Global Transcriptomic Profiling of Innate and Adaptive Immunity During Aspergillus flavus Endophthalmitis in a Murine Model.

Purpose: Fungal endophthalmitis is characterized by chronic inflammation leading to the partial or complete vision loss. Herein, we analyzed the transcriptomic landscape of Aspergillus flavus (A. flavus) endophthalmitis in C57BL/6 mice to understand the host-pathogen interactions. Methods: Endophthalmitis was induced by intravitreal injection of A. flavus spores in C57BL/6 mice and monitored for disease progression up to 72 hours. The enucleated eyeballs were subjected to histopathological analysis and mRNA sequencing using the Illumina Nextseq 2000. Pathway enrichment analysis was performed to further annotate the functions of differentially expressed genes (DEGs) and validation of cytokines was performed in vitreous of patients with fungal endophthalmitis using multiplex ELISA. Results: Transcriptomic landscape of A. flavus endophthalmitis revealed upregulated T-cell receptor signaling, PI3K-AKT, MAPK, NF-κB, JAK-STAT, and NOD like receptor signaling pathways. We observed significant increase in the T-cells during infection especially at 72 hours infection along with elevated expression levels of IL-6, IL-10, IL-12, IL-18, IL-19, IL-23, CCR3, and CCR7. Furthermore, host-immune response associated genes, such as T-cell interacting activating receptor, TNF receptor-associated factor 1, TLR1, TLR9, and bradykinin receptor beta 1, were enriched. Histopathological assessment validated the significant increase in inflammatory cells, especially T-cells at 72 hours post-infection along with increased disruption in the retinal architecture. Additionally, IL-6, IL-8, IL-17, TNF-α, and IL-1ß were also significantly elevated, whereas IL-10 was downregulated in vitreous of patients with Aspergillus endophthalmitis. Conclusions: Regulating T-cell influx could be a potential strategy to modulate the excessive inflammation in the retina and potentially aid in better vision recovery in fungal endophthalmitis.

A Novel and Low-cost Approach for Intravitreal Injection in an Experimental Model of Endophthalmitis.

Purpose: Animal models are necessary in understanding the pathogenesis of endophthalmitis and are also necessary to assist the development of new therapeutics for this sight-threatening ocular inflammation. Hamilton syringes are usually preferred to inject pathogens when performing experiments on test subjects, however, this method has technical and financial disadvantages. In this study, we report the findings and assess the related benefits of applying a novel low-cost intravitreal injection technique to initiate endophthalmitis in a mouse model while using the Eppendorf tip and a 26G needle. Methods: The 18-hr culture of clinical isolates of bacteria (Staphylococcus aureus and Pseudomonas aeruginosa) and fungus (Aspergillus flavus and Candida albicans) were resuspended to a final concentration of 10,000 colony forming units (CFU)/1 µL which were separately injected intravitreally into C57BL/6 mice (6-8 weeks) using a 0.1-2.5µL pipette attached to the modified Eppendorf tip with a 26G needle. The contralateral eye served as vehicle/uninjected control. Disease progression was determined by assessing the corneal haze, opacity, bacterial burden, and retinal histology of the eyes used in the model. Following euthanization, bacteria-infected mice were enucleated after 24 hr of the initial injection, and fungus-infected mice after 72 hr. Results: Of the 50 mice injected, the modified technique was successful in 48 mice. Two mice were excluded due to cataract formed by accidental injury to the lens. The experimental endophthalmitis mice model successfully mimicked the natural clinical course. Clinical assessment and histopathology confirmed the influx of inflammatory cells into the posterior segment of the eye along with dissolution of retinal architecture. Conclusion: Our novel method of injection using a modified Eppendorf tip and 26G needle yielded a cost-effective mouse model of clinical endophthalmitis, resulting in reproducible infection for understanding various aspects of its pathobiology.

Microbiological spectrum and antibiotic susceptibility patterns of non-viral conjunctivitis over 16 years at a tertiary eye care center in Southern India.

