Infectious Disease

Bacterial endophthalmitis is a sight threatening infection of the interior structures of the eye. Incidence in the US has increased in recent years, which appears to be related to procedures being performed on an aging population. The advent of outpatient intravitreal therapy for management of age-related macular degeneration raises yet additional risks. Compounding the problem is the continuing progression of antibiotic resistance. Visual prognosis for endophthalmitis depends on the virulence of the causative organism, the severity of intraocular inflammation, and the timeliness of effective therapy. We review the current understanding of the pathogenesis of bacterial endophthalmitis, highlighting opportunities for the development of improved therapeutics and preventive strategies.

PURPOSE: To define global transcriptional responses of Staphylococcus aureus and its codY mutant (CodY is a transcription regulator of virulence and metabolic genes in response to branched-chain amino acids) when growing in bovine aqueous (AH) and vitreous humor (VH) in vitro, and to investigate the impact of codY deletion on S. aureus virulence in a novel murine anterior chamber (AC) infection model. METHODS: For the in vitro model, differential transcriptomic gene expression of S. aureus and its codY mutant grown in chemically defined medium (CDM), AH, and VH was analyzed. Furthermore, the strains were inoculated into the AC of mice. Changes in bacterial growth, electroretinography and inflammation scores were monitored. RESULTS: Bovine AH and VH provide sufficient nutrition for S. aureus growth in vitro. Transcriptome analysis identified 72 unique open reading frames differentially regulated ≥10-fold between CDM, AH, and VH. In the AC model, we found comparable growth of the codY mutant and wild type strains in vivo. Average inflammation scores and retinal function were significantly worse for codY mutant-infected eyes at 24 h post-infection. CONCLUSION: Our in vitro bovine AH and VH models identified likely nutrient sources for S. aureus in the ocular milieu. The in vivo model suggests that control of branched-chain amino acid availability has therapeutic potential in limiting S. aureus endophthalmitis severity.

Staphylococcus aureus contains two distinct teichoic acid (TA) polymers, lipoteichoic acid (LTA) and wall teichoic acid (WTA), which are proposed to play redundant roles in regulating cell division. To gain insight into the underlying biology of S. aureus TAs, we used a small molecule inhibitor to screen a highly saturated transposon library for cellular factors that become essential when WTA is depleted. We constructed an interaction network connecting WTAs with genes involved in LTA synthesis, peptidoglycan synthesis, surface protein display, and D-alanine cell envelope modifications. Although LTAs and WTAs are synthetically lethal, we report that they do not have the same synthetic interactions with other cell envelope genes. For example, D-alanylation, a tailoring modification of both WTAs and LTAs, becomes essential when the former, but not the latter, are removed. Therefore, D-alanine-tailored LTAs are required for survival when WTAs are absent. Examination of terminal phenotoypes led to the unexpected discovery that cells lacking both LTAs and WTAs lose their ability to form Z rings and can no longer divide. We have concluded that the presence of either LTAs or WTAs on the cell surface is required for initiation of S. aureus cell division, but these polymers act as part of distinct cellular networks.

Microarrays were used to investigate the transcriptional response of Enterococcus faecalis to photostress. E. faecalis are Gram-positive bacteria used as indicators of water quality and have been shown to vary diurnally in response to sunlight. E. faecalis in filtered seawater microcosms were exposed to artificial sunlight for 12h and then placed in the dark for 12h. Transcript abundance was measured at 0, 2, 6, 12, and 24h in the sunlit microcosm and a dark control using microarrays. Culturable E. faecalis concentrations decreased 6-7 orders of magnitude within the first 6h of light exposure. After 12h in the dark, no evidence of dark-repair was observed. Expression data collected after 12h of sunlight exposure revealed a difference in transcript abundance in the light relative to dark microcosms for 35 unique ORFs, 33 ORFs showed increased transcript abundance and 2 ORFs showed reduced transcript abundance. A majority (51%) of the ORFs with increased transcript abundance in the sunlit relative to dark microcosms encoded hypothetical proteins; others were associated with protein synthesis, oxidative stress and DNA repair. Results suggest that E. faecalis exposed to sunlight actively transcribe RNA in response to photostress.

