An 18-Year Overview of Serratia marcescens Ocular Infection.

PURPOSE: Serratia marcescens is a frequent ocular bacterial pathogen implicated in keratitis, endophthalmitis, and conjunctivitis. We evaluated the risk factors and treatment outcomes of ocular infections due to S. marcescens. METHODS: In this retrospective observational study, all S. marcescens-positive cases between February 2002 and February 2020 were reviewed for ocular risk factors that included log of minimal angle of resolution visual acuity (VA), medical management, and time to epithelial defect closure. RESULTS: Fifty-one patients were identified (72.5% females, 46.8±23.3 years). Forty-six patients had complete medical records, and 5 had microbiology data available. The most prevalent ocular risk factors were, contact lens (CL) use (68.6%), corneal disease (52.9%), and history of ocular surgery (41.2%). Mean presenting VA was 1.3±1.0. About half of the patients presented with a central ulcer (49%, 25), large infiltrate (20.4±31.8 mm2 mean), and hypopyon (43.1%, 22). All cases were reported to be susceptible to ciprofloxacin. Defect closure occurred in 52.3±117.1 days and final VA was 0.86±0.88. Adjunctive treatments were required in 14 cases (27.5%). One patient underwent surgical intervention. Features associated with poor VA outcomes included, history of glaucoma (P=0.038), older age at presentation (P<0.001), presence of hypopyon (0.045), poor VA at presentation (0.0086), time to epithelial defect closure (0.0196), and large infiltrate size (P=0.0345). CONCLUSIONS: S. marcescens keratitis and conjunctivitis is associated with CL use and history of ocular surface disease. Worse outcomes were associated with older age, infiltrate size, presence of hypopyon, worse initial VA, longer time to epithelial defect closure, and history of glaucoma.

Bilateral Pseudomonas aeruginosa Keratitis in 7 Patients.

PURPOSE: To report 7 patients (14 eyes) with bilateral Pseudomonas aeruginosa keratitis (PAK). METHODS: Case records of patients with bilateral PAK were reviewed at a single academic medical center from April 2009 to December 2020. RESULTS: Seven patients aged 29 to 94 years presented with bilateral P. aeruginosa corneal ulcers. All patients were soft contact lens wearers except one elderly patient with a complex ocular history. Three patients wore specialty contact lens, including one cosmetic contact lens wearer, one multifocal contact lens wearer, and one extended-wear contact lens wearer. The remaining three patients were not overnight contact lens wearer but regularly showered in contact lens or used tap water for contact lens case cleaning. All patients presented with asymmetric disease, with visual acuity ranging from 20/20 to light perception. Ulcers were located centrally in 5 eyes (35.7%), diffusely over the entire cornea in 5 eyes (35.7%), temporally in 2 eyes (14.2%), and nasally in 1 eye (7.14%), and in 1 eye (8.3%), the ulcer location was not recorded. Corneal thinning was noted in 7 eyes (50%). Hypopyon was also noted in 7 eyes (50%). Two patients required procedures because of progressive stromal necrosis. All other patients were treated nonsurgically, with antibiotic drops, which resulted in ulcer resolution. Final visual acuity on last recorded follow-up ranged from 20/20 to no light perception. CONCLUSIONS: This is the largest case series of bilateral PAK, which occurred primarily in contact lens wearer. This case series reiterates the risk of contact lens infection related to poor hygiene.

A Case of Nocardia farcinica Keratitis in a Pediatric Contact Lens Wearer.

PURPOSE: To report a case of Nocardia farcinica keratitis in a pediatric contact lens wearer. METHODS: Case report and literature review. RESULTS: A pediatric contact lens wearer was initially misdiagnosed with a poorly healing corneal abrasion after swimming with his contact lenses. On examination at our center, he was found to have a 2.5 by 2.5 mm corneal infiltrate with feathery margins. Microbiology revealed N. farcinica keratitis, which was treated with topical amikacin. The patient returned to his baseline visual acuity after treatment. CONCLUSIONS: Nocardia keratitis can be misdiagnosed because of its low prevalence, especially in young patients. Prompt diagnosis is important for proper management in these cases. The infection resolved in our patient with prompt diagnosis and treatment with topical amikacin eye drops.

