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Objective: To characterize the oral microbiome of patients with head and neck squamous cell carcinoma (HNSCC) before and during radiotherapy (RT), compared to healthy individuals. Evaluating the impact of oral microbiome in the clinical outcomes one year following the end of RT., Methods: Oral samples were collected from HNSCC patients who underwent RT using the following regimens: no dose received (T0), dose 12-16 Gy (T1), dose 30-36 Gy (T2) and dose ≥ 60 Gy (T3). Samples from healthy individuals were also collected only once as a control group. Regions V1-V2 of the 16S rRNA were sequenced by Illumina and analyzed using Mothur., Results: 49 patients with HNSCC and 25 healthy individuals were included. At T0, HNSCC patients showed a lower abundance of Firmicutes and Streptococcus (p = 0.011, p = 0.002) and a higher abundance of Bacteroidetes (p = 0.005) compared to healthy individuals. During RT, Fusobacterium (p = 0.017) and Porphyromonas (p = 0.0008) decreased, while Streptococcus increased at T1 (p = 0.001). By T3, the differences in Firmicutes, Bacteroidetes, and Streptococcus between the control and HNSCC groups were no longer significant (p > 0.3). Patients with higher initial abundances of Porphyromonas (p = 0.012) and Fusobacterium (p = 0.017) had poorer outcomes, including recurrence, metastasis, and death. In contrast, disease-free patients had a higher abundance of Streptococcus (p = 0.004)., Conclusion: Oral microbiome dysbiosis was found in HNSCC patients. By the end of RT, the main initial differences in phylum and genus abundance observed at T0 between the control and HNSCC groups were no longer present. Higher abundances of Fusobacterium and Porphyromonas were associated with poor outcomes., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: To investigate the genomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) endophthalmitis and correlate it with the presenting clinical features and outcomes., Methods: Nine patients presenting with MRSA endophthalmitis from 2014 to 2022 were included. Phenotypic and genomic tests were used for strain characterization. Demographics, clinical presentation, treatment and outcomes were reviewed., Results: The MRSA population was dominated by multidrug-resistant (MDR) strains within the clonal complex 5 (CC5) carrying an SCC mec type II genetic element (USA100-like strains). These strains carried genes that confer resistance to five antibiotic classes, in addition to mutations in topoisomerase genes ( gyrA and parC ) that resulted in resistance to all fluoroquinolones tested. Patients were mostly male (56%), with a median age of 82.7 years, and most had no recent history of extensive healthcare exposure. All cases were exogenous following ocular surgery (67%) or intravitreal injection (33%). The main exam findings were visual acuity ≤ hand motion, hypopyon (89%), and vitreous opacity (89%). Five patients (56%) showed improvement in visual acuity at 1 month following presentation, three (33%) at 3 months, and two (22%) at 6 months. Complications included evisceration ( n  = 1) and phthisis ( n  = 1). Patients who had pars plana vitrectomy within 48 hours of presentation had better clinical outcomes compared to those who did not., Conclusion: Exogenous MRSA endophthalmitis is caused by MDR strains that resemble the hospital-acquired lineage USA100. These strains cause severe endophthalmitis in patients with no recent hospital/healthcare exposure.

Introduction: Ocular syphilis is a rare but potentially sight-threatening manifestation of infection with the spirochete Treponema pallidum subspecies pallidum . Molecular strain typing of clinical specimens obtained from patients with syphilis can provide useful epidemiological and clinical information. In this study, we assess the utility of non-ocular clinical samples in strain typing for patients with diagnosed ocular syphilis., Methods: We collected samples of excess blood, serum, and cerebrospinal fluid (CSF) from 6 patients with ocular syphilis treated in 2013-2016. DNA was extracted, purified, and then analyzed using an enhanced molecular typing method including sequence analysis of tp0548 , number of repeats in the arp gene, and restriction fragment length polymorphism of the tpr gene., Results: Molecular strain typing based on tp0548 gene sequence analysis revealed two cases of type F and two cases of type G in 3 of 6 (50%) cases with CSF samples, 1 of which was obtained after starting antibiotics. In a patient with 2 distinct episodes, the same tp0548 type (type G) was identified in both episodes using different sample types (CSF, whole blood). Serum samples were available in 6 cases, but none were successfully typed with any of the methods. Amplification of the tpr and arp genes was unsuccessful in all cases. Overall, strain types were identified in 4 of the 7 episodes., Conclusion: Treponema pallidum strain types F and G were detected in CSF or whole blood in 4 of 7 episodes in this series. We demonstrate moderate sensitivity of strain typing in ocular syphilis using non-ocular clinical specimens.