PURPOSE: To determine the prevalence and types of pathogens found in children with orbital cellulitis and to evaluate the utility of nonoperative cultures. METHODS: This was a retrospective cohort study of children with imaging-confirmed orbital cellulitis over a period of 8 years. Outcomes included prevalence and types of organisms, polymicrobial infection, mixed aerobic-anaerobic infection, effect of age, and culture utility. RESULTS: Of 220 children with orbital cellulitis, 112 (51%) had cultures taken; 69 (31%) had surgical intervention. Culture sources for the 112 children with cultures included blood (57 patients [51%]), sinus (53 [47%]), orbit (42 [38%]), brain (6 [5%]), and skin/conjunctiva/lacrimal sac (6 [5%]). Streptococcus anginosus group strains grew in cultures from 19 children (17%); methicillin-sensitive Staphylococcus aureus (MSSA), in 15 (13%); Streptococcus pyogenes, in 12 (11%); methicillin-resistant Staphylococcus aureus (MRSA), in 6 (5%); anaerobic/facultative gram negative rods, in 8 (7%); anaerobic Gram-positive cocci, other Viridans group streptococci, and Streptococci pneumoniae, in 3 (3%) each; and normal respiratory/skin flora, in 23 (21%). Polymicrobial infection (P = 0.08) and anaerobic organisms (P = 0.58) did not differ by age (range, 0.1-16.8 years). In all 220 (100%) children, nonoperative cultures were either not obtained (108 [49%]), not helpful in avoiding surgery (69 [31%]), showed no growth (39 [18%]), or grew an organism that did not change management from empiric therapy (4 [2%]). CONCLUSIONS: While many organisms may be cultured from children with orbital cellulitis, Streptococcus and MSSA were the most common in our study cohort. MRSA is uncommon, so initial empiric coverage is not necessary. Rates of polymicrobial and anaerobic infection were similar across ages. Our results indicate that nonoperative cultures are not indicated in the initial medical management of orbital cellulitis; in our cohort, they neither resulted in treatment changes nor helped avoid surgery.

Enterococci are gut microbes of most land animals. Likely appearing first in the guts of arthropods as they moved onto land, they diversified over hundreds of millions of years adapting to evolving hosts and host diets. Over 60 enterococcal species are now known. Two species, Enterococcus faecalis and Enterococcus faecium, are common constituents of the human microbiome. They are also now leading causes of multidrug-resistant hospital-associated infection. The basis for host association of enterococcal species is unknown. To begin identifying traits that drive host association, we collected 886 enterococcal strains from widely diverse hosts, ecologies, and geographies. This identified 18 previously undescribed species expanding genus diversity by >25%. These species harbor diverse genes including toxins and systems for detoxification and resource acquisition. Enterococcus faecalis and E. faecium were isolated from diverse hosts highlighting their generalist properties. Most other species showed a more restricted distribution indicative of specialized host association. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades, and the entry of genes associated with range expansion such as B-vitamin biosynthesis and flagellar motility to be mapped to the phylogeny. This work provides an unprecedentedly broad and deep view of the genus Enterococcus, including insights into its evolution, potential new threats to human health, and where substantial additional enterococcal diversity is likely to be found.