PURPOSE: To determine the trends in the microbial spectrum and antibacterial susceptibility patterns of non-viral conjunctivitis over 16 years. METHODS: Microbiology data were reviewed from 2006-2021 for all the patients with clinically and culture-proven infectious conjunctivitis. Conjunctival swabs and/or conjunctival scrapings were collected for microbiological workup, and the demographic and antibiotic susceptibility data were extracted from the EMR (Electronic Medical Record). For statistical analysis, χ2-test was performed. RESULTS: Of the 1711 patients, 814 (47.57%) were culture positive and 897 (52.43%) were culture negative. Of the total culture-proven conjunctivitis cases, 775/814 (95.20%) were bacteria, and 39/814 (4.80%) were fungi. Among these bacterial isolates, 75.74% were gram-positive bacteria, while 24.26% were gram negative. The predominant gram-positive pathogens isolated were S. epidermidis (16.7%), S. aureus (17.9%) (p < 0.05), and S. pneumoniae (18.2%), while Haemophilus spp. (36.2%) (p < 0.05) was the most often isolated gram-negative bacteria (36.2%), and Aspergillus spp. was the most commonly isolated fungus (50%). The susceptibility of gram-positive bacteria to cefazoline increased from 90.46 to 98% (p = 0.01), whereas the susceptibility for gatifloxacin decreased in both gram-positive (81-41%; p < 0.0001) and gram-negative bacteria (73-58%; p = 0.02). CONCLUSIONS: Increasing resistance of ocular isolates to mainstay antibiotics is a concern, and this data can assist healthcare practitioners in making informed choices regarding the treatment of ocular infections with ophthalmic antibiotics.

Aquaporin 5-in Extracellular Vesicles of Human Vitreous as a Potential Marker for Fungal Endophthalmitis.

PURPOSE: Extracellular vesicles (EVs) are lipid-bilayered nanoparticles that play an important role in cellular cross-talk, and as received attention for their role as diseases biomarker. Aquaporin-5 (AQP5) is a small integral membrane protein that help in the migration of cells, proliferation, and invasion. However, the association of AQP5 with fungal diseases is still unknown. The aim of this study was to evaluate the expression of AQP5 in EVs (EV-AQP5) extracted from the vitreous of patients with Fungal Endophthalmitis (FE). METHODS: Vitreous fluid was collected from 20 patients clinically suspected as FE, 10 patients from non-infectious conditions, and 10 patients with bacterial endophthalmitis as controls. EVs were isolated from human vitreous and characterized by dynamic light scattering, and scanning electron microscopy. Human Aquaporin-5 levels were evaluated using a commercial ELISA Kit. The Receiver Operating Characteristic (ROC) curves and its significance were correlated with microbiology data. RESULTS: Isolated EVs size were approx.250-380 nm in diameter. The measured levels of EV-AQP5 resulted significantly higher in FE patients (mean=216±15pg/ml; 95% confidence interval (CI): 182-250) in comparison to controls (mean=130±12pg/ml; 95%CI: 111-166)(p = .001). However, AQP5 levels in EVs derived from culture-proven bacteria patients were insignificant compared to controls (mean=169±4 pg/ml; 95%CI: 161-177). ROC curve was used to define the optimal cut-off level of the test at 180 pg/ml with an AUC of 98% (95%CI: 95-100) (p = .03), with a sensitivity of 100% and specificity of 90%. Additionally, the AQP5 level in EVs derived from culture-negative vitreous was above the threshold value (200 ± 10 pg/ml (95%CI: 180-230) in comparison to the control group (p < .001) However, no significant association was found between age or visual acuity and the level of AQP5 in FE. CONCLUSION: Our results reveal that the vitreous EV-AQP5 levels can aid in differentiating FE from non-infectious retinal conditions, mainly when the cultures are negative.

Evaluation of Vitreous Galactomannan and (1, 3) ß-D-Glucan Levels in the Diagnosis of Fungal Endophthalmitis in Southern India.

PURPOSE: To evaluate vitreous Galactomannan(GM) and 1,3 ß-D-Glucan (BDG) levels in the diagnosis of fungal endophthalmitis, with emphasis on culture-negative cases. METHODS: Vitreous from 31 clinically suspected fungal endophthalmitis patients and 11 controls were evaluated for GM and BDG using ELISA Kits. The Receiver Operating Characteristic (ROC) curves and diagnostic significance was calculated. RESULTS: The median vitreous GM in culture-positive (60.83pg/ml) and culture-negative (59.9pg/ml) samples were higher than the (51.2pg/ml) control group. The median vitreous BDG in culture-positive (1.47pg/ml) and culture-negative (1.52pg/ml) samples were also similar, and higher than the control group (1.18pg/ml). ROC analysis showed that at a cut-off of 51.35pg/ml, the sensitivity and specificity for GM were 0.88 and 0.73.Similarly, for BDG at a cut-off of 1.18pg/ml, the sensitivity and specificity were 0.94 and 0.82 respectively. CONCLUSION: Vitreous GM and BDG above the indicated threshold level could suggest a fungal infection, even when cultures are negative.