The Prevalence of Bacteria, Fungi, Viruses, and Acanthamoeba From 3,004 Cases of Keratitis, Endophthalmitis, and Conjunctivitis.

PURPOSE: The definitive identification of ocular pathogens optimizes effective treatment. Although the types of ocular pathogens are known; there is less definitive information on the prevalence of causative infections including viruses, fungi, and protozoa, which is the focus of this retrospective laboratory review. METHODS: Data used for laboratory certification were reviewed for the detection of bacteria, viruses, fungi, and protozoa, from patients with infectious keratitis, endophthalmitis, and conjunctivitis. The main outcome parameter was laboratory-positive ocular infection. RESULTS: The distribution of infectious agents for keratitis (n=1,387) (2004-2018) was bacteria 72.1% (Staphylococcus aureus 20.3%, Pseudomonas aeruginosa 18%, Streptococcus spp. 8.5%, other gram-positives 12.4%, and other gram-negatives 12.9%), Herpes simplex virus 16%, fungi 6.7%, and Acanthamoeba 5.2%. For endophthalmitis, (n=770) (1993-2018), the bacterial distribution was coagulase-negative Staphylococcus 54%, Streptococcus spp. 21%, S. aureus 10%, other gram-positives 8%, and gram-negatives 7%. The distribution for conjunctivitis (n=847) (2004-2018) was Adenovirus 34%, S. aureus 25.5%, Streptococcus pneumoniae 9%, Haemophilus 9%, other gram-negatives 8.8%, other gram-positives 6%, coagulase-negative Staphylococcus 4.5% and Chlamydia 3.2%. CONCLUSION: An updated monitoring of ocular pathogens creates an awareness of the different infectious etiologies and the importance of laboratory studies. This information can determine treatment needs for infectious ocular diseases.

Bacteria induce autophagy in a human ocular surface cell line.

Autophagy protects cells from intracellular pathogens, but can be exploited by some infectious agents to their benefit. Currently it is not known if bacteria induce autohpagy in cells of the cornea. The goal of this study was to develop an ocular surface autophagy reporter cell line and determine whether ocular bacterial pathogens influence host responses through autophagy induction. The cell line was made using lentivirus transduction of an LC3-GFP fusion protein in human corneal limbal epithelial (HCLE) cells. LC3-GFP puncta in HCLEs were induced by rapamycin and ammonium chloride treatments, and prevented by the autophagy inhibitors 3-methyladenine (3'MA) and bafilomycin. Importantly, secretomes from Escherichia coli, Serratia marcescens, Staphylococcus aureus, methicillin sensitive (MSSA) and resistant (MRSA), were found to induce autophagy, whereas other bacteria, including Acinetobacter baumannii, Achromobacter xylosoxidans, Enterococcus faecalis, Klebsiella pneumoniae, Moraxella sp., and Stenotrophomonas maltophilia, did not. Our data indicates differences between tested ocular isolates of MRSA and MSSA in the activation of autophagy. HCLEs treated with 3'MA were slightly more susceptible to cytotoxic factors produced by S. marcescens and MRSA keratitis isolates, by contrast, bafilomycin A1 treatment caused no difference. This work demonstrates the successful development and validation of an autophagy reporter corneal cell line and indicates differences between ocular bacterial isolates in the activation of autophagy.

AmpliVue Is a Practical and Timely Test for the Detection of HSV From Keratitis Specimens.