UNLABELLED: The polysaccharide capsule of Streptococcus pneumoniae is required for nasopharyngeal colonization and for invasive disease in the lungs, blood, and meninges. In contrast, the vast majority of conjunctival isolates are acapsular. The first serotype-specific gene in the capsule operon, cpsE, encodes the initiating glycosyltransferase and is one of the few serotype-specific genes that can tolerate null mutations. This report characterizes a spontaneously arising TIGR4 mutant exhibiting a reduced capsule, caused by a 6-nucleotide duplication in cpsE which results in duplication of Ala and Ile at positions 45 and 46. This strain (AI45dup) possessed more exposed phosphorylcholine and was hypersusceptible to C3 complement deposition compared to the wild type. Accordingly, the mutant was significantly better at forming abiotic biofilms and binding epithelial cells in vitro but was avirulent in a sepsis model. In vitro serial passaging of the wild-type strain failed to reproduce the AI45dup mutation but instead led to a variety of mutants with reduced capsule harboring single nucleotide polymorphisms (SNPs) in cpsE. A single passage in the sepsis model after high-dose inoculation readily yielded revertants of AI45dup with restored wild-type capsule level, but the majority of SNP alleles of cpsE could not revert, suppress, or bypass. Analysis of cpsE in conjunctival isolates revealed a strain with a single missense mutation at amino acid position 377, which was responsible for reduced encapsulation. This study supports the hypothesis that spontaneous, nonreverting mutations in cpsE serve as a form of adaptive mutation by providing a selective advantage to S. pneumoniae in niches where expression of capsule is detrimental. IMPORTANCE: While the capsule of Streptococcus pneumoniae is required for colonization and invasive disease, most conjunctival isolates are acapsular by virtue of deletion of the entire capsular operon. We show that spontaneous acapsular mutants isolated in vitro harbor mostly nonrevertible single nucleotide polymorphism (SNP) null mutations in cpsE, encoding the initiating glycosyltransferase. From a small collection of acapsular conjunctival isolates, we identified one strain with a complete capsular operon but containing a SNP in cpsE that we show is responsible for the acapsular phenotype. We propose that acapsular conjunctival isolates may arise initially from such nonreverting SNP null mutations in cpsE, which can be followed later by deletion of portions or all of the cps operon.

Acanthamoeba Keratitis (AK) is a severe corneal infection caused by the Acanthamoeba species of protozoa, potentially leading to permanent vision loss. AK requires prompt diagnosis and treatment to mitigate vision impairment. Diagnosing AK is challenging due to overlapping symptoms with other corneal infections, and treatment is made complicated by the organism's dual forms and increasing virulence, and delayed diagnosis. In this review, new approaches in AK diagnostics and treatment within the last 5 years are discussed. The English-language literature on PubMed was reviewed using the search terms "Acanthamoeba keratitis" and "diagnosis" or "treatment" and focused on studies published between 2018 and 2023. Two hundred sixty-five publications were initially identified, of which eighty-seven met inclusion and exclusion criteria. This review highlights the findings of these studies. Notably, advances in PCR-based diagnostics may be clinically implemented in the near future, while antibody-based and machine-learning approaches hold promise for the future. Single-drug topical therapy (0.08% PHMB) may improve drug access and efficacy, while oral medication (i.e., miltefosine) may offer a treatment option for patients with recalcitrant disease.

Acute conjunctivitis follows a seasonal pattern. Although its clinical course is typically self-limited, conjunctivitis epidemics incur a substantial economic burden because of missed school and work days. This study investigated seasonal and temporal trends of childhood conjunctivitis in the entire country of Burkina Faso from 2013 to 2016, using routine monthly surveillance from 2,444 government health facilities. A total of 783,314 cases were reported over the 4-year period. Conjunctivitis followed a seasonal pattern throughout the country, with a peak in April. A nationwide conjunctivitis outbreak with a peak in September 2016 was noted ( < 0.001), with an excess number of cases first detected in June 2016. Nationwide passive surveillance was able to detect an epidemic 3 months before its peak, which may aide in allocation of resources for containment and mitigation of transmission in future outbreaks.