Recent Advances and Ongoing Challenges in the Diagnosis of Culture Negative Endophthalmitis.

BACKGROUND: Culture negative (CN) but presumed infectious endophthalmitis poses a huge diagnostic challenge in terms of clinical management. This article outlines the current state of knowledge of infectious endophthalmitis with negative cultures and summarizes the recommendations for the work up of this condition along with providing a simple algorithm, by putting into context the recent concerns about over-diagnosing endophthalmitis. METHODS: We searched the PubMed and Scopus databases for large hospital based studies on diagnosis of endophthalmitis, with emphasis on culture-negative infections in October 2021. Only clinical studies written in English were included. Basic science studies, letters to the editor and case reports on endophthalmitis were excluded. RESULTS: Twenty studies were included in this study. The prevalence of CN endophthalmitis ranged from 40% to 70%. Recent advances in PCR along with high throughput sequencing have helped identify the etiological agent in most cases but these technologies are not easily available, requires advanced bioinformatic analysis and are not cost effective. Role of other inflammatory and relatively low-cost biomarkers in diagnosing a presumed infection is yet to be validated clinically but hold promise in helping ophthalmologists identify the causative agent. CONCLUSIONS: CN endophthalmitis is a relatively frequent finding and should not be labelled as sterile endophthalmitis. Recent advances provide a new perspective for ophthalmologist in diagnosis of presumed infectious endophthalmitis and further studies are needed to confirm their utility in clinical settings.

Proteomic profiling of extracellular vesicles derived from ARPE-19 cells challenged with Aspergillus flavus and Candida albicans: application in fungal endophthalmitis.

Extracellular vesicles (EVs) are nano-sized-particles that play an important role in cellular cross-talk. The aim of this study was to understand the proteomic cargo of EVs, released by Retinal Pigment Epithelial (RPE) cells challenged with Candida albicans (C-CA) and Aspergillus flavus (C-AF). EVs were isolated from culture supernatant of retinal cells infected with fungal pathogens and characterized by dynamic light scattering, SEM, and western blot. EV proteome was then evaluated by mass spectrometry (LC-MS/MS). Isolated EVs were approximately 120-150 nm and higher in number in infected group compared to control. Proteomic profiling of EVs from infected cells, showed a total of 419 and 254 differentially expressed proteins, of which 218 were upregulated in C-CA group and 81 proteins were upregulated in C-AF group. Gene ontology revealed majority of proteins associated with transport, cell migration, and in activation of innate immune response. Proteins identified were annexins, calpain, and Sorcin proteins. Additionally, KEGG analysis unveiled involvement of MAPK, HIF-1, and PI3K-AKT signalling pathways. Proteomic results indicate that EVs cargo derived from fungal-infected retinal cells can activate immune signalling pathways and might contribute to the pathogenesis of endophthalmitis, indicating the potential use of EVs as theranostic marker for management of fungal infections.

Proteomic profiling of aspergillus flavus endophthalmitis derived extracellular vesicles in an in-vivo murine model.