OBJECTIVE: The laboratory diagnostic detection of herpes simplex virus (HSV) from eye samples must be practical, timely, and definitive for appropriate therapy. Although polymerase chain reaction (PCR) and/or cell culture can be definitive, HSV results can be delayed. Enzyme Linked Virus Inducible System (ELVIS) is a test that can provide results within 24 to 48 hr. We evaluated "AmpliVue HSV 1+2 Assay" as a molecular colorimetric test that can detect HSV (1 or 2) DNA within 1 hr. METHODS: Cornea/conjunctival samples were tested retrospectively with AmpliVue against 53 true-positive and 20 true-negative specimens collected in chlamydial transport medium. All clinical specimens were tested by cell culture isolation, PCR, and ELVIS for routine patient care. RESULTS: The sensitivity of AmpliVue against ocular samples that were both culture-positive and PCR-positive was 84%. The specificity of AmpliVue was 100%. Only one clinical sample was HSV-2 positive, whereas all others tested positive for HSV-1. Based on PCR-positive and cell culture-negative samples, AmpliVue (11 of 17) tested more positive than ELVIS (0 of 17) (P=0.003, Fisher Exact). CONCLUSIONS: AmpliVue is moderately sensitive and highly specific as a practical and timely diagnostic test for detecting ocular HSV. Expertise is readily achieved and the test is straightforward with easy interpretation. Negative AmpliVue testing must be confirmed with PCR. AmpliVue has potential as an office-based diagnostic test.

In Vitro Evaluation of a Hypochlorous Acid Hygiene Solution on Established Biofilms.

OBJECTIVES: The purpose of this study was to determine whether a commercial formulation of hypochlorous acid hygiene solution (0.01%), Avenova, can destroy existing biofilms formed by ocular clinical bacterial isolates, including blepharitis isolates of Staphylococcus aureus and coagulase-negative staphylococci, and a keratitis isolate of Pseudomonas aeruginosa. METHODS: Biofilms grown in bacterial growth media on disposable contact lens cases were challenged with hypochlorous acid hygiene solution. At various time points, surviving bacteria were quantified by serial dilution and colony counts. Staphylococcus aureus biofilms formed on glass were challenged using a hypochlorous acid hygiene solution and imaged using vital staining and confocal laser scanning microscopy. RESULTS: Bactericidal activity (≥3 Log10; 99.9%) was observed for all tested bacterial species after a 30-min exposure. Staphylococcus aureus biofilms had a bactericidal level of killing by 10 min (P<0.01), Staphylococcus capitis by 5 min (P<0.001), Staphylococcus epidermidis by 30 min (P<0.001), and P. aeruginosa by 10 min (P<0.01). Confocal microscopy and crystal violet staining analysis of bacterial biofilms treated with hypochlorous acid solution both demonstrated that biofilm bacteria were readily killed, but biofilm structure was largely maintained. CONCLUSIONS: Hypochlorous acid (0.01%) hygiene solution was able to achieve bactericidal levels of killing of bacteria in biofilms but did not disrupt biofilm structures. Susceptibility of tested staphylococcal blepharitis isolates varied by species, with S. capitis being the most susceptible and S. epidermidis being the least susceptible.

Postsurgical Cataract Prophylaxis With Intravitreal "Triamcinolone-Moxifloxacin" May Not Be Optimal For Preventing Endophthalmitis.

PURPOSE: Intravitreal injections of antibiotics and anti-inflammatories are used by some cataract surgeons for surgical prophylaxis. To support this prophylaxis, intravitreal triamcinolone-moxifloxacin (TM) and triamcinolone-moxifloxacin-vancomycin (TMV) were tested for preventing Staphylococcus aureus (SA) endophthalmitis in rabbits. METHODS: Trademark formulations of TM (15/1 mg/mL) and TMV (15/1/10 mg/mL) were intravitreally injected into seven groups of eight rabbits each (A-G). Before intravitreal injection, the vitreous was first challenged with clinical SA endophthalmitis isolates (5,000 colony-forming unit) with varying minimum inhibitory concentrations (MICs in µg/mL) to moxifloxacin (denoted by the MIC at the end of each group listed): A) TMV-10, B) TM-10, C) Saline-10, D) TM-2, E) Saline-2, F) TM-0.032, and G) Saline-0.032. After 24 hr, the rabbit eyes were graded for clinical endophthalmitis and cultured for viable SA. RESULTS: Rabbits treated with TMV and challenged by SA with a moxifloxacin MIC of 10 µg/mL did not present with endophthalmitis (0/8, no eyes with endophthalmitis). For SA with moxifloxacin MICs of 10.0 and 2.0 µg/mL, TM did not prevent endophthalmitis (16/16, 100% of eyes with endophthalmitis). For SA with a moxifloxacin MIC of 0.032 µg/mL, endophthalmitis was prevented with TM (0/8, no eyes with endophthalmitis). All saline-treated eyes developed endophthalmitis (23/23, 100% of eyes with endophthalmitis). CONCLUSIONS: Intravitreal monotherapy with TM did not provide consistent prevention of SA endophthalmitis, whereas intravitreal TMV successfully prevented endophthalmitis because of SA with elevated MIC values to moxifloxacin. Cataract surgeons need to be aware that vancomycin seems to be essential for intravitreal prophylaxis to cover moxifloxacin resistance.

