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ARVO 2015 Annual Meeting Abstracts 413 Bacterial pathogenesis and infection Wednesday, May 06, 2015 8:30 AM–10:15 AM Exhibit Hall Poster Session Program #/Board # Range: 4043–4078/A0220–A0255 Organizing Section: Immunology/Microbiology Contributing Section(s): Clinical/Epidemiologic Research, Cornea, Physiology/Pharmacology, Retina Program Number: 4043 Poster Board Number: A0220 Presentation Time: 8:30 AM–10:15 AM Pseudomonas aeruginosa adaptation to the ocular surface: transcriptional changes and virulence determinants Matteo M. Metruccio1, Yvonne Wu1, David J. Evans1, 2, Suzanne M. Fleiszig1, 3. 1School of Optometry, University of California Berkeley, Berkeley, CA; 2College of Pharmacy, Touro University California, Vallejo, CA; 3Graduate Groups in Vision Science, Microbiology, and Infectious Disease & Immunity, University of California, Berkeley, Berkeley, CA. Purpose: Pseudomonas aeruginosa is a leading cause of contact lens-related corneal infection. Using a lens-wearing rodent model, we previously showed that bacteria could infect the cornea more efficiently if first pre-exposed to the ocular surface. This is consistent with the increased risk of infection with extended lens wear in humans. Here, we studied bacterial adaptions to the ocular surface that subsequently enable them to penetrate the corneal epithelium. Methods: RNA-seq was used to compare the transcriptional profile of P. aeruginosa strain PAO1 exposed to human tear fluid for 5 h at 37 °C to PBS controls. Tn-seq was used to identify bacterial genes required for traversal of human corneal epithelial cell multilayers in vitro. For the latter, a pooled transposon mutant library of PAO1 was generated, and ~106 cfu of bacterial mutants incubated with Transwell filter-grown human telomerase-immortalized corneal epithelial cells for 4 h at 37 °C. Transposon insertion sites were deep sequenced for the input and traversed populations. HiSeq 2000 (Illumina) was used for sequencing and data analysis performed with Galaxy and IGB. Results: RNA-sequencing showed many P. aeruginosa genes were deregulated (up- or down-regulated) by exposure to tear fluid (~180 genes ≥ 8 fold). They included; the phoP/Q two-component regulatory system (down-regulated), oprH for antimicrobial resistance (down-regulated), and algF for biofilm formation and antiphagocytic activity (up-regulated). Two small non-coding RNAs, rsmZ and phrS, associated with virulence factor regulation, response to oxygen availability and quorum sensing were also down- and up-regulated respectively. Tn-seq identified ~200 gene insertions differentially represented in traversed populations as compared to the input (cut-off ≥ 18 fold), showing roles in epithelial traversal. Insertions mapped in or near 150 genes belonging to numerous important virulence categories, including pcrV (type three secretion), motC (motility and attachment) and mexF (multidrug efflux pump). Conclusions: Use of an unbiased global genetic approach to study P. aeruginosa interaction with ocular surface components in vitro identified genes and genomic regions involved in bacterial adaptation to the host environment and ocular pathogenicity. Commercial Relationships: Matteo M. Metruccio, None; Yvonne Wu, None; David J. Evans, None; Suzanne M. Fleiszig, Allergan Inc. (C) Support: NIH EY024060 Program Number: 4044 Poster Board Number: A0221 Presentation Time: 8:30 AM–10:15 AM Molecular mechanisms of host-pathogen interactions in pseudomonas aeruginosa microbial keratitis Ahmad Elsahn1, 2, Maria del Mar Cendra-Gascon1, Pawez Hossain1, 2 , Myron Christodoulides1. 1Infection, Inflammation & Immunity, University of Southampton, Southampton, United Kingdom; 2 University Hospitals Southampton, Southampton, United Kingdom. Purpose: To examine the molecular mechanisms of interactions between P. aeruginosa bacteria and primary human corneal fibroblasts (hCF) in an invitro model of microbial keratitis Methods: Human CF were extracted from clinical samples, cultured to confluence in vitro and incubated with live PAO1 wild type bacteria as well as mutant strains deficient in type IV pilus (∆pilA), flagella (∆fliM) or double mutants (∆pilA∆fliM) for 3h. Bacterial association was quantified and compared across the strains. Confluent hCF were also pre-treated with SRC kinase inhibitors genstein (GST) and PP2 and actin microfilament inhibitor cytochalasin D (CD), and bacterial internalization was compared across pre-treated cells at 3h using a gentamicin protection assay. Wild type (WT) PA14 as well as mutant strains deficient in type III secretion system (T3SS) needle apparatus (∆popB) and flagella (∆flgK) were incubated with confluent hCF for 9h, and bacterial cytotoxicity was assessed by a lactate dehydrogenase (LDH) assay. Results: Mutant PAO1strains ∆pilA, ∆fliM and ∆pilA∆fliM adhered significantly less than WT to hCF (P<0.05). Bacterial internalization in hCF pre-treated with CD, GST and PP2 was significantly less than untreated cells (P<0.05). Mutant PA14 strains ∆popB and ∆flgK caused significantly less cytotoxicity to hCF than WT PA14 (P<0.05). Conclusions: PAO1 bacteria use type IV pilus and flagella to adhere to hCF. Bacterial internalization to hCF is dependent on the actin microfilament and SRC kinase systems. Bacteria use flagella to adhere to hCF and T3SS to induce cytotoxicity. Commercial Relationships: Ahmad Elsahn, None; Maria del Mar Cendra-Gascon, None; Pawez Hossain, None; Myron Christodoulides, None Program Number: 4045 Poster Board Number: A0222 Presentation Time: 8:30 AM–10:15 AM Correlation of P. aeruginosa Type III Secretion Profiles Diversity and Ocular Disease Syndromes Jorge Maestre, Edith Perez, Eduardo C. Alfonso, Harry W. Flynn, Darlene Miller. Ophthalmology, University of Miami, Miami, FL. Purpose: Pseudomonas aeruginosa associated ocular infections are among the most diverse and difficult to manage. Clinical presentations and pathology range from purulent conjunctivitis and ulcerative keratitis to invasive scleritis and persistent, destructive biomaterial centered infections. Virulence, disease course and patient outcomes are strain specific. We used a combination of molecular, metabolic and in vitro resistance markers to determine and correlate Pseudomonas aeruginosa Type lll effector protein (T3SS) profiles with biochemical patterns, ocular disease presentations and in vitro susceptibility. Methods: Type III Profiles: A multiplex PCR was used to characterize and compare the prevalence of exoS (invasive), exoT (invasive), exoU (cytotoxic) and exoY (adenyl cyclase) (genotypes for 155 Pseudomonas aeruginosa strains collected from ocular sources (cornea-n=96, contact lens-n=20, conjunctiva-n=23, lacrimal system-n=8, Intraocular fluids-n=8) from 2008 to 2014. Biochemicals and vitro susceptibility profiles were determined using the Vitek 2 system. Results: Exo Y and T effector proteins were detected in all 155 isolates, while exoS ( N=70) and exoU (N=63) were generally ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts mutually exclusive. Prevalence of the four identified genotypes were exoS+U- (45.2%), exoS-U+ (40.6%), exoS+U+ (7%) and exoU-S(9.7%). ExoU (cytotoxic strain) was the predominant profile of isolates recovered from cornea (46.9%) and contact lens cases (70%) but was evident in all sources. ExoS genotypes were documents in 50% or higher for isolates recovered from conjunctiva (56.5%), intraocular fluids (50%) and lacrimal system (50%). Strains with absence of T3SS effectors were seen most often in isolates associated with conjunctivitis (34.8%). No significant correlation of metabolic profiles (N=100) were associated with these isolates. Strains with genotype exoS-U+ had the higher MIC90 (1 ug/ml) for ciprofloxacin vs 0.5 ug/ml or less for exoS+U- and exoU-S- . Ciprofloxacin resistance ranged from 1% (cornea) to 6% (conjunctiva). MIC90 for moxifloxacin was 2 ug/ ml and ranged from 3% (cornea) to 8% (conjunctiva and lacrimal system). Conclusions: Ocular Pseudomonas aeruginosa strains are diverse and associated with specific disease syndromes and antibiotic profiles. Characterizing the T3SS profiles may serve as an important adjunct in understanding the pathology and management of these destructive and recalcitrant infections Commercial Relationships: Jorge Maestre, None; Edith Perez, None; Eduardo C. Alfonso, None; Harry W. Flynn, None; Darlene Miller, None Support: Core Grant Department Ophthalmology Bascon Palmer Eye Institute Program Number: 4046 Poster Board Number: A0223 Presentation Time: 8:30 AM–10:15 AM Photodynamic therapy to treat Methicillin-Resistant Staphylococcus Aureus (MRSA) keratitis: An in vitro study Heather A. Durkee1, Francisco Halili1, Mukesh Taneja2, Darlene Miller3, 4, Alejandro Arboleda1, Cornelis J. Rowaan1, Mariela C. Aguilar1, Guillermo Amescua4, Harry W. Flynn4, Jean-Marie A. Parel1, 5. 1Ophthalmic Biophysics Center, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL; 2 LV Prasad Eye Institute, Hyderabad, India; 3Ocular Microbiology Laboratory, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL; 4Anne Bates Leach Eye Hospital, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL; 5Brien Holden Vision Institute, UNSW, Sydney, NSW, Australia. Purpose: To assess the in vitro efficacy of rose bengal (RB) and riboflavin (Ribo) mediated photodynamic therapy (PDT) for the inhibition of a methicillin-resistant Staphylococcus aureus (MRSA) strain. Methods: A MRSA type II strain was isolated from the corneal scraping of a patient with confirmed bacterial keratitis. Twentyfour hours prior to experimentation, a parent culture was plated on nutrient agar. Next, a culture of MRSA was transferred into tryptic soy broth and adjusted to a concentration of 1.5x108 colony forming units per mL (cfu/mL). The MRSA suspension was diluted to a concentration of 1.5x107cfu/mL with the appropriate solution and 1mL aliquots were inoculated in triplicate onto nutrient agar plates. The eight groups were: (1) Control (high purity water) (2) UV-A irradiation (3) 0.1% Ribo (4) 0.1% Ribo + UV-A (5) 0.1% RB (6) 0.1% RB + 518nm light (7) 0.03% RB (8) 0.03% RB + 518nm light. All experiments were performed in minimal lighting conditions (4 lux) except for irradiation test plates. The UV-A and green lights are custom built LED sources. The UV-A light, activate Ribo, has a central wavelength of 375nm and an irradiance of 2.91mW/ cm2 over a 13.8cm2 surface. The 518nm light, activate RB, has a central wavelength of 518nm and an irradiance of 2.2mW/cm2 over a 28.3cm2 surface. Plates were either exposed to UV-A (Ribo) or 518nm (RB) irradiation for 20 minutes. All plates were immediately placed upside down, wrapped in foil, and placed in an incubator at 30°C. Plates were photographed every 24 hours for 6 days. Results: Riboflavin without irradiation did not inhibit MSRA growth; however in Ribo with irradiation it did inhibit MRSA growth. 0.1% RB with and without irradiation inhibited the growth of the MRSA. 0.03% RB inhibited MRSA growth with irradiation but did not inhibit MRSA growth in the non-irradiated group. Inhibition in all photosensitizer groups occurred as soon as 24 hours after irradiation. Conclusions: Rose Bengal strips of 1.0% concentration are clinically used to detect epithelial defects. Our study demonstrates MRSA can be inhibited with 0.1% RB without irradiation. The RB will be activated even when the patient is exposed to ambient light levels in daily activities. PDT could be an excellent adjunct treatment for MRSA keratitis as it provides a different mechanism of inhibition. Results of 0.1% Ribo and 0.1% RB Results of 0.03% Rose Bengal PDT at 72 hours Commercial Relationships: Heather A. Durkee, None; Francisco Halili, None; Mukesh Taneja, None; Darlene Miller, None; Alejandro Arboleda, None; Cornelis J. Rowaan, None; Mariela C. Aguilar, None; Guillermo Amescua, None; Harry W. Flynn, None; Jean-Marie A. Parel, None Support: Florida Lions Eye Bank, Drs KR Olsen and ME Hildebrandt, NIH P30EY1481 (Center Grant), Research to Prevent Blindness, Henri and Flore Lesieur Foundation (JMP). Technical support was provided by: Karam Alawa, Victor Hernandez, and Nidhi Relhan Batra. Program Number: 4047 Poster Board Number: A0224 Presentation Time: 8:30 AM–10:15 AM Interaction of Polymorphonuclear neutrophils (PMNs) with Pseudomonas aeruginosa in biofilms in corneal infections in the mouse Padmanabhan Saraswathi1, Roger Beuerman1, 2. 