Extracellular Vesicles (EVs) play pivotal roles in cell-to-cell communication, and are involved in potential pathological and physiological cellular processes. The aim of this study was to understand the proteomic cargo of these vesicles, in a murine model of Aspergillus flavus (AF) endophthalmitis. EVs were isolated from A. flavus infected C57BL/6 mice eyes by differential ultracentrifugation at 24 h post infection (p.i) and isolated EVs were characterized by Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), Exocet assay, and western blot. Proteomic profiling of EVs was then evaluated by mass spectrometry (LC-MS/MS) and compared it with control uninfected mice. The average size of the EVs were 180-280 nm by DLS and the number of EVs increased to 1.55 × 1010 in infected mice in comparison to EVs from uninfected eye (1.24 × 109). Western blot was positive for CD9, CD63, and CD81 confirming the presence of EVs. LC-MS/MS analysis, identified 81 differentially expressed proteins, of these 22 were up-regulated and 59 were down-regulated. Gene Ontology (GO) analysis revealed enrichment of lipid metabolism, protein complex binding, and transferase activity, and the proteins associated were Aquaporin-5, CD177 antigen, Solute carrier family-25, and Calcium/calmodulin-dependent protein kinase. Additionally, KEGG pathway analysis indicated that glucagon signalling, metabolic, and PPAR signalling pathway were significantly associated with EVs from A. flavus infected mice eyes. The protein cargo in EVs from A. flavus endophthalmitis provides new insights into the pathogenesis of fungal endophthalmitis and validation of these proteins can serve as diagnostic and/or prognostic biomarkers for patients with a clinical suspicion of fungal endophthalmitis. LAY SUMMARY: EVs play an important role in cell communication. In our study proteomic profiling of EVs isolated from A. flavus infected mice provided new insights into the understanding of the pathobiology of A. flavus endophthalmitis and validation of these proteins can serve as biomarkers.

Proteomic profiling of exosomes in a mouse model of Candida albicans endophthalmitis.

Exosomes play pivotal roles in intercellular communication, and pathophysiological functions. In this study, we aimed to understand the role of exosomal proteome derived from C. albicans infected mice (C57BL/6) eyeball. Exosomes were characterized by Dynamic Light Scattering and Western blot, quantified and subjected to LC-MS/MS and cytokine quantification by ELISA. The average size of exosomes was 170-200 nm with number of exosomes amounted to 1.42 × 1010 in infected set compared to control (1.24 × 109). Western blot was positive for CD9, CD63 and CD81 confirming the presence of exosomes. IL-6, IL1ß, TNF-α, and IFN-γ levels were significantly elevated in infected eye at 72 h.p.i. Proteomic analysis identified 42 differentially expressed proteins, of these 37 were upregulated and 5 were downregulated. Gene Ontology (GO) revealed enrichment of cell adhesion, cytoskeleton organisation, and cellular response proteins such as aquaporin-5, gasdermin-A, CD5 antigen-like, Catenin, V-ATPase, and vesicle associated protein. Additionally, KEGG pathway analysis indicated the association of metabolic and carbon signalling pathways with exosomes from C. albicans infected eye. The protein cargo in exosomes released during endophthalmitis with C. albicans seems to play a unique role in the pathogenesis of the disease and further validations with larger cohort of patients is required to confirm them as biomarkers.

Understanding the science of fungal endophthalmitis - AIOS 2021 Sengamedu Srinivas Badrinath Endowment Lecture.

Fungal endophthalmitis is a potentially blinding condition. It is more often reported from Asia, including India. The incidence is lower than bacterial endophthalmitis. But it is relatively more challenging to treat than bacterial endophthalmitis. Many eyes may need therapeutic keratoplasty and/or evisceration. The current mainstays of treatment are vitrectomy irrespective of the presenting vision, intravitreal antifungal agents, and systemic therapy; additionally, the patients could require prolonged treatment with repeat vitreous surgeries and intravitreal injections. Difficulty in clinical diagnosis, delay in microbiological culture, and limited options of antifungal drugs make the treatment more difficult and less rewarding. Three common fungi causing endophthalmitis are Aspergillus, Fusarium, and Candida. The former two are molds, often identified in exogenous endophthalmitis, postoperative and traumatic; the latter is yeast and is more often identified in endogenous endophthalmitis. A faster diagnosis with newer molecular microbiological technologies might help institute treatment earlier than it is currently possible. A target trial using big data from different regions of the world might emulate a randomized clinical trial to design a definite treatment strategy. Given fewer antifungal drugs, one must be mindful of antifungal stewardship to prevent resistance to the existing drugs.

Fungal Endophthalmitis: Analysis of 730 Consecutive Eyes from 7 Tertiary Eye Care Centers in India.