Objective: Antimicrobial resistance is a global pandemic that poses a major threat to vision health as ocular bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA), are becoming increasingly resistant to first-line therapies. Here we evaluated the antimicrobial activity of new synthetic lincosamides in comparison to currently used antibiotics against clinical ocular MRSA isolates., Methods: Antimicrobial susceptibility testing was performed by broth microdilution for two novel synthetic lincosamides (iboxamycin and cresomycin) and eight comparator antibiotics against a collection of 50 genomically characterised ocular MRSA isolates, including isolates harbouring erm genes (n = 25)., Results: Both drugs were active against widespread MRSA clonal complexes CC8 and CC5. The MIC 50 and MIC 90 of iboxamycin were 0.06 and 2 mg/L, respectively. Cresomycin (MIC 50 = 0.06 mg/L) also displayed good activity with an in vitro potency four-fold higher (MIC 90 = 0.5 mg/L) than iboxamycin. In isolates harbouring erm genes, MIC 90 were >16, 2, and 0.5 mg/L for clindamycin, iboxamycin, and cresomycin, respectively. The in vitro potencies of iboxamycin and cresomycin were similar or higher than that of comparator agents and were not impacted by multidrug-resistance phenotypes or by the presence of erm genes when compared with clindamycin., Conclusions: Our results demonstrate that iboxamycin and cresomycin display potent in vitro activity against ocular MRSA isolates, including multidrug-resistant isolates harbouring erm genes., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Bacterial keratitis is a vision-threatening infection mainly caused by Gram-positive bacteria (GPB). Antimicrobial therapy is commonly empirical using broad-spectrum agents with efficacy increasingly compromised by the emergence of antimicrobial resistance. We used a combination of phenotypic tests and genome sequencing to identify the predominant lineages of GPB causing keratitis and to characterize their antimicrobial resistance patterns. A total of 161 isolates, including Staphylococcus aureus ( n = 86), coagulase-negative staphylococci (CoNS; n = 34), Streptococcus spp. ( n = 34), and Enterococcus faecalis ( n = 7), were included. The population of S. aureus isolates consisted mainly of clonal complex 5 (CC5) (30.2%). Similarly, the population of Staphylococcus epidermidis was homogenous with most of them belonging to CC2 (78.3%). Conversely, the genetic population of Streptococcus pneumoniae was highly diverse. Resistance to first-line antibiotics was common among staphylococci, especially among CC5 S. aureus . Methicillin-resistant S. aureus was commonly resistant to fluoroquinolones and azithromycin (78.6%) and tobramycin (57%). One-third of the CoNS were resistant to fluoroquinolones and 53% to azithromycin. Macrolide resistance was commonly caused by erm genes in S. aureus , mphC and msrA in CoNS, and mefA and msr(D ) in streptococci. Aminoglycoside resistance in staphylococci was mainly associated with genes commonly found in mobile genetic elements and that encode for nucleotidyltransferases like ant(4')-Ib and ant(9)-Ia . Fluroquinolone-resistant staphylococci carried from 1 to 4 quinolone resistance-determining region mutations, mainly in the gyrA and parC genes. We found that GPB causing keratitis are associated with strains commonly resistant to first-line topical therapies, especially staphylococcal isolates that are frequently multidrug-resistant and associated with major hospital-adapted epidemic lineages., Competing Interests: The authors declare no conflict of interest.