1SERI, Singapore Eye Research Inst (SERI), Singapore, Singapore; 2Department of Ophthalmology, Yong Loo Lin School of Medicine, NUS, Singapore, Singapore. Purpose: Pseudomonas aeruginosa, is a common ocular pathogen found in cornea infections, often associated with contact lens wear. The survival of this bacteria as a “Biofilm” within a extrapolysaccharide substances sheltered from innate immune defence and antimicrobials. In this study, the interaction of polymorphonuclear neutrophils (PMNs) with Pseudomonas in planktonic and biofilm models was examined using a mouse model of experimental keratitis infection ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts Methods: A suspension (108 CFU/ml) of Pseudomonas aeruginosa (ATTC 9027) was used to infect the mouse cornea (C57BL/6). The infection was monitored by slit lamp. Infected eyes were enucleated at post infection (PI) day1 to describe the planktonic status and PI day3 and 5 for potential biofilm status. The response of polymorphonuclear neutrophils (PMNs) was characterised using histological, ultra-structural imaging and quantifying myeloperoxidase levels Results: Histology showed the expected large numbers of PMNs in the stroma at PI day1, with diverse morphologies. At later stages of infection they were seen on the corneal surface, but more spherical shape. Scanning Electron Microscopy (SEM) demonstrated the release of Neutrophil Extracellular Traps (NETs) as long fibrils and entangling many bacteria which were seen as free cells at the early stage of infection. However, at PI day3 and 5 the PMNs were seen to change their morphology and lacking pseudopodia appeared immobile, surrounding the clusters of Pseudomonas within a biofilm while other PMNs were on the corneal surface. Transmission Electron Microscopy (TEM) further confirmed the active phagocytosis at PI day1 and immotile PMNs settled beneath the layer of the biofilm at later stages of infection. Enhanced myeloperoxidase activity from 4.1 units at day1 to 16.9 units at day5 PI indicated the increased PMN activity in the course of the infection Conclusions: The murine neutrophils were seen to be highly activated during an infection and produce NETs as an early defence when the bacteria were planktonic. However, the PMNs were concomitantly sluggish and not successful in phagocytosis when the bacteria developed into biofilms at later times of infection. The early role of PMN NETs as basic host defence may perhaps be considered for novel drug targets to prevent biofilm formation and to augment bacteria killing Commercial Relationships: Padmanabhan Saraswathi, None; Roger Beuerman, None Program Number: 4048 Poster Board Number: A0225 Presentation Time: 8:30 AM–10:15 AM Interactions of tear-film neutrophils with clinical bacteria Maud Gorbet1, 2, Mark D. Willcox2. 1Systems Design Engineering, University of Waterloo, Waterloo, ON, Canada; 2School of Optometry and Vision Science, University of New South Wales, Sydney, NSW, Australia. Purpose: During sleep, in the closed-eye environment, a shift in tear film composition leads to the recruitment of leukocytes to the ocular surface. Neutrophils (also known as polymorphonuclear leukocytes; PMN) are considered our first line of defense and play an essential role in preventing infection. Following chemical stimulus, a lack of upregulation of cell activation markers has previously been observed on tear film neutrophils (TF-PMN). This pilot study was conducted to investigate the response of TF-PMN to clinical bacteria and assess whether overnight lens wear effected on their response to bacteria. Methods: Acuvue Oasys lens wearers and non-lens wearers were recruited to collect their cells upon awakening using an eye wash “at-home collection kit”. Collected cells were counted and resuspended in tubes containing either ATS-2% NHS (artificial tear solution supplemented with 2% of normal human serum) or PBS10% Heat Inactivated (HI) FBS. Collected cells were incubated with Pseudomona aeruginosa 6294 (an invasive clinical strain) and P. aeruginosa 6206 (a cytotoxic clinical strain) at bacteria to PMN ratios of 10:1 and 1:10. Following incubation, samples were serially diluted in PBS, plated on agar and incubated overnight at 35oC. The next day, bacteria colonies were counted. To further characterize the response to bacteria, TF-PMN were analysed by flow cytometry for receptor upregulation and oxidative burst. Results: With a bacteria:TF-PMN ratio of 10:1 in ATS-2% NHS, no reduction in bacteria growth was observed for 6206 and 6294. Fewer 6294 CFU were counted when interactions with TF-PMN occurred in PBS-HIFBS. Furthermore, regardless of incubation medium, bacteria:TF-PMN interactions at a ratio of 1:10 resulted in a significant increase in the number of 6206 cells (p<0.05). For both 6206 and 6294, interactions at the 1:10 ratio with TF-PMN collected following overnight lens wear led to a significant increase in CFU when compared to the 10:1 ratio (p<0.04). Flow cytometry results confirmed the differential TF-PMN response to 6206 and 6294. Conclusions: Our results suggest that at low bacteria to PMN ratio, a condition that may closely mimic the closed-eye environment, TF-PMN are unable to phagocytose clinical strains of Pseudomona aeruginosa and may be releasing factors that promote bacterial survival/growth. This may have an important impact during overnight lens wear and may contribute to the higher risks of microbial keratitis. Commercial Relationships: Maud Gorbet, None; Mark D. Willcox, None Support: American Optometric Foundation VISTAKON® Research Grant Program Number: 4049 Poster Board Number: A0226 Presentation Time: 8:30 AM–10:15 AM Pseudomonas aeruginosa Ocular Strains Resistant to Ceftazidime in Northeast of Mexico Pedro M. González, Alejandro F. Ibarra-Lozano, Jorge E. Valdez, Jaime Torres. Ophthalmology, Instituto Tecnológico de Monterrey, San Pedro Garza García, Mexico. Purpose: The purpose of this case series is to report Pseudomonas aeruginosa resistance to ceftazidime in keratitis isolates in northeast of Mexico. Concern exists around the growing antibiotic resistance in different ocular pathogens worldwide. Ceftazidime is known to be one of the antimicrobial agents used with a high sensitivity profile in bacterial keratitis caused by Pseudomonas aeruginosa. Methods: Patient records from January 2010 to November of 2014 were revised. Twenty eight patients were found with the diagnosis of infectious keratitis. Eight cases were culture positive for Pseudomona aeruginosa. variables studied included: gender, age, time of presentation to consultation, visual acuity, comorbidities, antibiotic sensitivity/resistance pattern and complications encountered. Results: Eight culture positive P. aeruginosa cases were found between 2010 to the end of 2014. There were 2 females and 6 males, the age range was from 19 to 67 years old. Ceftazidime resistance was reported in seven cases. Susceptibility was reported for ciprofloxacin, meropenem, amikacin and gentamicin. Ciprofloxaxin and meropenem were the ones with the lowest minimal inhibitory concentration. Final visual acuity had a strong relation with the initially reported. Two cases were associated to traumatism and one to soft contact lenses use. Two cases had corneal perforation. Conclusions: This study reviews a series of cases of P. aeruginosa keratitis in northeast of Mexico; the microbiologic profile showed 87.5% resistance to ceftazidime. Sensitivity to meropenem, ciprofloxacin, amikacin and gentamicin was of 100%. This data should influence the management of this entity in the geographic zone examined. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts Support: NIH Grant AI079192 Commercial Relationships: Pedro M. González, None; Alejandro F. Ibarra-Lozano, None; Jorge E. Valdez, None; Jaime Torres, None Program Number: 4050 Poster Board Number: A0227 Presentation Time: 8:30 AM–10:15 AM Corneal epithelial cells suppress vacuolar escape of P. aeruginosa Abby Kroken, David J. Evans, Suzanne M. Fleiszig. School of Optometry, University of California, Berkeley, Berkeley, CA. Purpose: Previously we reported that invasive strains of P. aeruginosa invade corneal epithelial cells wherein they replicate and induce formation of plasma membrane blebs to which they traffic. These events depend on the bacterial Type Three Secretion System (T3SS), specifically the T3SS toxin ExoS. Other investigators have studied ExoS using HeLa cells (not normally targeted by P. aeruginosa) and PA103 (does not natively express ExoS), and have not noted these phenomena. Here, we tested the hypothesis that the impact of ExoS depends on bacterial strain and cell type. Methods: Epithelial cell lines (HeLa or corneal) were infected with P. aeruginosa strain PAO1 (natively expresses ExoS) or PA103 (engineered to express ExoS). Intracellular bacteria were quantified using a gentamicin protection assay. A T3SS-driven GFP reporter was used to monitor T3SS expression in individual bacteria. Images were captured using time lapse wide-field or confocal microscopy. Results: In both cell types, PAO1 and PA103 expressing ExoS induced membrane blebbing, but only PAO1 invaded, replicated intracellularly, or trafficked to blebs. In contrast to corneal cells, HeLa cells even supported intracellular replication of PAO1 mutants lacking all known T3SS toxins (including ExoS). That replication remained dependent on the T3SS, which was expressed intracellularly, and occurred in the cytoplasm, without blebs, and in cells possessing acidified vacuoles - all differentiating it from ExoSdependent replication in corneal cells. T3SS machinery mutants remained in vacuoles in HeLa cells, showing that vacuolar escape is mediated by a T3SS component or unknown effector, but not ExoS. This contrasts with ExoS-dependent vacuolar escape in corneal cells. Conclusions: ExoS has been mostly studied using PA103 and HeLa cells. Our data show that neither accurately model how natively encoded ExoS influences P. aeruginosa interactions with corneal epithelial cells. This explains why its role in intracellular survival was overlooked by investigators using those tools. Our data show that; 1) corneal epithelial cells have innate defenses against internalized bacteria that are lacking in HeLa cells and which ExoS can overcome, and 2) unknown T3SS factors allow intracellular replication in cells lacking those innate defenses. Identifying mechanisms involved could lead to novel strategies to combat infection. Commercial Relationships: Abby Kroken, None; David J. Evans, None; Suzanne M. Fleiszig, Allergan (C) Program Number: 4051 Poster Board Number: A0228 Presentation Time: 8:30 AM–10:15 AM Pseudomonas aeruginosa induces autophagy in human corneal epithelial cells VIDYARANI MOHANKUMAR1, LakshmiPriya Jeganathan1, Lalitha Prajna1, Chidambaranathan Gowri Priya2. 