugía de catarata / Preoperative Conjunctival Microbiota of Diabetic Patients who are Candidates for Cataract Surgery

Introducción: La microbiota conjuntival en pacientes con catarata puede afectar el posoperatorio, lo que se puede hacer más complejo en pacientes con diabetes mellitus. Objetivos: Determinar la microbiota conjuntival en los pacientes diabéticos de la línea preoperatoria pendiente a cirugía de catarata en el Instituto Cubano Oftalmología "Ramón Pando Ferrer" desde septiembre de 2018 hasta diciembre de 2019. Método: Se realizó una investigación observacional, descriptiva, de corte transversal con 45 pacientes diabéticos diagnosticados de catarata senil, que fueran atendidos en el período y hospital antes mencionado. A todos los pacientes se les realizó hisopado conjuntival en cultivo correspondiente. Se realizó anamnesis para recoger datos sobre la diabetes. Los datos obtenidos (matriz en Excel) se analizaron con software SPSS 21, y los resultados se presentan en tablas de frecuencias. Se utilizó ji cuadrado para la correlación entre variables. Resultados: De los 45 pacientes, predominaron las mujeres (57,8 por ciento), con edades de 60 años y más (68,9 por ciento), y sin antecedentes patológicos oculares (82,2 por ciento). Hubo crecimiento bacteriano en el 66,7 por ciento (p < 0,002). El germen más frecuente fue Staphylococcus coagulasa negativa (48,3 por ciento). Conclusiones: El estudio de la microbiota conjuntival preoperatoria garantiza, en parte, el éxito de la cirugía de catarata(AU)

Introduction: The conjunctival microbiota in cataract patients may affect the postoperative course, which may become more complex in patients with diabetes mellitus. Objectives: To determine the conjunctival microbiota in diabetic patients in the preoperative line pending cataract surgery at the Cuban Ophthalmology Institute "Ramón Pando Ferrer" from September 2018-2019. Methods: An observational, descriptive, cross-sectional, descriptive research was conducted, 45 diabetic patients, diagnosed with senile cataract, who were treated in the period and hospital mentioned above. All patients underwent conjunctival swabbing and corresponding culture. Anamnesis was performed to collect data on diabetes. The data obtained (matrix in Excel) were analyzed with SPSS 21 software, and the results were presented in frequency tables. Chi-square was used for correlation between variables. Results: Out of the 45 patients, women predominated (57.8 percent), aged 60 years and older (68.9 percent), and with no ocular pathological history (82.2 percent). There was bacterial growth in 66.7 percent (p < 0.002). The most frequent germ was coagulase-negative Staphylococcus (48.3 percent). Conclusions: The study of the preoperative conjunctival microbiota, guarantees, in part, the success of cataract surgery(AU)

Cinnamon oil: A possible alternative for contact lens disinfection.

OBJECTIVE: To investigate the antibacterial activity of cinnamon oil alone and in combination with a multipurpose contact lens disinfectant solution (MPS) as well as tobramycin against multi drµg resistant conjunctival bacteria both in planktonic and sessile forms. METHODS: Minimum inhibitory concentrations (MIC) of tobramycin and cinnamon oil against 19 bacterial strains were investigated against planktonic and sessile cells by micro-dilution methods. Synergistic effects were determined by well diffusion and micro-dilution tissue culture plate methods for planktonic and sessile cells respectively. Time kill assay was performed to study the bactericidal effect of cinnamon oil in concentrations ranging from 5% to 0.312% combined with an MPS with respect to time. RESULTS: MICs of cinnamon oil against planktonic bacteria ranged from 0.04% to 1.25% versus 0.156% to 5% for sessile cells. Combination of cinnamon oil with tobramycin had a synergistic effect against most tested organisms. The MIC values of cinnamon oil in combination with tobramycin was significantly lower than cinnamon oil alone against biofilm production (P=0.004). Time kill assay revealed that combination of cinnamon oil and disinfectant successfully eradicated the tested microorganisms at all tested concentrations within 2h contact time except for 0.312% concentration (3h) versus 24h for MPS alone. CONCLUSION: Cinnamon oil has a promising antimicrobial effect. It could be a probable candidate for contact lens disinfection.