EepR Mediates Secreted-Protein Production, Desiccation Survival, and Proliferation in a Corneal Infection Model.

Serratia marcescens is a soil- and water-derived bacterium that secretes several host-directed factors and causes hospital infections and community-acquired ocular infections. The putative two-component regulatory system composed of EepR and EepS regulates hemolysis and swarming motility through transcriptional control of the swrW gene and pigment production through control of the pigA-pigN operon. Here, we identify and characterize a role for EepR in regulation of exoenzyme production, stress survival, cytotoxicity to human epithelial cells, and virulence. Genetic analysis supports the model that EepR is in a common pathway with the widely conserved cyclic-AMP receptor protein that regulates protease production. Together, these data introduce a novel regulator of host-pathogen interactions and secreted-protein production.

The verification of nucleic acid amplification testing (Gen-Probe Aptima Assay) for chlamydia trachomatis from ocular samples.

OBJECTIVE: Chlamydia trachomatis conjunctivitis may present with extended symptoms, and it can have social ramifications as a sexually transmitted disease. For appropriate therapy, C. trachomatis conjunctivitis should be diagnosed definitively. This study presents the verification of nucleic acid amplification testing (NAAT; Gen-Probe Aptima Combo 2 assay) for detection of C. trachomatis ribosomal RNA (rRNA) from direct ocular samples. DESIGN: Retrospective laboratory verification study. SUBJECTS: Patients with infectious conjunctivitis. METHODS: A battery of 25 true-positive specimens (direct ocular specimens from patients with symptoms consistent with C. trachomatis conjunctivitis and with previously demonstrated positive polymerase chain reaction [PCR] results for C. trachomatis DNA by Roche Amplicor) and 25 true-negative specimens (direct ocular specimens with culture-positive results for herpes simplex virus [n = 5], adenovirus [n = 5], Haemophilus influenzae [n = 5], and Streptococcus pneumoniae [n = 5]), and transport medium (n = 5) were tested for C. trachomatis rRNA by NAAT. These true-negative specimens have differential etiologic agents of infectious conjunctivitis. The 25 C. trachomatis specimens with PCR-positive results (obtained May 1994-May 2012) and 20 true-negative infectious ocular specimens (obtained December 2008-August 2013) were collected with soft-tipped applicators and placed in transport medium. All excess specimens were stored at -80°C. All samples were centrifuged at 13,000 rpm for 1 hour at 6°C. For each sample, using the Aptima Unisex collection blue swab, a specimen was collected from the conical apex of the storage tube where a pellet was formed. The Aptima Unisex collection swab was placed in a tube of Aptima swab transport medium for testing. All samples were tested in duplicate. MAIN OUTCOME MEASURES: Detection of C. trachomatis rRNA. RESULTS: Of 25 true-positive samples, 24 (96%) were positive by NAAT, whereas 25 of 25 true-negative samples (100%) showed negative results. The sensitivity, specificity, positive predictive value, negative predictive value, and efficiency were determined to be 96%, 100%, 100%, 96%, and 98%, respectively. CONCLUSIONS: The detection of C. trachomatis in ocular specimens by NAAT was verified for laboratory diagnosis. The test will be evaluated prospectively to determine future test performance precisely.

Comparison of Validated Polymerase Chain Reaction and Culture Isolation for the Routine Detection of Acanthamoeba From Ocular Samples.