1Ocular Microbiology, Aravind Medical Research Foundation, Madurai, India; 2Immunology and Stem Cell Biology, Aravind Medical Research Foundation, Madurai, India. Purpose: Keratitis caused by Pseudomonas aeruginosa is a serious ocular infection which may lead to corneal perforation if the intracellular bacteria are not cleared completely. Autophagy, a normal catabolic process, has been shown to play a major role in the clearance of intracellular pathogens. We propose that autophagy induced by P. aeruginosa in human corneal epithelial cells (HCET), may have a role in the clearance of intracellular bacteria. Methods: HCET cells transfected with LC3-GFP plasmid were infected with three different ocular isolates of P. aeruginosa and autophagy was monitored after 1h using a Leica TCS SP8 confocal microscope. EBSS treated (amino acid starvation) HCET cells were used as positive control for autophagy. Another set of HCET cells were infected with P. aeruginosa to study the mRNA expression of autophagy related protein, beclin1 by real time PCR. To study the intracellular survival and replication of the bacteria, HCET cells were infected with P. aeruginosa in the presence of EBSS or 3 –methyl adenine (3mM in EBSS), an inhibitor of autophagosome formation. After 3h, the extracellular bacteria were killed with gentamicin and the cells were incubated for another three hours to allow for intracellular bacterial replication. Diluted cell lysates were plated on to MacConkey agar, and the colonies were counted after an overnight incubation. Results: The HCET cells upon infection with three different P. aeruginosa isolates showed an increased LC3 punctation, which is a classical marker for autophagosome formation. The total number of LC3 positive cells and the relative number of LC3-puncta per cell varied upon infection with the different isolates which may be due to the difference in the intracellular bacterial load. Correspondingly, the mRNA expression of beclin1 was higher in P. aeruginosa infected cells compared to uninfected controls.The ocular isolates were able to efficiently invade and replicate inside the epithelial cells and the bacterial load was relatively higher when the cells were pretreated with 3-methyl adenine. Conclusions: Altogether, the results suggest that P. aeruginosa induces autophagy in human corneal epithelial cells, which inturn may limit the intracellular bacterial load. Since the ocular P. aeruginosa isolates can efficiently invade and replicate inside the epithelial cells, autophagy may represent a host defensive mechanism to curtail infection in P. aeruginosa keratitis. Commercial Relationships: VIDYARANI MOHANKUMAR, None; LakshmiPriya Jeganathan, None; Lalitha Prajna, None; Chidambaranathan Gowri Priya, None Program Number: 4052 Poster Board Number: A0229 Presentation Time: 8:30 AM–10:15 AM Role of IL-24 in Pseudomonas Aeruginosa Keratitis in a C57BL/6 Mouse Model Bing Xu, Nan Gao, Fushin X. Yu. Anatomy & Cell Biology and Ophthalmology, Wayne State University School of Medicine, Detroit, MI. Purpose: The cytokines in interleukin (IL)-10 family are known by their anti-infection and anti-inflammatory activities. However, ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts the biology of IL-24, a member of IL-10 family cytokines, is largely unknown, particularly in the ocular tissue. In this study, we investigated the role of IL-24 in a mouse Pseudomonas Aeruginosa (PA) keratitis model. Methods: Epithelium-injured B6 mouse corneas were pretreated with or without flagellin for 24h, followed by PA (ATCC 19660) inoculation. Samples were collected at various time points post infection and subjected to PCR and immunofluorescence. Mice were subconjunctivally injected with either IL-24 siRNA to knock down IL-24 expression or mouse IL-24 recombinant protein before the inoculation of PA to explore the biological functions of IL-24. Results: PA infection induced IL-24 transcription on corneal epithelial cells; flagellin pretreatment not only alleviated the infection, but also reduced the mRNA expression of IL-24, but not IL-19 and IL-20, the other two members of IL-10 family that share common receptors. STAT3, a signal mediator in IL-24 pathway, was phosphorated in response to PA infection, and p-STAT3 was mainly expressed in the infiltrated cells, indicating that immune cells were recruited and activated by the infection. IL-24 downregulation alleviated the severity of PA keratitis, and the application of IL-24 recombinant protein exacerbated PA infection on mouse cornea. Conclusions: These data demonstrate that IL-24 exhibits a detrimental effect on the host defense against PA infection, suggesting that IL-24 pathway may be a new intervention site for treating infectious keratitis. Commercial Relationships: Bing Xu, None; Nan Gao, None; Fushin X. Yu, None Support: NIH/NEI EY017960 Program Number: 4053 Poster Board Number: A0230 Presentation Time: 8:30 AM–10:15 AM Thrombomodulin protects against bacterial keratitis and is not angiogenic Linda D. Hazlett, Sharon A. McClellan, Cui Li. Anatomy & Cell Biology, Wayne State Univ Sch of Med, Detroit, MI. Purpose: Thrombomodulin (TMB), a cell surface glycoprotein composed of 5 domains, is expressed in a variety of cells. The N-terminal lectin-like domain (TMD1) interacts with Lewis Y antigen to inhibit angiogenesis and is responsible for TMB’s antiinflammatory properties; it also binds high mobility group box-1 (HMGB1), preventing its deleterious effects, while domains 2 and 3 (TMD23) induce neovascularization, with response regression within 24 days. Despite data regarding activities of specific domains of TMB, nothing has been reported regarding testing its protective effects in experimental microbial keratitis, and whether it induces corneal vascularity which is the purpose of this study. Methods: C57BL/6 mice were injected (subconjunctivally and i.p.) with recombinant (r)TMB and infected with P. aeruginosa. PBS controls were similarly treated. Clinical score, photography with a slit lamp, real time RT-PCR, MPO assay, and ELISA were used to assess the disease response. Results: Data show that treatment of C57BL/6 mice with rTMB reduced clinical disease scores, corneal opacity and the neutrophil infiltrate. mRNA levels for IL-1β, MIP-2, TLR4, and RAGE were decreased, while anti-inflammatory molecules, including SIGIRR and ST2 levels were increased when compared with controls. ELISA confirmed the PCR data showing significant reduction of both IL-1β and MIP-2 protein level (3 and 5 days post infection) after rTMB treatment. VEGF, VEGF-R1 and VEGF-R2 were no different, or reduced after TMB (5 days p.i.). Conclusions: TMB is protective in keratitis, reducing disease severity, and with no angiogenic effect . Commercial Relationships: Linda D. Hazlett, None; Sharon A. McClellan, None; Cui Li, None Support: NIH Grants EY016058 and P30EY04068 Program Number: 4054 Poster Board Number: A0231 Presentation Time: 8:30 AM–10:15 AM Conjunctival chemosis as a specific feature of Pseudomonas aeruginosa corneal ulcers Kaleena B. Michael. Tennents institute of ophthalmology, Glasgow, United Kingdom. Purpose: Corneal ulcer is a common ocular problem often complicated by delayed treatment from late diagnosis. We looked for presence of conjunctival chemosis in 44 consecutive cases of confirmed infective corneal ulcers. Methods: Retrospective examination of early ocular photographs of 44 consecutive cases of infective corneal ulcers. Results: Conjunctival chemosis was observed in 13 out of 44 cases. 12 of these were culture positive for pseudomonas aeruginosa, and 1 for colliform bacilli. Statistical analysis with Fishers Exact test was significant p>0.000001, Odds Ratio 112 (95% CI: 10.6 - 1190). Conclusions: Our findings suggest a strong association between conjunctival chemosis in pseudomonas aeruginosa corneal ulcers. This ocular feature could potentially help predict the presence of of pseudomonas aeruginosa in new corneal ulcers. This would enable individuals to receive the treatment of choice at an earlier stage, before microbiological confirmation. Commercial Relationships: Kaleena B. Michael, None Program Number: 4055 Poster Board Number: A0232 Presentation Time: 8:30 AM–10:15 AM Microbiota and P. aeruginosa Genotype adhesion difference on Worn Cosmetic Contact Lenses (CL) with Comparison of Disinfectant Sensitivity Elizabeth Shen1, Fung Rong Hu2. 1Ophthalmology, Taipei Tzu Chi Hospital, Taipei, Taiwan; 2Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan. Purpose: To analyze attached microbes found on worn cosmetic CL materials and compare difference in adhesion of two Type III secretion genotypes of P. aeruginosa. Disinfection ability of four disinfectants is compared between two genotypes. Methods: Healthy volunteers with myopia < -6 D and astigmatism < -1.0 D with signed informed consent were recruited in this prospective randomized study. Subjects wore nelfilcon (Alcon Freshlook Colorblends gray), hilafilcon (Bausch & Lomb Naturelle black),or etafilcon (Johnson & Johnson Acuvue 2 Define Accent style black) daily disposable CL for at least 8 hours/day for 2 weeks. The lenses are collected aseptically. Half of the lens is used to identify attached microbes. The other half is incubated for 2 hrs with108cfu/ ml of cytotoxic PA103 or invasive PAK strains. Viable cell culturing was done to determine number of bacteria attached. P. aeruginosa incubated lenses were soaked with various disinfectants (Renu, Aosept, Puremoist, and Replenish) at 25%, 50%, 75%, and 100% of the suggested disinfection time. The number of bacteria remaining on CL is counted and compared. Results: The most commonly identified microbes attached to nonsymptomatic volunteers were coagulase negative Staphylococcus, P. aeruginosa, and Streptococci. Adhesion of the cytotoxic strain PA103 was found to be greatest for hilfilcon lenses (34.8x104cfu/ ml) > etafilcon (33.1 x104cfu/ml) > nelfilcon (15.5x104cfu/ml) (P<0.05; ANOVA). Similar trend is noted for adhesion of the invasive strain PAK: hilfilcon (30.9x104 cfu/ml)>etafilcon(37.5x104 cfu/ml) >nelfilcon(10.1x104 cfu/ml) (P<0.05; ANOVA). At 100% suggested time, all tested disinfectants were able to kill all bacteria. However, ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts at less than 75% of suggested disinfection time, significant number of viable cytotoxic P. aeruginosa remained (P=0.03, t-test). Conclusions: With increasing popularity of cosmetic CL, clinicians are should be aware that different cosmetic materials attract different microbial adhesions. P. aeruginosa, a commonly identified pathogen in CL-associated microbial keratitis, is frequently found attached to CL of asymptomatic individuals. Cytotoxic strains are more resistant to MPS solution especially Renu. Proper lens care with strict adherence to suggested disinfection time is strongly advised to prevent sight threatening infections related to cosmetic lens wear. Commercial Relationships: Elizabeth Shen, None; Fung Rong Hu, None Support: NSC-102-2628-B-002-051-MY3 Program Number: 4056 Poster Board Number: A0233 Presentation Time: 8:30 AM–10:15 AM Effect of the interaction Fusarium solani-Staphylococcus aureus over limbocorneal fibroblasts Antonio Bautista-Hernandez1, 2, Beatriz Buentello-Volante3, Victor M. Bautista1, José Luis Gómez Olivares2. 1Microbiology and Ocular Proteomics, Inst of Ophthalmology “Conde de Valenciana”, Mexico City, Mexico; 2Department of Health Sciences, Autonomous Metropolitan University., Mexico City, Mexico; 3Genetics, Inst of Ophthalmology “Conde de Valenciana”, Mexico City, Mexico. Purpose: To study the effect of the interaction Fusarium solaniStaphylococcus aureus over limbocorneal fibroblasts. Methods: The limborcorneal fibroblasts were obtained from limbocorneal tissue and grown in media DMEM-F12 supplemented with serum fetal bovine 10%, were evaluated expression of vimentin and cytokeratin. F. solani and S. aureus were isolated from human corneal ulcers. The microorganisms were identified by classical microbiology. The biofilm formation was evaluated in media DMEM-F12 by the Christensen method and fluorescence microscopy. Fibroblasts were exposed to S. aureus and/or F. solani to evaluate the expression of CD34 and production of INFγ. Results: According to classical microbiology studies, bacterial strain corresponded to S. aureus and fungal strain to F. solani. Fluorescence microscopy showed a formed biofilm by the interactions between S. aureus and F. solani, that generated a extracellular matrix. The limbocorneal fibroblasts had characteristic morphology, vimentin positive and cytokeratin negative. The expression of CD34 decreased and increased content of INFγ with stimuli of S. aureus, F. solani and S. aureus-F. solani. Conclusions: Microorganism S. aureus and F. solani showed ability to form biofilm. The limbocorneal fibroblasts were vimentin positive and cytokeratin negative. Expression of CD34 was decreased and increased INFγ production with stimuli of S. aureus, F. solani and S. aureus-F. solani. Commercial Relationships: Antonio Bautista-Hernandez, None; Beatriz Buentello-Volante, None; Victor M. Bautista, None; José Luis Gómez Olivares, None Support: This work was supported by “Conde de Valenciana” Foundation Program Number: 4057 Poster Board Number: A0234 Presentation Time: 8:30 AM–10:15 AM Involvement of endoplasmic reticulum (ER) stress in the pathogenesis of bacterial endophthalmitis Ajay Kumar1, Pawan Kumar Singh1, Ashok Kumar1, 2. 