PURPOSE: To evaluate the cases of corneal graft rejection following SARS-CoV-2 vaccination reported to Centers for Disease Control and Prevention Vaccine Adverse Event Reporting System. METHODS: A descriptive analysis of the demographics, clinical history and presentation was performed. We evaluated the correlation between the vaccines and duration of vaccine-associated graft rejection (VAR) onset following vaccination using a one-way analysis of variance test. A post hoc analysis was performed between VAR onset-interval following vaccination dose and vaccine type. Finally, a 30-day cumulative incidence analysis was performed to assess the risk of VAR in short term following different doses, vaccines and type of corneal transplantation. RESULTS: A total of 55 eyes of 46 patients were diagnosed with VAR following vaccination with BNT162b2 (73.91%) and mRNA-1273 (26.09%). The mean age of the patients was 62.76±15.83 years, and 28 (60.87%) were female. The patients diagnosed with VAR had undergone penetrating keratoplasty (61.82%), Descemet membrane endothelial keratoplasty (12.73%), descemet stripping endothelial keratoplasty (18.18%), anterior lamellar keratoplasty (3.64%) and corneal limbal allograft transplantation (1.82%). The mean time for VAR since penetrating and endothelial keratoplasty was 8.42±9.23 years and 4.18±4.40 years, respectively. 45.65% of the cases of VAR were reported after the second dose of vaccine. The duration of VAR onset was significantly shorter after the second dose compared with the first and booster doses (p=0.0165) and in patients who underwent endothelial keratoplasty compared with penetrating keratoplasty (p=0.041). CONCLUSIONS: This study outlines a possible temporal relationship between corneal graft rejection and SARS-CoV-2 vaccination. An earlier onset of VAR was observed in patients who had a history of endothelial keratoplasty and following the second dose of vaccination.

Conjunctival hyperemia is one of the most common causes for visits to primary care physicians, optometrists, ophthalmologists, and emergency rooms. Despite its high incidence, the treatment options for patients with conjunctival hyperemia are restricted to over-the-counter drugs that provide symptomatic relief due to short duration of action, tachyphylaxis and rebound redness. As our understanding of the immunopathological pathways causing conjunctival hyperemia expands, newer therapeutic targets are being discovered. These insights have also contributed to the development of animal models for mimicking the pathogenic changes in microvasculature causing hyperemia. Furthermore, this progress has catalyzed the development of novel therapeutics that provide efficacious, long-term relief from conjunctival hyperemia with minimal adverse effects.

Bacterial keratitis continues to be one of the leading causes of corneal blindness in the developed as well as the developing world, despite swift progress since the dawn of the "anti-biotic era". Although, we have expeditiously developed our understanding about the different causative organisms and associated pathology leading to keratitis, extensive gaps in knowledge continue to dampen the efforts required for early and accurate diagnosis, and management in these patients, resulting in poor clinical outcomes. The ability of the causative bacteria to subdue the therapeutic challenge stems from their large genome encoding complex regulatory networks, variety of unique virulence factors, and rapid secretion of tissue damaging proteases and toxins. In this review article, we provide an overview of the established diagnostic techniques and therapeutics for keratitis caused by various bacteria. We extensively report the recent in-roads through novel tools for accurately diagnosing mono- and poly-bacterial corneal infections. Furthermore, we outline the recent progress by our groups and others in understanding the sub-cellular genomic changes that lead to antibiotic resistance in these organisms. Finally, we discuss in detail, the novel therapies and drug delivery systems in development for the efficacious management of bacterial keratitis.

Adenovirus E1A is the first viral protein expressed during infection. E1A controls critical aspects of downstream viral gene expression and cell cycle deregulation, and its function is thought to be highly conserved among adenoviruses. Various bioinformatics analyses of E1A from 38 human adenoviruses of species D (HAdV-D), including likelihood clade model partitioning, provided highly significant evidence of divergence of HAdV-Ds into two distinct groups for the conserved region 3 (CR3), present only in the E1A 13S isoform. This variance within E1A 13S of HAdV-Ds was not found in any other human adenovirus (HAdV) species. By protein sequence and structural analysis, the zinc finger motif of E1A CR3, previously shown as critical for transcriptional activation, showed the greatest differences. Subsequent codon usage bias analysis revealed substantial divergence in E1A 13S between the two groups of HAdV-Ds, suggesting that these two sub-groups of HAdV-D evolved under different cellular conditions. Hence, HAdV-D E1A embodies a previously unappreciated evolutionary divergence among HAdVs.