We used cultured human conjunctival goblet cells to determine (i) whether the toxigenic S. aureus- induced activation of the epithelial goblet cells requires two signals to activate the NLRP3 inflammasome, (ii) if one signal is mediated by TLR1, TLR2, or TLR6, and (iii) if the S. aureus toxin α toxin is another signal for the activation of the inflammasome and secretion of mature IL-1β. Cultured cells were incubated with siRNA to knock down the different TLRs. After stimulation with toxigenic S. aureus RN6390, pro-IL-1β synthesis, caspase-1 activity, and mature IL-1β secretion were measured. In a separate set of experiments, the cells were incubated with toxigenic S. aureus RN6390 or mutant S. aureus ALC837 that does not express α toxin with or without exogenous α toxin. A gentamicin protection assay was used to determine if intracellular bacteria were active. We conclude that α toxin from toxigenic S. aureus triggers two separate mechanisms required for the activation of the NLRP3 inflammasome and secretion of mature IL-1β. In the first mechanism, α toxin secreted from internalized S. aureus produces a pore, allowing the internalized bacteria and associated pathogen-associated molecular patterns to interact with intracellular TLR2 and, to a lesser extent, TLR1. In the second mechanism, α toxin forms a pore in the plasma membrane, leading to an efflux of cytosolic K + and influx of Ca 2+ . We conclude that α toxin by these two different mechanisms triggers the synthesis of pro-IL-1β and NLRP3 components, activation of capase-1, and secretion of mature IL-1β to defend against bacterial infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Hodges, AukrustNaqvi, Bair, Bispo, Gilmore, Gregory-Ksander and Dartt.)

Purpose: Ocular bacterial infections are important causes of morbidity and vision loss. Early antimicrobial therapy is necessary to save vision, but their efficacy is increasingly compromised by antimicrobial resistance (AMR). We assessed the etiology of ocular bacterial infections seen at Massachusetts Eye and Ear and investigated the molecular epidemiology and AMR profiles of contemporary isolates., Design: Laboratory investigation., Methods: We used a combination of phenotypic tests and genome sequencing to identify the predominant lineages of leading ocular pathogens and their AMR profiles., Results: A total of 1601 isolates were collected from 2014 to 2021, with Staphylococcus aureus (n = 621), coagulase-negative staphylococci (CoNS) (n = 234), Pseudomonas aeruginosa (n = 213), Enterobacteriaceae (n = 167), and Streptococcus pneumoniae (n = 95) being the most common. Resistance was high among staphylococci, with methicillin resistance (MR) detected in 28% of S aureus and 39.8% of CoNS isolates. Multidrug resistance (MDR) was frequent among MR staphylococci (MRSA 60%, MRCoNS 76.1%). The population of S aureus isolates consisted mainly of 2 clonal complexes (CCs): CC8 (26.1%) and CC5 (24.1%). CC5 strains carried a variety of AMR markers, resulting in high levels of resistance to first-line therapies. Similarly, the population of ocular Staphylococcus epidermidis was homogenous with most belonging to CC2 (85%), which were commonly MDR (48%). Conversely, ocular S pneumoniae, P aeruginosa, and Enterobacteriaceae were often susceptible to first-line therapies and grouped into highly diverse genetic populations., Conclusion: Our data showed that ocular bacterial infections in our patient population are disproportionately caused by strains that are resistant to clinically relevant antibiotics and are associated with major epidemic genotypes with both community and hospital associations., (Copyright © 2023 Elsevier Inc. All rights reserved.)

A large number of bacterial pathogens bind to host extracellular matrix (ECM) components. For example, many Gram-negative and Gram-positive pathogens express binding proteins for fibronectin (FN) on their cell surface. Mutagenesis studies of bacterial FN-binding proteins have demonstrated their importance in pathogenesis in preclinical animal models. However, means to draw on these findings to design therapeutic approaches that specifically target FN-bacteria interactions have not been successful because bacterial pathogens can elaborate several FN-binding proteins and also because FN is an essential protein and likely a nondruggable target. Here we report that select heparan compounds potently inhibit Streptococcus pneumoniae infection of injured corneas in mice. Using intact heparan sulfate (HS) and heparin (HP), heparinase-digested fragments of HS, HP oligosaccharides, and chemically or chemoenzymatically modified heparan compounds, we found that inhibition of S. pneumoniae corneal infection by heparan compounds is not mediated by simple charge effects but by a selective sulfate group. Removal of 2- O -sulfates significantly inhibited the ability of HP to inhibit S. pneumoniae corneal infection, whereas the addition of 2- O -sulfates to heparosan (H) significantly increased H's ability to inhibit bacterial corneal infection. Proximity ligation assays indicated that S. pneumoniae attaches directly to FN fibrils in the corneal epithelial ECM and that HS and HP specifically inhibit this binding interaction in a 2- O -sulfate-dependent manner. These data suggest that heparan compounds containing 2- O -sulfate groups protect against S. pneumoniae corneal infection by inhibiting bacterial attachment to FN fibrils in the subepithelial ECM of injured corneas. Moreover, 2- O -sulfated heparan compounds significantly inhibited corneal infection in immunocompromised hosts, by a clinical keratitis isolate of S. pneumoniae , and also when topically administered in a therapeutic manner. These findings suggest that the administration of nonanticoagulant 2- O -sulfated heparan compounds may represent a plausible approach to the treatment of S. pneumoniae keratitis., Competing Interests: CONFLICT OF INTEREST STATEMENT The authors declare no conflict of interest.