PURPOSE: Acanthamoeba keratitis should be definitively diagnosed for appropriate therapy. Our institution has validated polymerase chain reaction (PCR) as a routine diagnostic test to detect Acanthamoeba DNA from ocular samples. We compared PCR with culture isolation for detecting Acanthamoeba from ocular samples. METHODS: The microbiology records of patients that had specimens submitted (May 2012 to January 2014) for laboratory testing for Acanthamoeba keratitis were reviewed for (1) Acanthamoeba culture isolation, (2) Acanthamoeba DNA detection by PCR, and (3) non-Acanthamoeba culture results. For Acanthamoeba isolation, corneal samples were planted on nonnutrient agar overlaid with Enterobacter aerogenes. Validated PCR (May 2012) for Acanthamoeba DNA was processed at the Division of Molecular Diagnostics, UPMC, Pittsburgh, PA. Additional cultures were obtained for bacteria, fungus, and virus (i.e., herpes simplex virus) using standard techniques. RESULTS: Culture isolation and PCR were processed on 125 patients with a differential diagnosis of Acanthamoeba keratitis. Of these, 104 (83.2%) were culture negative, PCR negative; 14 (11.2%) were culture positive, PCR positive; 4 (3.2%) were culture negative, PCR positive; and, 3 (2.4%) were culture positive, PCR negative. Culture and PCR were statistically equivalent for detecting Acanthamoeba from ocular samples (P=1.0, McNemar's test). Nineteen of the culture-negative, PCR-negative corneal samples (18.3%) were positive for other pathogens such as bacteria, fungus, and virus. CONCLUSIONS: There is no clear advantage of PCR over culture isolation for detecting Acanthamoeba in ocular specimens. Other pathogens such as bacteria, fungus, and virus must still be considered in severe persistent keratitis. Polymerase chain reaction seems to be a complementary test for the clinical support of Acanthamoeba keratitis.

Can Serum Drops Containing Doxycycline Provide a Supplemental Anti-Bacterial Effect in the Treatment of Bacterial Keratitis?

PURPOSE: Systemic doxycycline has been prescribed to reduce inflammation and enhance corneal healing in bacterial keratitis. Topical autologous serum drops (ASD) containing doxycycline following oral supplementation may additionally confer an anti-bacterial effect. The potential of this supplementation was evaluated by determining the in vitro susceptibility of bacterial keratitis isolates to doxycycline. METHODS: The minimum inhibitory concentrations (MICs) of doxycycline against 100 bacterial keratitis isolates were determined using Etests. Twenty-seven Staphylococcus aureus, ten coagulase-negative Staphylococci, six Streptococcus pneumoniae, seven viridans group streptococci, seven other Gram-positive bacteria, nineteen Pseudomonas aeruginosa, eight Serratia marcescens, four Moraxella spp., two Haemophilus spp., and ten other Gram-negative bacteria isolates were tested. MICs of doxycycline were compared to a serum standard concentration of doxycycline (SSCD) of 4 µg/mL and concentrations that would be found in 50% and 20% serum component clinical preparations of ASD, corresponding to 50% SSCD (2 µg/mL) and 20% SSCD (0.8 µg/mL), respectively. MICs equal to or less than these values were used to deem a bacterial isolate susceptible. RESULTS: For Gram-positive bacteria, susceptibilities to SSCD, 50% SSCD, and 20% SSCD were 86%, 65%, and 60%, respectively. For Gram-negative bacteria, susceptibilities to SSCD, 50% SSCD, and 20% SSCD were 37.2%, 23.3%, and 11.6%, respectively. Chi-squared analyses comparing Gram-positive and Gram-negative susceptibilities showed significantly greater susceptibility of Gram-positive bacteria at all three tested MICs (<0.0001, <0.0001, <0.0001). CONCLUSIONS: Our data suggest that autologous serum drops containing theoretic concentrations of doxycycline may provide an additional anti-bacterial effect in the treatment of bacterial keratitis, especially for Gram-positive bacterial keratitis compared to Gram-negative bacterial keratitis.

Double Trouble: An Unusual Keratitis Case of Herpes Simplex Virus and Varicella-Zoster Virus Co-infection.