Defining the normal core microbiome of conjunctival microbial communities.

Bacterial ocular infections are common. Traditional culture and molecular biological methods have obvious limitations to identify the conjunctival microbiota, while metagenomic studies can avoid the defects of these methods. We used the Illumina high-throughput sequencing technology (MiSeq Illumina Sequencing Platform) to sequence the 16S rDNA V3-V4 hypervariable region of all bacteria in conjunctival swab samples. The operational taxonomic units were obtained from the sequences. The bioinformatic analyses of taxonomy, abundance and alpha diversity were performed. A total of 840 373 high-quality sequencing reads were generated from 31 conjunctival samples. The number of the species operational taxonomic units ranged from 159 to 2042, indicating high microbial diversity. The combined bacterial community was classified into 25 phyla and 526 distinct genera. At the genus level, Corynebacterium (28.22%), Pseudomonas (26.75%), Staphylococcus (5.28%), Acinetobacter (4.74%), Streptococcus (2.85%), Millisia (2.16%), Anaerococcus (1.86%), Finegoldia (1.68%), Simonsiella (1.48%) and Veillonella (1.00%) accounted for over 76% of the microbial community, possibly representing the core genera in normal conjunctival microbiota. The composition and diversity of microbiota in the normal adult human conjunctiva were characterized using high-throughput sequencing. A framework for investigating potential roles played by the diverse microbiota in disease related with the ocular surface was provided.

Tear production, intraocular pressure and conjunctival microbiota, cytology and histology of New Zealand rabbits (Oryctolagus cuniculus) / Produção lacrimal, pressão intraocular, microbiota, citologia e histologia conjuntival de coelhos Nova Zelândia

The purpose of this study was to establish reference values for selected ophthalmic diagnostic tests in New Zealand rabbits (Oryctolagus cuniculus). A total of 22 adult male rabbits were used. The ophthalmic tests included evaluation of tear production with Schirmer tear test 1(STT1) and Endodontic absorbent paper point tear test (EAPPTT) using two different commercial brand materials. Applanation tonometry, Culture of the conjunctival bacterial flora, , conjunctival cytology and conjunctival histology were also performed. Mean (±SD) for STT1, EAPPTTa, EAPPTTb and IOP was 7.27±2.51mm/min, 12.43±1.69mm/min, 15.24±2.07mm/min, 12.89±2.80mm Hg, respectively. Staphylococcus epidermidis, Staphylococcus sp. and Bacillus sp. were predominant. The cytological evaluation revealed the presence columnar epithelial cells, superficial squamous keratinized cells, lymphocytes, heterophils, red blood cells, mucus and bacteria. The histological analysis revealed a stratified epithelium, characterized by the presence of columnar epithelial cells with a large number of goblet cells. The reported data can be used for therapeutic or experimental purposes.

O objetivo deste estudo foi estabelecer valores de referência para testes diagnósticos oftálmicos em coelhos da raça Nova Zelândia (Oryctolagus cuniculus). 22 coelhos, machos, adultos foram utilizados. Foi mensurada a produção lacrimal através do teste lacrimal de Shirmer 1 (TLS1) e da Tira endodôntica de papel absorvente (EAPPTT) de duas marcas comerciais distintas. Tonometria de aplanação, identificação da microbiota conjuntival, , citologia e histologia conjuntival também foram realizadas. A média e desvio padrão do TLS1, EAPPTT1, EAPPTT2 e pressão intraocular foi 7,27±2,51 mm/min, 12,43±1,69 mm/min, 15,24±2,07 mm/min e 12,89±2,80 mmHg, respectivamente. Staphylococcus epidermidis, Staphylococcus sp. e Bacillus sp. mostraram-se predominantes. A citologia conjuntival evidenciou a presença de células epiteliais colunares, células escamosas superficiais queratinizadas, linfócitos, heterofilos, células sanguíneas, muco e bactérias. A histologia revelou epitélio estratificado caracterizado pela presença de células epiteliais colunares com grande número de células caliciformes. Os achados deste estudo poderão ser utilizados com fins terapêuticos ou experimentais.