Purpose: Intraocular infections are sight-threatening conditions that can lead to vision loss. Rapid identification of the etiologies plays a key role in early initiation of effective therapy to save vision. However, current diagnostic modalities are time consuming and lack sensitivity and inclusiveness. We present here a newly developed comprehensive ocular panel designed to improve diagnostic yields and provide a tool for rapid and sensitive pathogen detection., Design: Experimental laboratory investigation., Methods: A panel containing 46 pathogens and 2 resistance/virulence markers that are commonly detected in intraocular infections was developed. Genomic targets were scrutinized for stretches predicted to be specific for a particular species while being conserved across different strains. A set of primers for sample enrichment, and two 50mer NanoString compatible probes were then designed for each target. Probe-target hybrids were detected and quantified using the NanoString nCounter SPRINT Profiler. Diagnostic feasibility was assessed in a pilot clinical study testing samples from infectious retinitis (n = 15) and endophthalmitis (n = 12) patients, for which the etiologies were confirmed by polymerase chain reaction (PCR) or culture., Results: Analytical studies demonstrated highly sensitive detection of a broad spectrum of pathogens, including bacteria, viruses, and parasites, with limits of detection being as low as 2.5 femtograms per reaction. We also found excellent target specificity, with minimal cross-reactivity detected. The custom-designed NanoString ocular panel correctly identified the causative agent from all clinical specimens positive for a variety of pathogens., Conclusion: This highly multiplexed panel for pathogen detection offers a sensitive, comprehensive, and uniform assay run directly on ocular fluids that could significantly improve diagnostics of sight-threatening intraocular infections., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: The purpose of this study was to assess the potential of new lipoglycopeptides as novel topical therapies for improved treatment of recalcitrant ocular infections. We evaluated the in vitro antimicrobial activity of oritavancin, dalbavancin, and telavancin compared with vancomycin (VAN) against a large collection of ocular staphylococcal isolates and their cytotoxicity on human corneal epithelial cells (HCECs)., Methods: Antimicrobial susceptibility testing was performed by broth microdilution against 223 Staphylococcus spp. clinical isolates. Time-kill kinetics were determined for methicillin-resistant strains of Staphylococcus aureus (MRSA) (n = 2) and Staphylococcus epidermidis (MRSE) (n = 1). In vitro cytotoxicity assays were performed with AlamarBlue and live/dead staining on HCECs., Results: All new lipoglycopeptides showed strong in vitro potency against ocular staphylococci, including multidrug-resistant MRSA strains, with dalbavancin showing a slightly higher potency overall [minimum inhibitory concentration (MIC) 90 0.06 μg/mL] compared with telavancin and oritavancin (MIC 90 0.12 μg/mL), whereas VAN had the lowest potency (MIC 90 2 μg/mL). Oritavancin exerted rapid bactericidal activity within 1 h for MRSA and 2 h for MRSE. All other drugs were bactericidal within 24 h. At a concentration commonly used for topical preparations (25 mg/mL), cytotoxicity was observed for VAN after 5 min of incubation, whereas reduction in HCEC viability was not seen for telavancin and was less affected by oritavancin and dalbavancin. Cytotoxicity at 25 mg/mL was seen for all drugs at 30 and 60 min but was significantly reduced or undetected for lower concentrations., Conclusions: Our study demonstrates that new lipoglycopeptides have substantially better in vitro antimicrobial activity against ocular staphylococcal isolates compared with VAN, with a similar or improved toxicity profile on HCECs., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.)