PURPOSE: The aim of this study was to describe a case of herpes simplex virus (HSV) and varicella-zoster virus (VZV) corneal co-infection in a patient with systemic immunosuppression. METHODS: A 77-year-old White man who was recently administered pembrolizumab present with reduction in visual acuity in his left eye from 20/25 to 20/50. There was a known history of ocular HSV keratitis. Slit-lamp examination showed superficial dendritic lesions suggestive of VZV. RESULTS: Viral polymerase chain reaction testing was positive for both HSV and VZV, confirming clinical diagnosis of VZV keratitis in the setting of recurrent HSV keratitis. The infection responded to treatment with topical trifluridine. Two months later, he had another episode of keratitis based on his symptoms reported through telephone encounter which resolved with trifluridine. Unfortunately, the patient committed suicide 4 months after onset. CONCLUSIONS: This is the first case of keratitis with HSV and VZV co-infection likely related to systemic immunosuppression. Clinicians should have a high suspicion for viral co-infections in the setting of systemic immunosuppression.

Clinical Characterization and Outcomes of Culture- and Polymerase Chain Reaction-Negative Cases of Infectious Keratitis.

PURPOSE: To examine the clinical presentation, management, and outcomes of culture and polymerase chain reaction (PCR) negative cases of infectious keratitis. METHODS: In this retrospective case series, we evaluated the laboratory and medical records of culture- and PCR-negative cases (2016-2020) reported to a tertiary care center, which were presumed to be infectious keratitis on the basis of clinical history and presentation. RESULTS: A total of 121 cases with culture-negative keratitis were included in this study. The mean age of the patients was 48.42 ± 1.89 years, and 53.72% were female. At presentation, the presumed etiology was viral in 38.01%, bacterial in 27.27%, fungal in 8.26%, Acanthamoeba in 6.61%, and unlisted in 28.92% of cases. The most common risk factors were a previous history of ocular surface diseases (96.69%) and contact lens use (37.19%). In total, 61.98% of the patients were already on antimicrobial medication at presentation. The initial management was altered in 79 cases (65.29%) during the treatment course. Average presenting and final (post-treatment) visual acuities (VA) were 0.98 ± 0.04 (LogMAR) and 0.42 ± 0.03 (LogMAR), respectively. A significantly higher frequency of patients with a final VA worse than 20/40 (Snellen) had worse VA at initial presentation (p < 0.0001). A history of ocular surface disease, cold sores, and recurrent infection (p < 0.05) were more commonly associated with a presumed diagnosis of viral keratitis. The patients with presumed bacterial etiology were younger and had a history of poor contact lens hygiene (p < 0.05). CONCLUSIONS: We observed a distinct difference in clinical features among patients with culture-negative and PCR-negative keratitis managed for presumed viral and bacterial infections. Although there was significant variability in presentation and management duration in this cohort, the visual outcomes were generally favorable.

The Short-chain Fatty Acid Propionic Acid Activates the Rcs Stress Response System Partially through Inhibition of d-Alanine Racemase.

The Enterobacterial Rcs stress response system reacts to envelope stresses through a complex two-component phosphorelay system to regulate a variety of environmental response genes, such as capsular polysaccharide and flagella biosynthesis genes. However, beyond Escherichia coli, the stresses that activate Rcs are not well-understood. In this study, we used a Rcs system-dependent luminescent transcriptional reporter to screen a library of over 240 antimicrobial compounds for those that activated the Rcs system in Serratia marcescens, a Yersiniaceae family bacterium. Using an isogenic rcsB mutant to establish specificity, both new and expected activators were identified, including the short-chain fatty acid propionic acid, which is found at millimolar levels in the human gut. Propionic acid did not reduce the bacterial intracellular pH, as was hypothesized for its antibacterial mechanism. Instead, data suggest that the Rcs-activation by propionic acid is due, in part, to an inactivation of alanine racemase. This enzyme is responsible for the biosynthesis of d-alanine, which is an amino-acid that is required for the generation of bacterial cell walls. Consistent with what was observed in S. marcescens, in E. coli, alanine racemase mutants demonstrated elevated expression of the Rcs-reporter in a d-alanine-dependent and RcsB-dependent manner. These results suggest that host gut short-chain fatty acids can influence bacterial behavior via the activation of the Rcs stress response system. IMPORTANCE The Rcs bacterial stress response system responds to envelope stresses by globally altering gene expression to profoundly impact host-pathogen interactions, virulence, and antibiotic tolerance. In this study, a luminescent Rcs-reporter plasmid was used to screen a library of compounds for activators of Rcs. Among the strongest inducers was the short-chain fatty acid propionic acid, which is found at high concentrations in the human gut. This study suggests that gut short-chain fatty acids can affect both bacterial virulence and antibiotic tolerance via the induction of the Rcs system.