PURPOSE: To evaluate the clinical and microbiological features of a large cohort with culture-confirmed fungal endophthalmitis across India. DESIGN: Cross-sectional, hospital-based, retrospective medical record review. PARTICIPANTS: Seven large tertiary eye care centers from different regions of India. METHODS: Patient data were pooled from electronic or physical medical records of each participating center. Fellowship-trained vitreoretinal specialists clinically managed all patients, and in-house microbiology laboratories performed all microbiological workups. The clinical and microbiological procedures were broadly uniform across all participating centers. The essential treatment consisted of vitreous surgery as well as intravitreal and systemic therapies with antifungal agents. MAIN OUTCOME MEASURES: Clinical outcome of the causative event and causative fungus. RESULTS: In the period from 2005 to 2020, 7 centers treated 3830 cases of culture-proven endophthalmitis, and of these, 19.1% (n = 730) were cases of culture-confirmed fungal endophthalmitis. It included 46.9% cases of postoperative (87.4% postcataract surgery), 35.6% of traumatic, and 17.5% of endogenous endophthalmitis. The fungi included 39.0% of Aspergillus (high prevalence in central, east, and south zones), 15.1% of Candida (high prevalence in west zone), and 15.9% of Fusarium (high prevalence in north and west zones). The time to symptom development was between 1 week and 4 weeks in more than one third of the patients, except in patients with traumatic endophthalmitis. Less than half of the patients had hypopyon on presentation. The presenting visual acuity (PVA) in most patients was <20/400. Nearly all patients needed vitrectomy and an average of 2 intravitreal injections of antifungal agents. At least 10% of eyes needed therapeutic keratoplasty, and up to 7% of eyes were eviscerated. After treatment, the final (best corrected) visual acuity (FVA) was >20/400 in 30.5% (n = 222) of eyes and >20/40 in 7.9% (n = 58) of eyes, and 12% (n = 88) of eyes lost light perception. A post hoc analysis showed the male sex to be significantly more associated with traumatic endophthalmitis than with postoperative (P < 0.0001) and endogenous (P = 0.001) endophthalmitis, more isolation of Candida species in patients with endogenous endophthalmitis than in those with postoperative (P = 0.004) and traumatic (P < 0.0001) endophthalmitis, better PVA in eyes with Candida species infection (P < 0.0001), and poorer FVA in eyes with Aspergillus species infection. CONCLUSIONS: Fungal endophthalmitis is not uncommon in India. The inclusion of antifungal agents with antibiotics as the first empirical intravitreal therapy before microbiological confirmation should be considered when a fungal infection is suspected.

Differential inflammatory response to Aspergillus flavus and Candida albicans infection in human retinal pigment epithelial cells: Role in treatment of endophthalmitis.

OBJECTIVE: Fungal endophthalmitis is an emerging vision-threatening complication in tropical countries and the Retinal pigment epithelial cells (RPE) are said to play a major role in the retinal pathology. The aim of this study was to compare the immune response of Retinal pigment epithelial cells (RPE) challenged with A. flavus and C. albicans strains, isolated from patients with fungal endophthalmitis. MATERIAL AND METHODS: Retinal Pigment epithelial cells (ARPE-19) cells were infected with A. flavus and C. albicans, and gene expression were assessed for TLRs, immune-mediators, and matrix metalloproteinases (MMPs). RESULTS: We observed a time-dependent gene expression of TLRs (TLR-2,-7 and -9); IL-8 and TNF-α in RPE cells challenged with A. flavus and C. albicans. Additonally, IL-6 (3.3 fold), IL-10 (15.2 fold), and IL-17 (5.6 fold) were significantly up-regulated only in cells infected with A. flavus. Additionally, MMP-9 gene expression was up-regulated in both A.flavus as well as C.albicans infected cells, while MMP- 2 gene expression was induced only in cells infected with C.albicans. A total of 9 upregulated differential expression of genes (DEGs) in A. flavus infected cells and 7 DEGs in C. albicans infected cells were used to construct Protein-protein interaction (PPI) network. CONCLUSION: RPE cells induced a differential innate immune response depending on fungal species involved (A.flavus and C.albicans) and may provide clues for novel treatment targets and better prognosis.

Microglial Response to Aspergillus flavus and Candida albicans: Implications in Endophthalmitis.