Implantable tubes, shunts, and other medical conduits are crucial for treating a wide range of conditions from ears and eyes to brain and liver but often impose serious risks of device infection, obstruction, migration, unreliable function, and tissue damage. Efforts to alleviate these complications remain at an impasse because of fundamentally conflicting design requirements: Millimeter-scale size is required to minimize invasiveness but exacerbates occlusion and malfunction. Here, we present a rational design strategy that reconciles these trade-offs in an implantable tube that is even smaller than the current standard of care. Using tympanostomy tubes (ear tubes) as an exemplary case, we developed an iterative screening algorithm and show how unique curved lumen geometries of the liquid-infused conduit can be designed to co-optimize drug delivery, effusion drainage, water resistance, and biocontamination/ingrowth prevention in a single subcapillary-length-scale device. Through extensive in vitro studies, we demonstrate that the engineered tubes enabled selective uni- and bidirectional fluid transport; nearly eliminated adhesion and growth of common pathogenic bacteria, blood, and cells; and prevented tissue ingrowth. The engineered tubes also enabled complete eardrum healing and hearing preservation and exhibited more efficient and rapid antibiotic delivery to the middle ear in healthy chinchillas compared with current tympanostomy tubes, without resulting in ototoxicity at up to 24 weeks. The design principle and optimization algorithm presented here may enable tubes to be customized for a wide range of patient needs.

Antimicrobial resistance poses a major threat to public health. Given the paucity of novel antimicrobials to treat resistant infections, the emergence of multidrug-resistant bacteria renewed interest in antimicrobial peptides as potential therapeutics. This study designed a new analog of the antimicrobial peptide Plantaricin 149 (Pln149-PEP20) based on previous Fmoc-peptides. The minimal inhibitory concentrations of Pln149-PEP20 were determined for 60 bacteria of different species and resistance profiles, ranging from 1 mg/L to 128 mg/L for Gram-positive bacteria and 16 to 512 mg/L for Gram-negative. Furthermore, Pln149-PEP20 demonstrated excellent bactericidal activity within one hour. To determine the propensity to develop resistance to Pln149-PEP20, a directed-evolution in vitro experiment was performed. Whole-genome sequencing of selected mutants with increased MICs and wild-type isolates revealed that most mutations were concentrated in genes associated with membrane metabolism, indicating the most likely target of Pln149-PEP20. Synchrotron radiation circular dichroism showed how this molecule disturbs the membranes, suggesting a carpet mode of interaction. Membrane depolarization and transmission electron microscopy assays supported these two hypotheses, although a secondary intracellular mechanism of action is possible. The molecule studied in this research has the potential to be used as a novel antimicrobial therapy, although further modifications and optimization remain possible.

Candida species are the most common causes of sight-threatening fungal ocular infections in temperate regions of the world. Despite their relevance, little is known about the emergence of novel species and the molecular epidemiology of these infections. Here, we molecularly characterized 38 yeast isolates collected from patients diagnosed with endophthalmitis or keratitis at Massachusetts Eye and Ear from 2014 to 2021. Sequencing of the ITS1-5.8S-/ITS2 regions demonstrated that this population of yeasts was dominated by Candida spp. (37 out of 38; 97%), with 58% of the cases caused by C. albicans (n = 22) and the remaining by emerging non-albicans species, predominantly by C. parapsilosis (n = 8) and C. dubliniensis (n = 6). One isolate each was identified as C. tropicalis and Clavispora lusitaniae. Interestingly, all C. dubliniensis were isolated from endophthalmitis and most C. parapsilosis from keratitis. Multilocus sequence typing analysis of C. albicans showed a prevalence of CC-1 isolates that has DST69 as the putative founder, with 64% of them belonging to this clonal complex (CC). Isolates grouped within this cluster were more predominant in endophthalmitis (10 out of 14; 71%). One C. albicans CC-1 isolate was multi-azole resistant. In conclusion, we observed that nearly half of the ocular infections caused by yeasts are associated with C. albicans, with evidence for the emergence of non-albicans species that are differentially enriched in distinct ocular niches. Candida albicans isolates clustered within the predominant CC-1 group were particularly more common in endophthalmitis, demonstrating a potential pattern of ocular disease enrichment within this clade., (© The Author(s) 2022. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Infectious uveitis is a sight-threatening infection commonly caused by herpesviruses. Vitreous humor is often collected for molecular confirmation of the causative agent during vitrectomy and mixed in large volumes of buffered saline, diluting the pathogen load. Here, we explore affinity-capture hydrogel particles (Nanotrap ® ) to concentrate low abundant herpesviruses from diluted vitreous. Simulated samples were prepared using porcine vitreous spiked with HSV-1, HSV-2, VZV and CMV at 10 5 copies/mL. Pure undiluted samples were used to test capturing capability of three custom Nanotrap particles (red, white and blue) in a vitreous matrix. We found that all particles demonstrated affinity to the herpesviruses, with the Red Particles having both good capture capability and ease of handling for all herpesviruses. To mimic diluted vitrectomy specimens, simulated-infected vitreous were then serially diluted in 7 mL TE buffer. Diluted samples were subjected to an enrichment protocol using the Nanotrap Red particles. Sensitivity of pathogen detection by qPCR in diluted vitreous increased anywhere between 2.3 to 26.5 times compared to non-enriched specimens. This resulted in a 10-fold increase in the limit of detection for HSV-1, HSV-2 and VZV. These data demonstrated that Nanotrap particles can capture and concentrate HSV-1, HSV-2, VZV and CMV in a vitreous matrix.

Introduction: Since 2009, the Antibiotic Resistance Monitoring in Ocular Microorganisms (ARMOR) surveillance study has been assessing in vitro antibiotic resistance for bacterial isolates sourced from ocular infections in the US. The main goal of this systematic review was to compare in vitro resistance data for ocular pathogens from published US studies with the most recently published data from the ARMOR study (2009-2018) and, where possible, to evaluate trends in bacterial resistance over time over all studies., Methods: A literature search was conducted using MEDLINE®, BIOSIS Previews®, and EMBASE ® databases (1/1/1995-6/30/2021). Data were extracted from relevant studies and antibiotic susceptibility rates for common ocular pathogens (Staphylococcus aureus, coagulase-negative staphylococci [CoNS], Streptococcus pneumoniae, Pseudomonas aeruginosa, and Haemophilus influenzae), longitudinal changes in susceptibility, and multidrug resistance (MDR) were compared descriptively., Results: Thirty-two relevant studies were identified. High in vitro resistance was found among S. aureus and CoNS to fluoroquinolones, macrolides, and methicillin/oxacillin across studies, with high rates of MDR noted, specifically among methicillin-resistant staphylococci. Data from studies pre-dating or overlapping the early years of ARMOR reflected increasing rates of S. aureus resistance to fluoroquinolones, macrolides, methicillin/oxacillin, and aminoglycosides, while the ARMOR data suggested slight decreases in resistance to these classes between 2009 and 2018. Overall, methicillin-resistant S. aureus (MRSA) prevalence peaked from 2005 to 2015 with a possible decreasing trend in more recent years., Discussion and Conclusions: Data from local and regional US datasets were generally consistent with data from the national ARMOR surveillance study. Continued surveillance of ocular bacterial pathogens is needed to track trends such as methicillin resistance and MDR prevalence and any new emerging antibiotic resistance phenotypes. Susceptibility data from ARMOR can inform initial choice of therapy, especially in practice areas where local antibiograms are unavailable., (© 2022. The Author(s).)