1 Ophthalmology, Wayne State University School of Medicine, Detroit, MI; 2Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI. Purpose: Endoplasmic Reticulum (ER), through the unfolded protein response (UPR), regulates various cellular functions, including inflammation. Here, we have investigated the role of UPR signaling, specifically IRE1α in regulating intraocular inflammation in an experimental model of Staphylococcus aureus (SA) endophthalmitis Methods: Endophthalmitis was induced by intravitreal injection of S. aureus in WT (C57BL/6), TLR2-/-, and MyD88-/- mice. After 24 hours of infection, retinal tissue was collected; RNA was extracted, and RT-PCR was performed to assess ER stress marker genes (XBP-1s, IRE1α, PD1, CHOP, WSF, BiP, and ERDj4) using specific primers. To determine the role of IRE1α, inhibition studies were performed using IRE1α specific inhibitor, 4μ8C, given prior to S. aureus challenge. Secretion of inflammatory mediators and the activation of TLR-downstream signaling pathways (JNK1/2, MAPK, and NF-kB) were assessed using ELISAs and western blot analyses respectively. Mechanistic studies were performed using cultured BV2 microglial cells. Results: S. aureus did not induce the classical UPR response as evidenced by upregulation of ER stress markers (PD1, CHOP, WSF, BiP, and ERDj4) in the infected retina. However, S. aureus caused the splicing of XBP1 in both the mouse retina and the BV2 microglia. Concomitant with increased XBP-1s levels, the level of IRE1α was also increased in infected cells/tissue. The IRE1 inhibitor (4μ8C) reduced the levels of both XBP-1s and IRE1α in WT mice and microglia, but not in TLR2-/- and MyD88-/- mice. Similarly, the expression and secretion of inflammatory cytokines were attenuated in 4μ8C-treated WT mice and microglia cells, while no reduction was observed in TLR2-/- and MyD88-/- mice. Furthermore, S. aureusinduced the activation of ERK1/2, p38 MAPK, and NF-kB signaling was down regulated by the IRE1α inhibition. Conclusions: Taken together, our study, for the first time, provides evidence of IRE1α-mediated UPR stress signaling in generating retinal innate responses in bacterial endophthalmitis. Furthermore, these findings suggest a potential cross-talk between TLR and UPRsignaling in orchestrating inflammatory responses in the retina. Commercial Relationships: Ajay Kumar, None; Pawan Kumar Singh, None; Ashok Kumar, None Support: NIH EY019888 Program Number: 4058 Poster Board Number: A0235 Presentation Time: 8:30 AM–10:15 AM Bacterial Impediment of Corneal Cell Migration Kimberly Brothers, Nicholas A. Stella, Kristin M. Hunt, Regis P. Kowalski, Jes Klarlund, Robert M. Shanks. Ophthalmology, University of Pittsburgh, Pittsburgh, PA. Purpose: The loss of corneal epithelium in patients with microbial keratitis is well known but the underlying mechanisms are poorly understood. This study set out to determine the impact of bacterial secreted factors on ocular wound healing. Methods: Bacterial secretomes from ocular keratitis isolates were prepared from overnight cultures and filtered to remove bacteria. Using a plate based cell migration assay, secretomes were added to stratified human corneal limbal epithelial (HCLE) cells and incubated. Calcein AM viability stained cell layers were imaged by confocal microscopy. Porcine corneal organ culture was used to assess epithelial wound healing phenotypes ex vivo. Transposon mutagenesis of the Serratia marcescens genome was conducted to identify bacterial genes responsible for corneal cell migration inhibition. LPS was purified by the hot phenol method. The waaG lipopolysaccharide (LPS) gene was cloned using yeast homologous recombination into vector pMQ131. Results: Corneal cells treated with bacterial secretomes from 4/5 Pseudomonas aeruginosa, 26/27 S. marcescens, and 2/14 ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts Staphylococcus aureus isolates showed dose dependent inhibition of HCLE migration. No inhibition was seen with secretomes from 4 other ocular pathogens. Using an ex vivo porcine corneal wound model, we have recapitulated S. marcescens inhibition of wound healing. A transposon mutation in the S. marcescens LPS biosynthetic locus that prevented bacterial inhibition of epithelial cell migration was identified and complemented with the wild-type gene on a plasmid. Supporting the importance of LPS in the cell migration phenotype, depletion of LPS with polymyxin B agarose inactivated the inhibitory ability of the bacterial secretomes. Purified S. marcescens LPS, but not E. coli LPS was able to inhibit corneal cell migration. Conclusions: Together these data support that multiple ocular pathogens secrete factors able to inhibit corneal epithelial wound healing. Genetic and biochemical data using S. marcescens as a model organism indicate that S. marcescens LPS is sufficient to prevent corneal epithelial cell migration and wound healing. This study presents a novel host-pathogen interaction with implications for corneal ulcers and other medical problems where bacteria impact wound healing, such as chronic wounds and provides evidence that LPS may be a key factor in the inhibitory mechanism. Commercial Relationships: Kimberly Brothers, None; Nicholas A. Stella, None; Kristin M. Hunt, None; Regis P. Kowalski, None; Jes Klarlund, None; Robert M. Shanks, None Support: NIH 2T32 EY017271-06A1 NIH EY08098 NIH AI085570 Program Number: 4059 Poster Board Number: A0236 Presentation Time: 8:30 AM–10:15 AM Toxicity and Virulence of Viridans Group Streptococci Isolated from Endophthalmitis Mary E. Marquart, Hannah R. Rice. Microbiology, Univ of Mississippi Med Ctr, Jackson, MS. Purpose: Endophthalmitis due to viridans group streptococci (VGS) has been shown to result in poor visual prognosis despite antibiotic therapy. We hypothesized that VGS possess toxins that are involved in ocular pathogenesis. The purpose of this study was to identify the virulence characteristics of endophthalmitis strains of VGS. Methods: Concentrated extracellular milieu from 20 endophthalmitis strains of VGS, and concentrated culture media (controls), were subjected to 3 assays: lysis of sheep erythrocytes, retinal pigmented epithelial (RPE) cell toxicity, and Western blot for detection of pneumolysin (the major toxin of S. pneumoniae). Each strain was assigned a profile based on assay results. Four strains with different profiles were chosen for intravitreous injection of 100 colonyforming units (CFU) of each strain into the left eyes of each of 3 rabbits, followed by clinical examination and bacterial recovery from the vitreous. The concentrated extracellular milieu from each of the 4 strains was also tested for protease activity by gelatin zymography. Results: Eight of 20 strains (40%) lysed sheep erythrocytes and 15 (75%) were cytotoxic at a level of ≥2 on a scale from 0 (no activity) to 4 (full activity). Pneumolysin was detected in 6 (30%). Two of the 4 strains tested in vivo caused anterior chamber inflammation in addition to vitreous haze, with severity increasing with time up to 72 hours after infection. The most severe eyes, which were infected with strain E664, had clinical scores with a mean of 25.33 ± 2.08 (scale of 0 to 32) and bacterial recovery with a mean of 6.95 ± 0.53 log10 CFU/mL at 72 hours. Interestingly, E664 was not hemolytic nor did it produce pneumolysin, but was highly toxic to RPE cells. The secondmost virulent strain in vivo, 144065, was negative for all of the in vitro assays. The least virulent strain in vivo, E618, was positive for all of the in vitro assays. Zymography of the 4 strains tested in vivo showed protease activity in E664 and 144065, but not in the other 2 strains. Conclusions: VGS from endophthalmitis are diverse in toxin activity, and toxin activity cannot necessarily predict virulence in the rabbit eye. Strains with protease activity induce more damage in the rabbit eye than those without detectable protease. Determination of whether protease activity contributes to pathogenesis will aid in the first steps of characterizing the virulence of VGS in the eye. Commercial Relationships: Mary E. Marquart, None; Hannah R. Rice, None Program Number: 4060 Poster Board Number: A0237 Presentation Time: 8:30 AM–10:15 AM Measuring Severe Inflammatory Trachoma (TI) when prevalence is low provides indirect data on infection with C. trachomatis in endemic communities Andrea I. Zambrano2, Beatriz E. Munoz1, Laura Dize2, Harran Mkocha4, Charlotte Gaydos3, Thomas Quinn2, 3, Sheila K. West1. 1 Ophthalmology, Wilmer Eye Inst Johns Hopkins Univ, New York, NY; 2Infectious Disease, International Chlamydia Laboratory, Johns Hopkins School of Medicine, Baltimore, MD; 3National Institute of Allergy and Infectious Disease, Bethesda, MD; 4Kongwa Trachoma Project, Kongwa, United Republic of Tanzania. Purpose: Inflammatory trachoma (TI) is not measured when assessing the impact of trachoma programs because it is felt to indicate non-trachoma disease; only follicular trachoma (TF) is measured. We tested the supposition that TI was not associated with infection when disease prevalence is low, and does not add to understanding the effect of programs. Methods: In each of 52 communities in Kongwa Tanzania, 100 children ages 1-9 years were randomly selected for survey at baseline, 6, and 12 months. In 17 communities, Mass Drug Administration (MDA) was stopped at baseline because infection rates were 1% or less; 35 had MDA. Both eyelids were graded for evidence of TF and TI and a swab for detection of infection was taken. The swabs were tested using Aptima (GenProbe/Hologic) for presence of C. trachomatis. Overall prevalence rate of active trachoma in communities with and without MDA at each time point were compared, and the proportion of active trachoma cases that were TI alone was estimated. Proportion of active trachoma cases that were TI alone were compared among the treated and not treated communities. All comparisons were analyzed using Fisher’s exact test. Results: Overall prevalence of active trachoma at baseline was 6% (318 cases); 15% were TI alone. The prevalence of infection in TF cases was 36% and 37% in TI alone. At 6 months, the prevalence of active trachoma was 5.5% in communities where MDA was stopped; 18% of the cases were TI alone, for whom the rate of infection was 41.2%. In treated communities, prevalence of active trachoma was 3.9% and 10% of cases were TI alone for whom the rate of infection was 30.8%. At 12 months, prevalence of active trachoma in communities with MDA stopped was 5.8% and 17% of cases were TI alone; 59% had infection with C. trachomatis. In treated communities, prevalence rate was 5.1% with 22% of cases being TI alone for whom the rate of infection was 37.5%. Infection in the TI cases in communities where MDA was stopped was significantly greater than in treated communities (p=0.02). Conclusions: Despite low prevalence, the clinical sign of TI was better correlated with infection than TF, and particularly so when the survey was a year or more after MDA. TI should be measured as part of impact surveys and particularly at surveys several years after the last MDA. Commercial Relationships: Andrea I. Zambrano, None; Beatriz E. Munoz, None; Laura Dize, None; Harran Mkocha, None; ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts Charlotte Gaydos, None; Thomas Quinn, None; Sheila K. West, None Support: National Eye Institute EY022584 Program Number: 4061 Poster Board Number: A0238 Presentation Time: 8:30 AM–10:15 AM Evaluation of effectiveness of real-time PCR for bacterial keratitis diagnosis Daisuke Shimizu, Dai Miyazaki, Keiko Yakura, Tomoko Haruki, Yoshitsugu Inoue. Ophthalmology and Visual Science, Tottori University Faculty of Medicine, Yonago city, Japan. Purpose: To determine the effectiveness of measurement of bacterial DNA amount by real-time PCR for the diagnosis of bacterial keratitis, we characterized the sensitivity and specificity profile and determined cut-off value for diagnosis by using Receiver Operating Characteristic (ROC) analysis. Methods: Consecutive case series (241 eyes of 241 cases), suspected of infectious keratitis and measured for the amount of bacterial DNA in corneal tissue samples, were retrospectively analyzed. The measurement of bacterial DNA amount (16S rDNA by real-time PCR), smear examination by Gram and Fungiflora