Differentiating methicillin resistant and susceptible Staphylococcus aureus from ocular infections using photoacoustic labeling.

Introduction: Antibiotic resistance in bacterial species constitutes a growing problem in the clinical management of infections. Not only does it limit therapeutic options, but application of ineffective antibiotics allows resistant species to progress prior to prescribing more effective treatment to patients. Methicillin resistance in Staphylococcus aureus is a major problem in clinical infections as it is the most common hospital acquired infection. Methods: We developed a photoacoustic flow cytometer using engineered bacteriophage as probes for rapid determination of methicillin resistance in Staphylococcus aureus with thirteen clinical samples obtained from keratitis patients. This method irradiates cells under flow with 532 nm laser light and selectively generates acoustic waves in labeled bacterial cells, thus enabling detection and enumeration of them. Staphylococcus aureus isolates were classified from culture isolation as either methicillin resistant or susceptible using cefoxitin disk diffusion testing. The photoacoustic method enumerates bacterial cells before and after treatment with antibiotics. Decreasing counts of bacteria after treatment indicate susceptible strains. We quantified the bacterial cells in the treated and untreated samples. Results: Using k-means clustering on the data, we achieved 100% concordance with the classification of Staphylococcus aureus resistance using culture. Discussion: Photoacoustics can be used to differentiate methicillin resistant and susceptible strains of bacteria from ocular infections. This method may be generalized to other bacterial species using appropriate bacteriophages and testing for resistance using other antibiotics.

Herpes zoster ophthalmicus: frequency and risk factors for developing uncommon ocular manifestations.

OBJECTIVE: To determine the frequency of herpes zoster ophthalmicus (HZO) and assess risk factors for developing uncommon ocular manifestations of laboratory-verified HZO. DESIGN: Retrospective cohort study. METHODS: The frequency of HZO out of all herpes zoster cases was calculated using International Classification of Diseases codes for patients seen at the University of Pittsburgh Medical Center from January 1, 2004 to October 31, 2021. We also collected demographic and clinical data of patients with HZO identified by polymerase chain reaction (PCR) detection of varicella zoster virus from January 1, 2011 to December 31, 2020. RESULTS: The frequency of HZO from 2004 to 2021 in all ages was 4.2% and ranged from 2.7% to 6.7% annually, with a consistent increase of 2.9% from 2012 to 2021. After the live zoster vaccine became available in 2008, the frequency of HZO decreased by 5.1% from 2008 to 2012 in patients aged 60 and older. Among 50 cases of PCR-verified HZO, 62% represented clinically-common ocular manifestations, mostly comprised of 13 cases of keratitis and 10 cases of anterior uveitis. Fifteen cases of acute retinal necrosis (ARN) represented the majority of uncommon HZO manifestations (38%), which were significantly more likely to occur in immunosuppressed patients (unadjusted odds ratio 4.55, 95% confidence interval 1.29-13.83). CONCLUSIONS: The overall frequency of HZO from 2004 to 2021 was 4.2% and has increased annually since 2012. Uncommon ocular manifestations of PCR-verified HZO, mostly comprised of ARN, were more likely to occur in immunosuppressed patients.

Coagulase-negative staphylococcal endophthalmitis: clinical severity and outcomes based on speciation.