Aspergillus flavus is the most common etiology of fungal endophthalmitis in India, while Candida albicans is the causative agent in the West. In this study, we determined the role of microglial cells in evoking an inflammatory response following an infection with A. flavus and C. albicans strains isolated from patients with endophthalmitis. Microglia (CHME-3) cells were infected with A. flavus and C. albicans and the expression of Toll-Like Receptors (TLRs), cytokines and Matrix metalloproteinases (MMPs) were assessed at various time intervals. A. flavus infected cells induced higher expressions of TLR-1, -2, -5, -6, -7 and -9 and cytokines such as IL-1α, IL-6, IL-8, IL-10 and IL-17. In contrast, C. albicans infected microglia induced only TLR-2 along with the downregulation of IL-10 and IL-17. The expression of MMP-9 (Matrix metalloproteinase-9) was however upregulated in both A. flavus and C. albicans infected microglia. These results indicate that microglial cells have the ability to incite an innate response towards endophthalmitis causing fungal pathogens via TLRs and inflammatory mediators. Moreover, our study highlights the differential responses of microglia towards yeast vs. filamentous fungi.

Targeted High-Throughput Sequencing Identifies Predominantly Fungal Pathogens in Patients with Clinically Infectious, Culture-Negative Endophthalmitis in South India.

To evaluate the clinical utility of high-throughput sequencing (HTS) approach-based analysis of the bacterial and fungal genome in vitreous fluids from patients clinically diagnosed as endophthalmitis, we subjected 75 vitreous fluids from clinically presumed infectious endophthalmitis patients to high-throughput sequencing (Illumina HiSeq 2500) after DNA extraction and amplification of the 16S rRNA for the detection of bacteria, and ITS 2 region for detection of fungal pathogens. As controls, we included vitreous biopsies from 70 patients diagnosed with other non-infectious retinal disorders. Following the construction of the curated microbial genome database and filtering steps to reduce ambiguousness/contaminants from the environment, the paired reads were analyzed. Our HTS reads revealed in almost all cases the same organism that was grown in culture (bacterial-14/15, fungal 3/3) by conventional microbiological workup. HTS additionally diagnosed the presence of microbes in 42/57 (73.7%) patients who were conventionally negative (fungal pathogens in 36/57, bacterial pathogens in 11/57, including five cases that showed the presence of both bacterial and fungal organisms). Aspergillus sp., Fusarium sp., Exserohilum sp., and Candida sp. were the most predominant genera in our cohort of culture-negative endophthalmitis cases. Heat map based microbial clustering analysis revealed that these organisms were taxonomically similar to the species identified by conventional culture methods. Interestingly, 4/70 control samples also showed the presence of bacterial reads, although their clinical significance is uncertain. HTS is useful in detecting pathogens in endophthalmitis cases that elude conventional attempts at diagnosis and can provide actionable information relevant to management, especially where there is a high index of suspicion of fungal endophthalmitis, particularly in tropical countries. Outcome analyses and clinical trials addressing the success and cost savings of HTS for the diagnosis of endophthalmitis are recommended.

Trends in microbiological spectrum of endophthalmitis at a single tertiary care ophthalmic hospital in India: a review of 25 years.

PURPOSE: To determine trends in the microbial spectrum of endophthalmitis over the past 25 years and to review its antibiotic susceptibility patterns over the last 10 years. METHODS: Microbiology records of culture-positive endophthalmitis cases from 1991 to 2015 were reviewed. Additionally, data between 2005 and 2015 was also analyzed for trends in antibiotic susceptibility. RESULTS: Of the total of 9278 patients, 3319 (35.7%) were culture positive and included bacteria (2840/3319, 85.56%), fungi (387/3319, 11.66%), and mixed cultures (92/3319, 2.7%). Gram-positive bacteria accounted for 67.68% (1922/2840) of the total bacteria seen, with the most prevalent pathogen being Streptococcus pneumoniae and Staphylococcus epidermidis. Among the gram-negative organisms Pseudomonas aeruginosa was the most prevalent while. Aspergillus flavus was the most common fungus isolated and Candida sp. accounted for 6.9% of the total fungi isolated. There was no significant change in the trends of bacteria isolated during the study period. Overall susceptibility patterns showed that gram-positive bacteria were most susceptible to vancomycin (96%) and fluoroquinolones (89%). The resistance to ceftazidime increased from 31% in 2005 to 62% in 2015 (P = 0.006) and amikacin decreased from 36% in 2005 to 33% in 2015 (P = 0.782). Although a significant trend (P < 0.001) toward increasing microbial resistance against cephalosporins and fluoroquinolones was observed, decreasing microbial resistance against glycopeptides and aminoglycosides was also detected. CONCLUSION: The spectrum of pathogens causing endophthalmitis at our institute remained similar over the study period. These findings impact the empiric treatment and choice of antibiotics in patients with endophthalmitis.