staining, and culture testing were evaluated for diagnostic efficacy by ROC analysis. Results: One hundred and nine eyes were diagnosed as definitive bacterial keratitis. Eighty four eyes were diagnosed as keratitis of other causes. In the definitive bacterial keratitis eyes, average bacterial DNA copy number was 1.6x106, culture positive rate was 54%, and the smear positive rate was 45%. In the non bacterial keratitis eyes, they were 6.1x103, 0.0%, and 7.0%, respectively. Area under the curve (AUC) was calculated to show diagnostic efficacy based on ROC analysis of these testing. The AUC for definitive bacterial keratitis was 0.73, 0.65, and 0.60 for smear testing, bacterial DNA copy measurement, and culture testing, respectively. To determine the most useful combination of each testing, AUC of combined outcome was calculated using propensity scoring analysis. Combination of smear testing and bacterial DNA amount indicated highly efficacious diagnostic value (AUC: 0.80), and cut off value of bacterial DNA copy for diagnosis of bacterial keratitis was 1.0X103. Conclusions: Combined testing of bacterial DNA quantification and smear testing was highly efficacious to definitively diagnose bacterial keratitis at initial visit. Commercial Relationships: Daisuke Shimizu, None; Dai Miyazaki, None; Keiko Yakura, None; Tomoko Haruki, None; Yoshitsugu Inoue, Alcon Japan Inc. (F) Program Number: 4062 Poster Board Number: A0239 Presentation Time: 8:30 AM–10:15 AM Evaluation of real-time PCR for the diagnosis of intra-ocular tuberculosis Soumyava Basu, Manas R. Barik, Praveen K. Balne, Soveeta S. Rath, Mamatha Reddy, Savitri Sharma. LV Prasad Eye Institute, Bhubaneswar, India. Purpose: Definitive diagnosis of intraocular tuberculosis has remained challenging despite recent advances in molecular diagnostic techniques. Here we report the development of a realtime polymerase chain reaction (PCR) assay for detection of Mycobacterium tuberculosis complex in aqueous and vitreous samples from eyes with intraocular tuberculosis. Methods: Aqueous or vitreous humor samples were collected from patients with clinically suspected ocular tuberculosis (based on previously published diagnostic criteria; Gupta et al, Surv Ophthalmol’ 2007) and non-uveitis eyes undergoing vitrectomy or cataract surgeries (controls). mpb64 gene of M. tuberculosis genome and human RPPH1 (RNase P RNA component H1) were amplified from the extracted DNA and detected real-time by customized FAM-labeled probes. The ratio of copy numbers of mpb64 and RPPH1, obtained from each test and control sample was used to generate Receiver Operating Characteristic (ROC) curves. The optimum cut-off value of real-time PCR was identified from the experimental data that had the highest Youden index (Youden index = sensitivity+specificity-1). Results: M. tuberculosis complex genome was detected in 33 of 47 test samples (70.2%) and 2 of 18 healthy controls (11.2%) based on optimum cutoff value of copy number ratios (0.025) obtained from ROC curve having highest Youden index number, 0.727. At this cutoff value the sensitivity was 81.0% and specificity 91.7%. The copy number ratios varied widely in different clinical samples, with the highest median value seen in intermediate uveitis sub-group (0.387±0.664). The numbers were not sufficient to compare aqueous and vitreous samples. Conclusions: We could develop a highly specific and sensitive PCR assay for detection of M. tuberculosis complex in aqueous and vitreous samples. There was wide variation in copy numbers in different disease sub-types that need to be analyzed in larger studies. Commercial Relationships: Soumyava Basu, None; Manas R. Barik, None; Praveen K. Balne, None; Soveeta S. Rath, None; Mamatha Reddy, None; Savitri Sharma, None Program Number: 4063 Poster Board Number: A0240 Presentation Time: 8:30 AM–10:15 AM Models of microbial keratitis in ex vivo rabbit and human corneas Abigail Pinnock1, Nagaveni Shivshetty2, Sanhita Roy2, Stephen Rimmer1, Ian Douglas1, Sheila MacNeil1, Prashant Garg2. 1University of Sheffield, Sheffield, United Kingdom; 2LV Prasad Eye Institute, Hyderabad, India. Purpose: To study microbial keratitis, cultured cells and in vivo animal models are commonly used but, these are either not representative of the in vivo situation or involve the use of many animals. Recently, there is increased use of ex vivo corneal models to replace or reduce animal use but there is no direct comparison of bacterial and fungal keratitis in these models. Accordingly, we aimed to establish reproducible models of bacterial and fungal infections in rabbit and human ex vivo corneal models to aid studies of keratitis. Methods: Wild brown rabbit and donated human corneas were maintained in corneal organ culture as previously described (Deshpande et al., Biomater. 2013). Corneas were wounded with a scalpel, and exposed to, or injected intrastromally with, 108Staphylococcus aureus, Pseudomonas aeruginosa or Candida albicans for 24 or 48h at 37°C. Corneas were lysed, the resulting suspension serially diluted and spotted onto agar plates for colony enumeration. Corneal tissue was histologically processed and sections were Gram-stained. Corneas not exposed to microbes were used as controls. Results: After 24h, using the scalpel wounding method, S.aureus, P.aeruginosa and C.albicans were recovered at 6.4±1.5x105, 5.5±0.8x106 and 2.2±0.8.1x104 CFU/rabbit cornea respectively and 3.8±0.8x106, 4.4±0.6x108 and 1.9±0.3x105 CFU/human cornea respectively. No difference in the CFU/cornea after 48h was observed compared with 24h. The injection method yielded a 10-fold increase (p<0.05) in detectable organisms for P.aeruginosa but no difference for S.aureus or C.albicans, compared with the scalpel method. Histology of the scalpel-wounded and injection models indicated extensive infiltration of P.aeruginosa, throughout the entire cornea, with less infiltration observed for S.aureus and C.albicans. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts Conclusions: Bacterial and fungal infections were initiated in ex vivo corneal models after 24 and 48h, with both scalpel wounding and injection methods suitable for inducing infection. Differences between the CFU/cornea for rabbit and human corneas may be due to the expression of different antimicrobial peptides or surface receptors influencing colonisation of the tissue. These simple and reproducible ex vivo models will be useful as an alternative to monolayer cells and in vivo models for investigating microbial keratitis. Commercial Relationships: Abigail Pinnock, None; Nagaveni Shivshetty, None; Sanhita Roy, None; Stephen Rimmer, None; Ian Douglas, None; Sheila MacNeil, None; Prashant Garg, None Support: Wellcome-DBT grant 0998800/B/12/Z Program Number: 4064 Poster Board Number: A0241 Presentation Time: 8:30 AM–10:15 AM The use of pediatric blood culture bottles in the diagnosis of acute postoperative endophthalmitis Tatiana Tanaka1, Joao N. Almeida2, Thais S. Di Gioia2, Flavia Rossi2, Juliana M. Kato3, Bruno F. Ferreira1, Aline D. Ruppert1, Yoshitaka Nakashima1, Sergio L. Pimentel1, Joyce H. Yamamoto1. 1 Ophthalmology, University of São Paulo, São Paulo, Brazil; 2 Microbiology, University of São Paulo, São Paulo, Brazil; 3Faculty of Medicine, University of São Paulo, São Paulo, Brazil. Purpose: Sample culture is an essential laboratory procedure necessary to confirm the microbiological etiology and to prompt and appropriate treatment of endophthalmitis. Techniques for culturing vitreous samples vary. The traditional method for culturing the undiluted vitreous uses solid media plates and tioglicolate. Direct inoculation of blood culture bottles may be alternative.1 Pediatric blood culture bottles may be suitable for samples of small amount.2 The present study evaluated the culture yield in the diagnosis of endophthalmitis using for inoculation conventional methodology (CM) or pediatric blood culture bottle (PBCB). Methods: This retrospective study included cases of clinically suspected acute postoperative endophthalmitis treated between January 2010 and December 2013 at Department of Ophthalmology, University of São Paulo, SP, Brazil. Undiluted vitreous were cultivated in CM from January 2010 to December 2011 and in PBCB from January 2012 to December 2013. The isolated agents and culture yield were analysed for each methodology. The study was approved by the Institutional Ethics Committee. Results: Fourty two cases were included during this 4-year period. These cases were associated with phacoemulsification (n=20, 47.6%), trabeculectomy (n=9, 21.4%), extracapsular cataract extration (n=6, 14.3%), pars plana vitrectomy (n=4, 9.5%), phacoemulsification/ trabeculectomy (n=2, 4,8%) and intravitreal bevacizumab injection (n=1, 2.4%). The most prevalent agents were Staphylococcus epidermidis (n=5, 23.8%), Streptococcus viridans (n=3, 14.3%), coagulase-negative Staphylococus (n=2, 9.5%) and Enterococcus faecalis (n=2, 9.5%). The culture yield of the 23 eyes cultured in CM was 36.4 % and of the 20 eyes cultured in PBCB was 65% (Table). Conclusions: In spite of this non-comparative and retrospective study, pediatric blood culture bottle yielded substantially high positivity and seems a good alternative to CM if access to microbiological facilities is suboptimal. PBCB had some advantages over conventional methodology: easy inoculation, reduce the risk of contamination with transport and possibility of storage at room temperature. 1 Joondeph BC et a. A new culture method for infectious endophthalmitis. Arch Ophthlamol 1989;107:1334-7; 2 Heggers JP et al. The efficacy of pediatric bllod culture sets in the determinations of burn bacteremia. J Burn Care Rehabil 1990; 11:419-22 Table Commercial Relationships: Tatiana Tanaka, None; Joao N. Almeida, None; Thais S. Di Gioia, None; Flavia Rossi, None; Juliana M. Kato, None; Bruno F. Ferreira, None; Aline D. Ruppert, None; Yoshitaka Nakashima, None; Sergio L. Pimentel, None; Joyce H. Yamamoto, None Program Number: 4065 Poster Board Number: A0242 Presentation Time: 8:30 AM–10:15 AM Gene expression analysis of severe bacterial, fungal and acanthamoeba keratitis: role of immune and inflammatory biomarkers of disease Jaya D. Chidambaram1, 2, Namperumalsamy Venkatesh Prajna2, 3 , Palepu Srikanthi2, Manisha Shah3, Lalitha Prajna3, Elakkiya Shanmugam3, Julien Bauer4, Martin Holland1, Matthew J. Burton1, 5 1 . Clinical Research Dept, London School of Hygiene & Tropical Medicine, London, United Kingdom; 2Cornea Department, Aravind Eye Hospital, Madurai, India; 3Aravind Medical Research Foundation, Madurai, India; 4Pathology Dept, University of Cambridge, Cambridge, United Kingdom; 5London School of Hygiene & Tropical Medicine, International Centre for Eye Health, London, United Kingdom. Purpose: Microbial keratitis (MK) is a leading cause of blindness worldwide. Excessive host inflammatory responses are thought to be the cause of tissue damage in MK. In this study, we investigated the expression of 45 genes involved in immune and inflammatory pathways, and extracellular matrix (ECM) modulation in corneal cells taken from late stage human bacterial (BK), fungal (FK) and acanthamoeba keratitis (AK), as compared to normal cadaver corneal tissue. Methods: Corneal swab samples from 239 patients presenting to Aravind Eye Hospital Cornea Clinic in India from Feb 2012 to Feb 2013 with MK (>=3mm diameter stromal infiltrate). Final outcome was recorded at 21 days post-enrolment (i.e. “nonhealing”=perforation or descemetocoele or intervention, e.g. corneal glue, transplant or intrastromal antifungal). Cadaver corneal tissue (n=13) was collected from Aravind Eye Bank. Total RNA was extracted from swabs/tissue (Qiagen, Netherlands), and RTqPCR performed with custom Taqman Low Density Arrays (Life Technologies, USA). Genes were normalized to HPRT1. Pairwise comparisons between BK, FK or AK versus Controls, BK versus FK and FK healed versus FK non-healed were performed and statistical significance of differential expression assessed with Wilcoxon rank sum test in Stata 12.1 (Bonferroni adjusted p-values). ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts Results: 218 patients were microbiologically positive for fungus (n=185, mainly Fusarium sp. or Aspergillus flavus), bacteria (n=20, mainly Streptococcus pneumoniae) and Acanthamoeba sp. (n=13). Differential expression (FC >2 or <2, p<0.0005) was found in 15, 9 and 3 genes in FK, BK and AK versus Controls, with no significant difference between BK and FK expression. Upregulated genes included ECM modifiers MMP9 (BK), MMP10 (FK, BK) and COL5A1 (BK, FK, AK); cytokines IL8 (BK, FK), IL18 (FK), and neutrophil antimicrobial effectors S100A9 and PI3 (all 3). In FK, PI3 was upregulated in non-healing ulcers (FC 3.6, p=0.002). Conclusions: Several MMPs and immune effectors are significantly upregulated in BK, FK and AK. PI3, expressed in healing epithelia and known to be an immune modulator, correlates with poor prognosis in other diseases (e.g. cutaneous graft-vs-host disease). Further research is needed into PI3 as a possible prognostic biomarker for FK. Commercial Relationships: Jaya D. Chidambaram, None; Namperumalsamy Venkatesh Prajna, None; Palepu Srikanthi, None; Manisha Shah, None; Lalitha Prajna, None; Elakkiya Shanmugam, None; Julien Bauer, None; Martin Holland, None; Matthew J. Burton, None Support: Wellcome Trust PhD Research Fellowship Grant no. 097437/Z/11/Z Program Number: 4066 Poster Board Number: A0243 Presentation Time: 8:30 AM–10:15 AM Microbiome of contact lens cases following corneal infiltrative events Ajay Kumar Vijay1, Jacqueline Tan1, Lily Ho1, Anahit Penesyan2, Ian Paulsen2, Mark D. Willcox1. 1School of Optometry & Vision Science, University of New South Wales, Sydney, NSW, Australia; 2 Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia. Purpose: Contact lens cases become contaminated with bacteria during use and this can lead to corneal infiltration or infection. Many bacterial species remain non-culturable with standard laboratory techniques. We performed culturing and next-generation DNA sequencing to identify both culturable and non-culturable organisms that reside on contact lens cases of patients during corneal infiltrative events. Methods: Contact lens cases were collected from patients with corneal infiltrative events and both wells were individually swabbed. Swabs from the right wells were cultured to isolate and identify viable microbes using standard culture techniques. Microbial DNA was extracted from the swabs of the left wells, and 16S rRNA amplicon sequencing performed using PCR primers 515/806 targeting the V4 variable region of 16S rRNA gene. Results: Six lens cases were collected from five subjects with corneal infiltrative events: microbial Keratitis (n=1), contact lens peripheral ulcer (n=1) and infiltrative keratitis (n=3). All six lens cases were culture positive for Gram-negative bacteria; no Gram-positive bacteria, fungi or Acanthamoeba were grown. Several bacterial strains were cultured from 5/6 of the lens case wells; S. marcescens (n=5), A. xylosoxidans (n=2), S. maltophilia (n=1), A. faecalis (n=1) and P. fluorescens (n=1). 16S rRNA sequencing of DNA from lens cases identified multiple microbial species (median = 30, max = 79, min = 21) including members of Proteobacteria (95.9%), Bacteroidetes (2.2%), Actinobacteria (1.8%), Firmicutes (0.1%), Cyanobacteria (0.01%), Candidate division OP11 and Deinococcus thermos (combined 0.01%). Conclusions: The results of this study confirm that a large number of microbes remain non-culturable and DNA analysis of lens cases can provide valuable information that may help understand the pathogenesis of contact lens related microbial keratitis and corneal infiltrative events. Commercial Relationships: Ajay Kumar Vijay, Bausch+Lomb (F); Jacqueline Tan, None; Lily Ho, None; Anahit Penesyan, None; Ian Paulsen, None; Mark D. Willcox, Bausch+Lomb (F) Program Number: 4067 Poster Board Number: A0244 Presentation Time: 8:30 AM–10:15 AM Human Ocular Surface Microbiome Composition Revealed By Next-Generation Sequencing Thuy Doan1, Lakshmi Akileswaran1, Dallin Andersen1, Narae Ko1, Angira Shrestha1, Cecilia S. Lee1, Aaron Lee2, Russell Van Gelder1. 1Ophthalmology, University of Washington, Seattle, WA; 2 Ophthalmology, University of British Columbia, Vancouver, BC, Canada. Purpose: Human mucosal surfaces are thought to be colonized by a diverse community of microorganisms that help shape the immune system and when altered may lead to infections or cause inflammation in the host. Conventional culture techniques have failed to identify the composition and to characterize the diversity of these communities because a majority of these microbes are unculturable. In this study, we sought to characterize the ocular surface bacterial community in healthy subjects by using 16S rRNA gene deep sequencing on the Illumina platform. Methods: Conjunctiva samples of the upper and lower fornices of both eyes were collected using forensic DNA recovery swabs from 35 healthy volunteers. Along with appropriate negative control samples, the conjunctiva samples underwent DNA extraction, library preparation, and 16S rRNA gene deep sequencing on the Illumina platform. Quantitative PCR for Torque Teno Virus (TTV) was also performed. Data were analyzed in R. Results: Sequencing resolution to the genus and species levels were obtained for 140 conjunctiva samples from 35 healthy volunteers. Propionibacterium acnes, Arthrospira fusiformis, Corynebacterium tuberculostearicum, Enterobacter hormaechei, and Chryseobacterium indologenes were the most abundant species across all samples. Principal component analyses showed that the genera responsible for the majority of the variance across all conjunctiva samples were Corynebacterium, Propionibacterium, and Staphylococcus. TTV was detected in 63% of the patients (17/27). Subgroup analyses revealed that the TTV load was statistically higher in men compared to women (0.012 TTV copy/epithelial cell ± 0.0028 TTV copy/epithelial cell vs. 0.001 TTV copy/epithelial cell ± 0.0006 TTV copy/epithelial cell, mean ± SEM, p = 0.0078). Conclusions: The ocular surface microbiome bacterial composition in healthy volunteers is diverse. The variability across samples is largely determined by Corynebacterium, Propionibacterium, and Staphylococcus. Low TTV levels are found in the majority of the samples and TTV viral load is dependent on gender. Future experiments using unbiased next-generation sequencing to characterize the bacterial, fungal, and viral composition of the ocular surface will further our understanding of ocular infectious and inflammatory diseases. Commercial Relationships: Thuy Doan, None; Lakshmi Akileswaran, None; Dallin Andersen, None; Narae Ko, None; Angira Shrestha, None; Cecilia S. Lee, None; Aaron Lee, None; Russell Van Gelder, None Support: EY022038, P30EY001730, Unrestricted Research Departmental Grant From Research To Prevent Blindness ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts Program Number: 4068 Poster Board Number: A0245 Presentation Time: 8:30 AM–10:15 AM Mucosal microbiome in Sjögren Syndrome Stephen C. Pflugfelder1, Cintia S. De Paiva1, Dan B. Jones2, Quianta Moore1, Shani Corbiere5, Joseph Petrosino3, Diane Smith3, Michael E. Stern4, 1, Nadim Ajami3. 1Ophthal-Ocular Surf Ctr, Baylor College of Medicine, Houston, TX; 2Ophthalmology, Baylor College of Medicine, Houston, TX; 3Alkek Center for Metagenomics and Microbiome Research, Baylor College of Medicine, Houston, TX; 4 Biological Sciences, Allergan, Irvine, CA; 5Sjogren Syndrome Support Group, Houston, TX. Purpose: To compare the ocular, oral and fecal microbiome in patients with Sjögren syndrome (SS) with control subjects Methods: Conjunctival, tongue and fecal samples were obtained from 10 patients with primary SS meeting revised ACR criteria and controls (normal eyes and fecal samples, and tongue samples from patients with rosacea). Severity of oral and ocular surface disease was graded. Conjunctival goblet cell density was counted in impression cytology. 16s ribosomal DNA gene sequencing was performed by 454 (conjunctival) and MiSeq sequencing and sequences were mapped to microbial databases. Relative abundance of phyla and genera and alpha and beta diversity of observed OTUs between groups were compared. Results: A low abundance ocular surface microbiome consisting of core phyla Actinobacteria,, Bacteroidetes, Proteobacteria and Firmicutes was identified. There were no differences in alpha or beta diversity between normal and dry eyes; however, there was greater abundance of Firmicutes in the SS group. Compared to control, alpha diversity was greater in the tongue and reduced in the stool in the SS group. Between group differences in relative abundance were observed with greater Streptococcus and Hemophilus and reduced Neisseria and Fusobacterium genera in the SS tongue and greater abundance of Blautia, Escherichia/Shigella, and Streptococcus and reduced Akkermansia, Subdoligranulum, Faecalibacterium and Prevotella in the SS stool. Distinct clustering of OTUs was seen in SS stool and subjects with most severe clinical severity scores had the least diversity of observed OTUs in the stool. Conclusions: Minimal differences in abundance and diversity were found in the SS ocular microbiome. In contrast, SS stool showed a less diverse microbiome that contained a greater number of inflammatogenic and lower number of homeostatic flora. Commercial Relationships: Stephen C. Pflugfelder, None; Cintia S. De Paiva, None; Dan B. Jones, None; Quianta Moore, None; Shani Corbiere, None; Joseph Petrosino, None; Diane Smith, None; Michael E. Stern, None; Nadim Ajami, None Support: Sjögren Syndrome Metagenomics Research Fund, NIH Grant EY11915 (SCP), Research to Prevent Blindness, Oshman Foundation, William Stamps Farish Fund, Hamill Foundation Program Number: 4069 Poster Board Number: A0246 Presentation Time: 8:30 AM–10:15 AM Metaproteomic analysis in infected ocular surface Diana Gabriela Ponce-Angulo1, 2, Antonio Bautista-Hernandez1, 3, Gerardo Aparicio-Ozores2, Victor M. Bautista1. 11. Research Unit/ Microbiology and Ocular Proteomics, Institute of Ophthalmology “Fundación de Asistencia Privada Conde de Valenciana”, Mexico City, Mexico; 22. Laboratory of Medical Bacteriology, Department of Microbiology, National School of Biological Sciences- IPN, Mexico City, Mexico; 33. Doctoral Program in Biological Sciences and health, Autonomous Metropolitan University (UAM), Mexico city, Mexico. Purpose: To realize a comparative metaproteomic analysis of the ocular surface in patients with ocular infection. Methods: The samples were obtained from patients with healthy and infected ocular surface. The human tears samples were taken following the internal protocols in both groups. Steril saline solution was applied over the ocular surface and collected by capillar tubes. Microorganisms were identified by automated microbiology methods. Chocolate agar, blood agar, Sabouraud agar were seeded with samples from both groups, samples also were added to Brain Heart Infusion. The bacterial identification was determined by Vitek 2 Compact System. The fungi were identificated by morphologycal caracteristics. The tears samples of each patient were analysed by 2D electrophoresis and differential protein expression between groups was performed using Dymensión 2 Software. Results: Microbiological analysis showed the presence of Staphylococus aureus, S. epidermidis, S. lentus, Kokuraria rosea, Streptococcus thoraltensis, Rothia mucilaginosa, Granulicatella adiacens and Candida guillermondi in healthy group; S. capitis, S. epidermidis and C. albicans were isolated and identified in infected group, three patients not shown microbiology growth. Proteomic analysis showed In the results of healthy group, there were differential growt of Staphylococus aureus, S. epidermidis, S. lentus, Kokuraria rosea, Streptococcus thoraltensis, Rothia mucilaginosa, Granulicatella adiacens and Candida guillermondi. In the infection group, the findings in the isolated microorganism were S. capitis, S. epidermidis and C. albicans and in three of all them had not microbiology grown. At the end of the analysis of differential expression in 2D gels without infection group compared with the control group the following results: in the first sample were obtained 5 differentially expressed spots, in the second sample 4 spots, en the third sample 2 spots, in the fourth sample 3 spots, in the fifth sample 2 spots and finally in the sixth sample 2 spots. The metaproteomic analysis shows the relevant expression of diferential proteins. The diferential proteins will be identificated by mass spectrometry. Conclusions: The bacterial species were obtained in both groups were Staphylococcus epidermidis and Staphylococcus capitis. The analysis have difencial protein expression in helthy group in compararising with infection group. Commercial Relationships: Diana Gabriela Ponce-Angulo, None; Antonio Bautista-Hernandez, None; Gerardo Aparicio-Ozores, None; Victor M. Bautista, None Support: This work was supported by “Conde de Valenciana” Foundation Program Number: 4070 Poster Board Number: A0247 Presentation Time: 8:30 AM–10:15 AM Microbiologic spectrum of post-injection endophthalmitis by indication for intravitreal anti-VEGF therapy Charles Calvo1, Nadim Rayess1, Ehsan Rahimy1, Chirag Shah2, Jeremy D. Wolfe3, Eric Chen4, Francis DeCroos5, Sunir J. Garg1, Jason Hsu1. 