Post-keratoplasty infectious keratitis (PKIK) represents a unique clinical entity that often poses significant diagnostic and therapeutic challenges. It carries a high risk of serious complications such as graft rejection and failure, and less commonly endophthalmitis. Topical corticosteroids are often required to reduce the risk of graft rejection but their use in PKIK may act as a double-edged sword, particularly in fungal infection. The increased uptake in lamellar keratoplasty in the recent years has also led to complications such as graft-host interface infectious keratitis (IIK), which is particularly difficult to manage. The reported incidence of PKIK differs considerably across different countries, with a higher incidence observed in developing countries (9.2-11.9%) than developed countries (0.02-7.9%). Common risk factors for PKIK include the use of topical corticosteroids, suture-related problems, ocular surface diseases and previous corneal infection. PKIK after penetrating keratoplasty or (deep) anterior lamellar keratoplasty is most commonly caused by ocular surface commensals, particularly Gramme-positive bacteria, whereas PKIK after endothelial keratoplasty is usually caused by Candida spp. Empirical broad-spectrum antimicrobial treatment is the mainstay of treatment for both PKIK, though surgical interventions are required in medically refractory cases (during the acute phase) and those affected by visually significant scarring (during the late phase). In this paper, we aim to provide a comprehensive overview on PKIK, encompassing the epidemiology, risk factors, causes, management and outcomes, and to propose a treatment algorithm for systematically managing this challenging condition.

PURPOSE: To compare the rates of infectious endophthalmitis following intravitreal injection of ranibizumab using prefilled syringes vs conventional preparation. DESIGN: Multicenter retrospective cohort study. METHODS: All eyes receiving intravitreal injection of 0.5 mg ranibizumab for retinal vascular diseases at 10 retina practices across the United States (2016 to 2017) and Japan (2009 to 2017) were included. The total numbers of eyes and injections were determined from billing codes. Endophthalmitis cases were determined from billing records and evaluated with chart review. Primary outcome was the rate of postinjection acute endophthalmitis. Secondary outcomes were visual acuity and microbial spectrum. RESULTS: A total of 243 754 intravitreal 0.5 mg ranibizumab injections (165 347 conventional and 78 407 prefilled) were administered to 43 132 unique patients during the study period. In the conventional ranibizumab group, a total of 43 cases of suspected endophthalmitis occurred (0.026%; 1 in 3845 injections) and 22 cases of culture-positive endophthalmitis occurred (0.013%; 1 in 7516 injections). In the prefilled ranibizumab group, 12 cases of suspected endophthalmitis occurred (0.015%; 1 in 6534 injections) and 2 cases of culture-positive endophthalmitis occurred (0.0026%; 1 in 39 204 injections). Prefilled syringes were associated with a trend toward decreased risk of suspected endophthalmitis (odds ratio 0.59; 95% confidence interval 0.31-1.12; P = .10) and a statistically significant decreased risk of culture-positive endophthalmitis (odds ratio 0.19; 95% confidence interval 0.045-0.82; P = .025). Average logMAR vision loss at final follow-up was significantly worse for eyes that developed endophthalmitis from the conventional ranibizumab preparation compared to the prefilled syringe group (4.45 lines lost from baseline acuity vs 0.38 lines lost; P = .0062). Oral-associated flora was found in 27.3% (6/22) of conventional ranibizumab culture-positive endophthalmitis cases (3 cases of Streptococcus viridans, 3 cases of Enterococcus faecalis) compared to 0 cases in the prefilled ranibizumab group. CONCLUSION: In a large, multicenter, retrospective study the use of prefilled syringes during intravitreal injection of ranibizumab was associated with a reduced rate of culture-positive endophthalmitis, including from oral flora, as well as with improved visual acuity outcomes.