OBJECTIVE: To identify characteristics and visual outcomes of coagulase-negative staphylococcal (CoNS) endophthalmitis in the era after the Endophthalmitis Vitrectomy Study. DESIGN: Single-centre retrospective analysis. PARTICIPANTS: Forty-two samples from 40 patients with documented CoNS endophthalmitis. METHODS: Visual acuity outcomes of CoNS endophthalmitis were assessed in relation to species and type of treatment instituted (i.e., pars plana vitrectomy [PPV] versus vitreous tap and injection of intravitreal antibiotics [T&I]) on 42 samples from 40 patients. RESULTS: Staphylococcus epidermidis was the most prevalent CoNS in our study. Cataract surgery and intravitreal injections were the most common sources for acute CoNS endophthalmitis. Eyes presenting with hand motion or better vision had similar mean final vision after either intravitreal antibiotics or PPV, whereas those with light perception or worse vision at onset had better outcomes after PPV only. Subanalysis showed that patients with S. epidermidis endophthalmitis (n = 39 eyes) had similar visual outcomes with either intravitreal injections or PPV regardless of visual acuity. Hypopyon and vitritis are not always present. CONCLUSIONS: Patients with S. epidermidis endophthalmitis may benefit similarly from either early vitrectomy or intravitreal antibiotic injections regardless of visual acuity. This finding may be a supplement to the complements the management standards set forth by the Endophthalmitis Vitrectomy Study.

A rise in the frequency of lasR mutant Pseudomonas aeruginosa among keratitis isolates between 1993 and 2021.

Introduction: Pseudomonas aeruginosa causes vision threatening keratitis. The LasR transcription factor regulates virulence factors in response to the quorum sensing molecule N-3-oxo-dodecanoyl-L-homoserine lactone. P. aeruginosa isolates with lasR mutations are characterized by an iridescent high sheen phenotype caused by a build-up of 2-heptyl-4-quinolone. A previous study demonstrated 22% (n=101) of P. aeruginosa keratitis isolates from India between 2010 and 2016 were sheen positive lasR mutants, and the sheen phenotype correlated with worse clinical outcomes for patients. In this study, a longitudinal collection of P. aeruginosa keratitis isolates from Eastern North America were screened for lasR mutations by the sheen phenotype and sequencing of the lasR gene. Methods: Keratitis isolates (n=399) were classified by sheen phenotype. The lasR gene was cloned from a subset of isolates, sequenced, and tested for loss of function or dominant-negative status based on an azocasein protease assay. A retrospective chart review compared outcomes of keratitis patients infected by sheen positive and negative isolates. Results: A significant increase in sheen positive isolates was observed between 1993 and 2021. Extracellular protease activity was reduced among the sheen positive isolates and a defined lasR mutant. Cloned lasR alleles from the sheen positive isolates were loss of function or dominant negative and differed in sequence from previously reported ocular lasR mutant alleles. Retrospective analysis of patient information suggested significantly better visual outcomes for patients infected by sheen positive isolates. Discussion: These results indicate an increase in lasR mutations among keratitis isolates in the United States and suggest that endemic lasR mutants can cause keratitis.

Rise in frequency of lasR mutant Pseudomonas aeruginosa among keratitis isolates between 1993 and 2021.

Pseudomonas aeruginosa causes severe vision threatening keratitis. LasR is a transcription factor that regulates virulence associated genes in response to the quorum sensing molecule N-3-oxo-dodecanoyl-L-homoserine lactone. P. aeruginosa isolates with lasR mutations are characterized by an iridescent high sheen phenotype caused by a build-up of 2-heptyl-4-quinolone. A previous study indicated a high proportion (22 out of 101) of P. aeruginosa keratitis isolates from India between 2010 and 2016 were sheen positive and had mutations in the lasR gene, and the sheen phenotype correlated with worse clinical outcomes for patients. In this study, a longitudinal collection of P. aeruginosa keratitis isolates from Eastern North America were screened for lasR mutations by the sheen phenotype and sequencing of the lasR gene. A significant increase in the frequency of isolates with the sheen positive phenotype was observed in isolates between 1993 and 2021. Extracellular protease activity was lower among the sheen positive isolates and a defined lasR mutant. Cloned lasR alleles from the sheen positive isolates were loss of function or dominant negative and differed in sequence from previously reported ocular lasR mutant alleles. Insertion elements were present in a subset of independent isolates and may represent an endemic source from some of the isolates. Retrospective analysis of patient information suggested significantly better visual outcomes for patients with infected by sheen positive isolates. Together, these results indicate an increasing trend towards lasR mutations among keratitis isolates at a tertiary eye care hospital in the United States.