1Retina Service, Wills Eye Hospital, Philadelphia, PA; 2Ophthalmic Consultants of Boston, Boston, MA; 3Associated Retinal Consultants at William Beaumont Hospital, Royal Oak, MI; 4 Retina Consultants of Houston, Houston, TX; 5Southeastern Retina Associates, Chattanooga, TN. Purpose: To describe the microbiologic spectrum of endophthalmitis following intravitreal anti-VEGF injections for treatment of neovascular age-related macular degeneration (AMD), diabetic eye disease, and retinal vein occlusion (RVO). Methods: A multicenter, retrospective, consecutive case series. Results: Between January 1, 2011 and September 30, 2013, a total of 503,890 intravitreal anti-VEGF injections were performed at the five participating clinical sites. Presumed infectious endophthalmitis occurred in 159 of 416,133 injections performed for neovascular ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts AMD (1/2617), 16 of 40,982 for diabetic eye disease (1/2561), and 8 of 46,775 for RVO (1/5846). For patients with neovascular AMD, 61 of the 159 cases (38%) of endophthalmitis were culture positive, corresponding to a culture positive rate of 1/6822. The cultured organisms included 13 cases of coagulase-negative Staphylococcus, 12 of S. epidermidis, 5 of S. pneumonia, 5 of S. mitis, 5 of methicillin-sensitive S. aureus, 4 of Staphylococcus lugdunesis, 4 of Enterococcus fecalis, 3 of S. viridans, 3 of non-differentiated gram-positive cocci, and 1 case of each of the following organisms: Staphylococcus auricularis, Staphylococcus homininis, Streptococcus sanguis, alpha-hemolytic Streptococcus, Candida parapsicolosis, Propionibacterium, and Lactobacillus. For patients treated for diabetic eye disease, 8 of the 16 cases (50%) were culture positive, providing a culture positive rate of 1/5123. Four eyes grew coagulase-negative Staphylococcus, 2 had S. epidermidis, and there was 1 case of S. pneumonia and 1 case of H. influenzae. For patients with RVO, 4 of the 8 cases (50%) were culture positive, resulting in an overall culture positive rate of 1/11,694. There was 1 case of S. pneumoniae, methicillin-sensitive S. aureus, S. mitis, and coagulasenegative Staphylococcus. Conclusions: Gram-positive coagulase-negative Staphylococcus was the most common bacteria isolated in cases of post-injection endophthalmitis. A greater proportion of coagulase-negative Staphylococcus was found in endophthalmitis following injections for diabetic eye disease (75%) compared to AMD (51%) and RVO (25%). Oral flora pathogens accounted for 22% of the cases. Commercial Relationships: Charles Calvo, None; Nadim Rayess, None; Ehsan Rahimy, None; Chirag Shah, None; Jeremy D. Wolfe, None; Eric Chen, None; Francis DeCroos, None; Sunir J. Garg, None; Jason Hsu, None Program Number: 4071 Poster Board Number: A0248 Presentation Time: 8:30 AM–10:15 AM Microbiologic Analysis of Dacryocystitis at LAC + USC Medical Center Mica Bergman1, 2, Jesse berry1, 2. 1USC Eye Institute, Los Angeles, CA; 2Ophthalmology, LAC + USC Medical Center, Los Angeles, CA. Purpose: To investigate the microbiologic profile of dacryocystitis at a major county hospital in Southern California. Methods: IRB approved retrospective review of dacryocystorhinostomy (DCR) operations performed from 1/1/2000 – 11/3/2014. Patients with a diagnosis of dacryocystitis in whom at least one culture was performed during the course of their care were included in this study. Results: Sixteen cases of dacryocystitis were identified in fifteen patients. Of these, eleven underwent culture one time, four underwent culture two times, and one underwent culture three times, yielding a total of 22 cases. Of the 22 cases, 19 cases had a positive yield, 12 cases were monomicrobial, 6 cases were bimicrobial, and 1 case was trimicrobial. In lacrimal sacs undergoing culture two or more times, the same organism was found in multiple specimens two out of five times. In total, there were 14 isolates of gram-positive cocci, 10 isolates of gram-negative bacilli, 1 isolate of gram-negative diplococci, and 2 fungal isolates. The most commonly identified bacteria was klebsiella pneumoniae (4 isolates), followed by staphylococcus aureus and streptococcus viridans (3 isolates each). Bacteria thought to be nonpathogenic (eg staphylococcus epidermidis and diphtheroids) were excluded. Conclusions: This series demonstrates that there is a wide range of pathogens implicated in dacryocystitis in our county hospital in Southern California. The most commonly isolated bacteria were klebsiella pneumoniae, staphylococcus aureus, and streptococcus viridans. One third of patients had polymicrobial culture results. Given that gram-negative and gram-positive pathogens were found in similar numbers, it is advisable to treat initially with a broadspectrum antibiotic. Commercial Relationships: Mica Bergman, None; Jesse berry, None Support: An Unrestricted grant from Research to Prevent Blindness, New York, NY 10022 Program Number: 4072 Poster Board Number: A0249 Presentation Time: 8:30 AM–10:15 AM 15 Years of Microbial Keratitis at an Urban University Practice in Saint Louis: The Isolates Hugo Y. Hsu1, 2, Sean Edelstein3. 1Doheny Eye Institute, Los Angeles, CA; 2Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, CA; 3Ophthalmology, Saint Louis University School of Medicine, Saint Louis, MO. Purpose: The spectrum of micro-organisms causing infectious keratitis varies between geographic areas and through time. We wish to identify the isolated micro-organisms from infectious keratitis cases and to observe any trends in the spectrum of causative organisms over a 15-year period at Saint Louis University’s Department of Ophthalmology. Methods: We searched the database of the microbiology department and the diagnosis database of the ophthalmology department at Saint Louis University to identify cases of microbial keratitis from 1999-2013. Records of culture-positive cases were reviewed retrospectively. Non-contaminant isolates were tabulated into three 5-year periods (1999-2003; 2004-2008; and 2009-2013) and compared. Results: 229 non-contaminant isolates were identified: 45 from 1999-2003; 84 from 2004-2008; and 100 from 2009-2013. Overall, Gram-positive organisms were the most commonly isolated (47%) followed by Gram-negative organisms (34%). Fungi represented 18% of the overall isolates. Separated into the three 5-year periods, Gram+ organisms represented 53%, 44%, and 46% of the total; Gramrepresented 38%, 27%, and 33% of the total; and fungal organisms represented 9%, 20%, and 20% of the total. Pseudomonas was the most commonly isolated organism overall (21%) as well as in each of the 3 time periods. Streptococcus species were the next most common isolates overall (15%) followed by coagulase-negative staphylococcus and Staphylococcus aureus (14% each). The percentage of Staphylococcus aureus isolates that were oxacillinresistant (ORSA) increased in each of the three 5-year periods from 22% to 44% to 69%. Conclusions: The number of non-contaminant isolates increased in each of the three 5-year periods. Pseudomonas was the most common isolate found in infectious keratitis at Saint Louis University over the 15-years period. The two biggest changes we observed were in the number and percentage of fungal isolates as well as the increase in the proportion of ORSA isolates over time. Fungal organisms accounted for 9% of isolates at the start of the 15-year period but then doubled to 20% during the mid 2000’s and continued through the end of the 15-year period in 2013. The percentage of ORSA isolates tripled over the 15 years period. Commercial Relationships: Hugo Y. Hsu, None; Sean Edelstein, None ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts Program Number: 4073 Poster Board Number: A0250 Presentation Time: 8:30 AM–10:15 AM Clinical and Microbiological Characteristics of Infectious Keratitis in Mexico Alejandro F. Ibarra-Lozano, Pedro M. Gonzalez, Jaime Torres, Jorge E. Valdez. Ophthalmology Residence, Instituto Tecnológico y de Estudios Superiores de Monterrey, San Pedro Garza Garcia, Mexico. Purpose: Geographical differences play a major role to the clinical and microbiological characteristics of infectious keratitis. The purpose of this observational retrospective study is to present these aspects of the infectious keratitis found in Mexico to be able to establish empiric treatment guidelines. Methods: Patient records from January 2010 to November of 2014 were revised. Cases in which corneal scrape cultures were made were selected. Variables studied included were: gender, age, time of presentation to medical attention, visual acuity, microbiologic agent encountered, comorbidities, and complications. Results: Twenty-eight cases of microbial keratitis with corneal scrape culture were found. Male:Female ratio was 18:10 respectively and age at presentation varied from 2 to 85 years. The most commonly encountered microbiologic agent was Pseudomona aeruginosa, found in 8 patients; 5 cases were positive to fungal agents, 2 for Fusarium spp and 2 for Candida spp; S. pneumonie was found in two patients, Acantamoeba spp in one patient, and Corynebacerium matruchotti in another patient. Other isolated microorganisms included M. catharralis, H. influenzae, A. xyloxidans, Aspergillus, and S. marcescens. Twenty one percent of the cultures were negative. Ocular trauma was associated to 32.1% of the cases, including vegetal and metallic objects. Diabetes Mellitus was found as a comorbidity in 32.1% of the cases and one case was HIV positive. Only 3 contact lens users were identified in the total of cases. Time from the beginning of symptoms to time of medical attention ranged from 1 to 60 days. Patients who waited more time to get medical attention were those with fungal and parasitic infections. Gramnegative bacteria dominated the microbiologic pattern being the encountered organism in 36.3% of the positive cultures. Four patients had corneal perforation, two of them associated to Pseudomona aeruginosa. Conclusions: This study revised the epidemiology of microbial keratitis in northern Mexico. The microbiologic pattern encountered varies importantly from that in other geographic zones, especially in the unusually high Gram-negative predominance. There was a tight relation between microbial keratitis with trauma and Diabetes Mellitus. This study will aid the generation of new treatment guidelines in the geographic zone studied. Commercial Relationships: Alejandro F. Ibarra-Lozano, None; Pedro M. Gonzalez, None; Jaime Torres, None; Jorge E. Valdez, None Program Number: 4074 Poster Board Number: A0251 Presentation Time: 8:30 AM–10:15 AM Gram-negative Isolates from Patients with Endophthalmitis: Incidence Rates and Antibiotic Susceptibilities Benjamin D. Wilson, Darlene Miller, Harry W. Flynn. Bascom Palmer Eye Institution, Miami, FL. Purpose: To report incidence rates and antibiotic susceptibilities among gram-negative isolates from patients with endophthalmitis. Methods: Microbiology reports were reviewed to identify the incidence rates of gram negative pathogens (Enterobacteriaceae vs NonEnterobacteriaceae) recovered from endophthalmitis patients during a 25 year period (1990-2014). A combination of disk diffusion, Vitek 2 and Etests were used to susceptibility trends for amikacin, ceftazidime, and ciprofloxacin. Etests were used to evaluate moxifloxacin. Identifications