Graduate medical education (GME) in ophthalmology has faced and overcome many challenges over the past years, and 2020 has been a game-changer. Although the severe acute respiratory syndrome coronavirus pandemic disrupted medical education globally, ophthalmic educators rapidly transformed their curricula to novel and effective virtual learning formats. Thus, while the COVID-19 outbreak has been one of the most significant challenges faced in the history of medical education, it has also provided an impetus to develop innovative teaching practices, bringing with it unprecedented success in allowing medical students to continue their education in ophthalmology despite these challenges. We review and appraise novel educational interventions implemented by various institutions in response to the COVID-19 pandemic, highlighting their effectiveness, challenges and proposing future directions beyond the pandemic. Many of these innovations will persist even after the end of the pandemic because they have proven that face-to-face learning is not required for all aspects of the ophthalmic GME curriculum. As ophthalmic educators harness the power of educational technology it is critical that their novel educational initiatives are incorporated into competency-based curricula with assessments mapped to the competencies. Future research should focus on evaluating the impact of this transformation to virtual learning environments on student performances as well as implementing longitudinal assessment strategies for clinical competence in workplace-based practice.

FasL was recently shown be required for bacterial clearance in C57BL/6 mice that express the FasL.1 allotype. The FasL.2 allotype is expressed in BALB/c mice and exhibits increased binding affinity to and increased cytotoxic activity against Fas(+) target cells. Therefore, we hypothesized that BALB/c mice would be more resistant to Staphylococcus aureus-induced endophthalmitis. To test this hypothesis, C57BL/6, BALB/c, and BALB(gld) mice received intravitreal injections of 2,500 CFU of S. aureus (RN6390). Clinical examinations, electroretinography (ERG), histology, and bacterial quantification were performed at 24, 48, 72, and 96 h postinjection. The myeloperoxidase (MPO) assay was used to quantitate neutrophil infiltration. At 96 h postinfection, 86% of C57BL/6 mice presented with complete destruction of the eye, compared to only 29% of BALB/c mice with complete destruction. To our surprise, in the absence of Fas ligand, BALB(gld) mice showed no difference in bacterial clearance compared to BALB/c mice. However, histology and ERG analysis revealed increased retinal damage and significant loss of retinal function. MPO analysis revealed equal numbers of neutrophils in BALB(gld) and BALB/c mice at 24 h postinfection. However, at 48 h, the neutrophil numbers remained significantly elevated in BALB(gld) mice, correlating with the increased retinal damage observed in BALB(gld) mice. We conclude that the increased resistance to S. aureus induced endophthalmitis in BALB/c mice is not dependent upon the FasL. However, in contrast to C57BL/6 mice, FasL is required for resolution of inflammation and protecting host tissue from nonspecific damage in BALB/c mice.

OBJECTIVES: Polyanionic polymers, including lipoteichoic acid and wall teichoic acid, are important determinants of the charged character of the staphylococcal cell wall. This study was designed to investigate the extent to which teichoic acid contributes to protection from anionic azo dyes and to identify barriers to drug penetration for development of new antibiotics for multidrug-resistant Staphylococcus aureus infection. METHODS: We studied antimicrobial activity of azo dyes against S. aureus strains with or without inhibition of teichoic acid in vitro and in vivo. RESULTS: We observed that inhibition of wall teichoic acid expression resulted in an ∼1000-fold increase in susceptibility to azo dyes such as Congo red, reducing its MIC from >1024 to <4 mg/L. Sensitization occurred when the first step in the wall teichoic acid pathway, catalysed by TarO, was inhibited either by mutation or by chemical inhibition. In contrast, genetic blockade of lipoteichoic acid biosynthesis did not confer Congo red susceptibility. Based on this finding, combination therapy was tested using the highly synergistic combination of Congo red plus tunicamycin at sub-MIC concentrations (to inhibit wall teichoic acid biosynthesis). The combination rescued Caenorhabditis elegans from a lethal challenge of S. aureus. CONCLUSIONS: Our studies show that wall teichoic acid confers protection to S. aureus from anionic azo dyes and related compounds, and its inhibition raises the prospect of development of new combination therapies based on this inhibition.