were confirmed using conventional methods and Vitek 2. Results: In period I (1990-1999, period I) Gram negative pathogens represent 11.4% (n=125/1095) of the cases. Organism spectrum included: Enterobacteriaceae (32%, n=40/125). Top pathogens were ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts Proteus mirabilis (n=12/30%), Serratia marcescens (n=8/20%), and Klebsiella pneumoniae (n=7/17.5%). NonEnterobacteriaceae were the predominant group -68% (n= 85/125). Top pathogens: Pseudomonas aeruginosa (41/48%), Haemophilus influenzae (n=15/17.6%) and Moraxella species (n=8/9.4%). In period II (2000-2014), gram negative pathogen frequency was 11.6% (n=121/1039), 75/62% were nonEnterobacteriaceae. Top pathogens were: Pseudomonas aeruginosa (n=37/49.3%), Haemophilus influenzae (15/20%), Moraxella osloensis (8/10.7%). For Enterobacteriaceae, the rate was 38% (n= 46). Serratia marcescens (n=16/34.8%), Enterobacter cloacae (9/19.6%), and Klebsiella pneumoniae (5/10.9%). Antimicrobial susceptibilities for Enterobacteriaceae from period I were: amikacin-80%; ceftazidime-83% and ciprofloxacin-89%. Non-Enterobacteriaceae: amikacin-100%; ceftazidime-90%; and ciprofloxacin-98%. Susceptibilities for Enterobacteriaceae from period II: amikacin, 95%; ceftazidime, 97%; and ciprofloxacin- 89%. Non-Enterobacteriaceae: amikacin, 84%; ceftazidime, 91%; and ciprofloxacin-98%. Moxifloxacin susceptibility for the Enterobacteriaceae (N=12) was 83% versus 41% for the Non-Enterobacteriaceae (N=17). The highest resistance and/or nonsusceptible trends for all pathogens were observed for moxifloxacin-41% (n=12/29), followed by amikacin (10%), ceftazidime (6%) and ciprofloxacin (4%). Conclusions: Gram negative isolates from patients with endophthalmitis have remained stable over the 25 year period. Emerging resistance trends to commonly used antibiotics amikacin, ceftazidime, ciprofloxacin and moxifloxacin have been identified. Commercial Relationships: Benjamin D. Wilson, None; Darlene Miller, BPEI (E); Harry W. Flynn, BPEI (E) Support: RPB Unrestricted Award, NIH Core Grant P30EY014801 Program Number: 4075 Poster Board Number: A0252 Presentation Time: 8:30 AM–10:15 AM Detection of virulence factors with multiplex PCR Technique of Conjunctival Coagulase Negative-Staphylococcus (CNS) from patients undergoing cataract surgery Veronica E. Castillo1, Yolanda Lopez2, Margarita Samudio2, Norma Farina2, Sonia Abente2, Nilsa Gonzalez2, Florentina Laspina2, Agustin Carron1, Diogenes Cibils1, Herminia Mino de Kaspar3. 1 Ophthalmology, Hospital de Clínicas, National University of Asunción, Asuncion, Paraguay; 2Research Institute for Health Sciences, National University of Asunción, Paraguay, Asuncion, Paraguay; 3Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. Purpose: We performed a prospective study to identify by multiplex PCR (Polymerase Chain Reaction), genes encoding virulence factors (ica, Atle and mecA) in CNS isolates from the ocular microbiota of patients undergoing cataract surgery and to investigate possible changes in the Coagulase Negative-Staphylococcus (CNS) profile due to antibiotic prophylaxis. Methods: After approval of the Institutional Review Board of our institution had been obtained, patients undergoing cataract surgery were recruited for this study at the Department of Ophthalmology, National University of Asuncion, Paraguay. Patients (n= 162) received in the eye to be operated moxifloxacin 0.5% eye drops, four times the day before surgery and a last drop one hour before surgery (T1). The other eye remained as control (T0). Conjunctival swabs were taken from both eyes one hour after the last drop. Presence of genes encoding biofilm formation (ica and atlE) and gene encoding methicillin resistance (mecA) were detected by a multiplex PCR in CNS isolates from both timepoints. Results: Of the 162 patients, 87 eyes were positive for CNS in T0, yielding 96 CNS isolates and in T1, 70 eyes were positive, yielding 77 isolates CNS. We used for this study 43 CNS isolates from T0 and 45 from T1. Of the total CNS isolates, 81,8% (72/88) had at least one virulence factor gene (37/43 from T0 and 35/45 from T1 (p=0.314), simultaneous detection of ica and Atle genes was more frequent in T0 (n=25, 58.0%) than T1 (n=21, 46.7%), but the difference was not significant (p=0.28). Conclusions: A high frequency of genes encoding virulence factors was observed in the coagulase-negative Staphylococcus isolates. The use of moxifloxacin did not modify significantly the CNS virulence factor profiles. Commercial Relationships: Veronica E. Castillo, None; Yolanda Lopez, None; Margarita Samudio, None; Norma Farina, None; Sonia Abente, None; Nilsa Gonzalez, None; Florentina Laspina, None; Agustin Carron, None; Diogenes Cibils, None; Herminia Mino de Kaspar, None Program Number: 4076 Poster Board Number: A0253 Presentation Time: 8:30 AM–10:15 AM Comparison of two different evaluation methods for biofilm formation of S.epidermidis isolated from ocular surface Berna Akova Budak1, Sertac Argun Kivanc1, Meral Yildiz1, Merih Kivanc2, Gulay Gullulu3. 1Uludag University, Bursa, Turkey; 2 Anadolu University, Eskiehir, Turkey; 3Armedica Eye Center, Izmit, Turkey. Purpose: To compare of two different evaluation methods for biofilm formation of Staphylococcus epidermidis isolated from ocular surface. Methods: S.epidermidis strains that were isolated from ocular surface previously were studied. Microtiter plate method (MPA) and Congo red agar (CRA) method were used for measuring biofilm formation. Ica A, ica D, bap genes positivity and multi-antibiotic resistance were determined. Accurracy of measuring biofilm production capacity of MPA and CRA were compared. Results: 8 of the strains produced strong biofilm according to MPA method, and 4 of these strains also produced strong biofilm according to CRA method. 8 strains produced strong biofilm according to CRA method. 11 strains were biofilm negative with MPA however 7 (64 %) of these were positive with CRA. 15 strains were resistant to 3 or more antibiotics, 7 (46%) and 6 (40 %) of these strains produced strong biofilm according to MPA and CRA methods respectively. 8 strains were resistant to 4 or more antibiotics, 6 (75 %) and 3 (37 %) of these strains produced strong biofilm according to MPA and CRA methods respectively. Conclusions: Strains that produced strong biofilm with MPA had more multi-antibiotic resistance than with CRA.These two methods may not be consistent with each other. Commercial Relationships: Berna Akova Budak, None; Sertac Argun Kivanc, None; Meral Yildiz, None; Merih Kivanc, None; Gulay Gullulu, None Program Number: 4077 Poster Board Number: A0254 Presentation Time: 8:30 AM–10:15 AM Microbiology and Biofilm Growth on Clinically Infected Silicone Implants within the Lacrimal System: A Thirty Year Review Lilangi S. Ediriwickrema1, David Samimi2, 1, Brett Bielory2, Darlene Miller2, Thomas V. Johnson2. 1Ophthalmology, USC Eye Institute, Los Angeles, CA; 2Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL. Purpose: To investigate the pathogens and biofilms responsible for clinically significant infection of silicone stents implanted within the lacrimal system. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected]. ARVO 2015 Annual Meeting Abstracts Methods: Retrospective review of culture results for silicone lacrimal stents removed early for clinically significant infection over a thirty year period. As a control, routinely removed, clinically noninfected stents were prospectively sent for culture over a six month period. Four clinically infected stents and six clinically non-infected stents showing mucus within the lumen at removal were sent for scanning electron microscopy to grade the presence of organisms, matrix deposits, organisms within a matrix, and a significant biofilm by a masked electron micrographer. Results: Nineteen stents were included in the study; 100% of clinically non-infected stents (n=9) and of those removed for infection (n=10) were culture positive. None of the non-infected stents were culture positive for mycobacteria compared with 90% of infected specimens (p < 0.001). Of non-infected stents, 89% grew gram-positive organisms compared with 50% of infected stents (p=0.07). Sixty-seven percent of non-infected stents had gramnegative organisms versus 50% of infected stents (p=0.46). Electron microscopy of the four infected stents revealed organisms consistent with mycobacteria (size, shape) encased within a matrix. Of the six non-infected stents examined, organisms were identified within the lumens that were consistent with culture results but were without clear biofilm formation. A masked electron micrographer was able to identify and grade the presence of organisms, matrix deposits, organisms within a matrix, and a significant biofilm all with statistical significance. Conclusions: In our study population, atypical mycobacteria comprise the primary pathogen responsible for clinically significant infection of silicone stents in the lacrimal system. Robust biofilm production by this organism likely plays a role in pathogenesis. Development of biofilm resistant implant material, targeted liposomal antibiotic delivery, and physical or chemical disruption strategies are potential methods towards reducing rates of infection. Commercial Relationships: Lilangi S. Ediriwickrema, None; David Samimi, None; Brett Bielory, None; Darlene Miller, None; Thomas V. Johnson, None Support: Research to Prevent Blindness, New York, NY 10022. Results: After 6 hours exposure, MPS-1, MPS-2 and MPS-6 showed >2.4 log kill against combined inoculum of all ISO bacteria and clinical isolates and did not exhibit reduced efficacy or regrowth over 14 days. MPS-3 showed <1 log kill against combined inoculum of all ISO bacteria and clinical isolates after 6 hours exposure, and exhibited regrowth over 14 days. MPS-4 and MPS-5 exhibited <1 log kill against combined inoculum of all ISO bacteria and Serratia marcescens and all ISO bacteria and Achromobacter xylosoxidans clinical isolates after 6 hours exposure and showed reduced efficacy over 7 days storage. MPS-4 and MPS-5 exhibited <1.5 log kill against combined inoculum of all ISO bacteria and Achromobacter sp. clinical isolate after 6 hours exposure and showed reduced efficacy after 24 hours. Low numbers of ISO bacteria S. aureus and S. marcescens were observed for MPS-3, MPS-4 and MPS-5 after 6 hours exposure when combined inoculum of these bacteria and Achromobacter sp. was used. Conclusions: MPS-1, MPS-2 and MPS-6 showed high ability to reduce microbial load after 14 days storage, while MPS-4 and MPS-5 exhibited reduced disinfection efficacy. MPS-3 exhibited low disinfection efficacy at 6 hours and regrowth over 14 days. Commercial Relationships: Marina Milenkovic, Abbott Meducal Optics (E); James Cook, Abbott Medical Optics (E) Program Number: 4078 Poster Board Number: A0255 Presentation Time: 8:30 AM–10:15 AM Antimicrobial Efficacy of Multipurpose Disinfecting Solutions against Combined Inoculum of ISO Bacteria and Clinical Isolates Marina Milenkovic1, James Cook2. 1Corneal R&D Microbiology, Abbott Medical Optics, Santa Ana, CA; 2Corneal R&D, Abbott Medical Optics, Santa Ana, CA. Purpose: To compare antimicrobial efficacy of multipurpose disinfecting solutions (MPS) against combined inoculum of ISO bacteria and Gram-negative clinical isolates during 14 days storage. Methods: The multipurpose disinfecting solutions studied were MPS-1: polyquaternium (PQ1)+alexidine dihydrochloride (ALX), MPS-2: polyhexamethylene biguanide (PHMB)+poloxamer, MPS-3: PQ1+myristamidopropyl dimethylamine (ALDOX)+nonanoylEDTA, MPS-4: PQ1+ALDOX+polyoxyethylene-polyoxybutylene (EOBO-41), MPS-5: PQ1+ALDOX and MPS-6: PHMB+PQ1. Test solutions were inoculated with combined inoculum of ISO bacteria and Gram-negative clinical isolates in the test tube according to ISO 14729. Test solution efficacy was evaluated at minimum recommended manufacturer disinfection time of 6 hours, 24 hours, 7 days and 14 days. ISO bacteria tested were: Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027 and Serratia marcescens ATCC 13880. Clinical isolates tested were: Achromobacter sp., Serratia marcescens and Achromobacter xylosoxidans. ©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].