Streptococcus pneumoniae is a leading cause of ocular infections including serious and sight-threatening conditions. The use of pneumococcal conjugate vaccines (PCV) has substantially reduced the incidence of pneumonia and invasive pneumococcal diseases, but has had limited impact on ocular infections. Additionally, widespread vaccine use has resulted in ongoing selective pressure and serotype replacement in carriage and disease. To gain insight into the population structure of pneumococcal isolates causing ocular infections in a post-PCV-13 time period, we investigated the genomic epidemiology of ocular S. pneumoniae isolates ( n =45) collected at Massachusetts Eye and Ear between 2014 and 2017. By performing a series of molecular typing methods from draft genomes, we found that the population structure of ocular S. pneumoniae is highly diverse with 27 sequence types (grouped into 18 clonal complexes) and 17 serotypes being identified. Distribution of these lineages diverged according to the site of isolation, with conjunctivitis being commonly caused by isolates grouped in the Epidemic Conjunctivitis Cluster-ECC (60 %), and ST448 (53.3 %) being most frequently identified. Conversely, S. pneumoniae keratitis cases were caused by a highly diverse population of isolates grouping within 15 different clonal complexes. Serotyping inference demonstrated that 95.5 % of the isolates were non-PCV-13 vaccine types. Most of the conjunctivitis isolates (80 %) were unencapsulated, with the remaining belonging to serotypes 15B, 3 and 23B. On the other hand, S. pneumoniae causing keratitis were predominantly encapsulated (95.2 %) with 13 different serotypes identified, mostly being non-vaccine types. Carriage of macrolide resistance genes was common in our ocular S. pneumoniae population (42.2 %), and usually associated with the mefA +msrD genotype ( n =15). These genes were located in the Macrolide Efflux Genetic Assembly cassette and were associated with low-level in vitro resistance to 14- and 15-membered macrolides. Less frequently, macrolide-resistant isolates carried an ermB gene ( n =4), which was co-located with the tet M gene in a Tn-916-like transposon. Our study demonstrates that the population structure of ocular S. pneumoniae is highly diverse, mainly composed by isolates that escape the PCV-13 vaccine, with patterns of tissue/niche segregation, adaptation and specialization. These findings suggest that the population structure of ocular pneumococcus may be shaped by multiple factors including PCV-13 selective pressure, microbial-related and niche-specific host-associated features.

Purpose: To characterize diphtheroid corneal infections in eyes in the chronic phase of Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN)., Methods: Observational case series., Results: Four eyes of 3 patients were included in this review. Each eye presented with persistent corneal epithelial defect with corneal thinning in the chronic phase of SJS/TEN. None of the epithelial defects were associated with stromal infiltration. The corneas were cultured at the time of workup of persistent epithelial defect (3 eyes) or at time of tectonic penetrating keratoplasty after perforation (1 eye). Cultures yielded abundant growth of Corynebacterium spp., including Corynebacterium jeikeium (n = 2), Corynebacterium glucuronolyticum (n = 1), and a multidrug-resistant Corynebacterium striatum isolate (n = 1). The ocular surface was stabilized with surgical intervention (1 eye) or with introduction of fortified topical antibiotic based on laboratory identification and susceptibility testing of the isolated organisms (3 eyes). Numerous risk factors for microbial keratitis were present in all 4 eyes., Conclusions: In eyes with a persistent corneal epithelial defect in the chronic phase of SJS/TEN, even in the absence of an infiltrate, corneal culture should be undertaken. Recognition and treatment of Corynebacterium spp. as opportunistic pathogens may lead to favorable outcomes in cases of clinically sterile ulceration during the chronic phase of SJS/TEN., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: To investigate a cluster of corneoscleral rim cultures positive for Achromobacter species over a 6-month period at Massachusetts Eye and Ear., Methods: An increased rate of positive corneal donor rim cultures was noted at Massachusetts Eye and Ear between July and December 2017. Positive cultures were subjected to identification and antimicrobial susceptibility testing by phenotypic (MicroScan WalkAway) and genotypic (16S rDNA sequencing) methods. Samples of the eye wash solution (GeriCare) used in the eye bank were also evaluated. Antimicrobial activity of Optical-GS against Achromobacter spp. at 4°C and 37°C was assessed by time-kill kinetics assay., Results: Of 99 donor rims cultured, 14 (14.1%) grew bacteria with 11 (78.6%) due to uncommon nonfermenting Gram-negative bacilli. These had been identified by standard automated methods as Achromobacter (n = 3), Alcaligenes (n = 3), Ralstonia (n = 2), Pseudomonas (n = 2), and Stenotrophomonas (n = 1). Eight of these 11 isolates were subsequently available for molecular identification, and all were identified as Achromobacter spp. Six bottles of eyewash solution were evaluated and were positive for abundant Achromobacter spp. (3.4 × 105 ± 1.1 CFU/mL). Optisol-GS had no bactericidal activity against Achromobacter spp. at 4°C after 24-hour incubation but was bactericidal at 37°C. None of the patients who had received the contaminated corneas developed postoperative infection., Conclusions: An eyewash solution arising from a single lot was implicated in the contamination of donor rims by Achromobacter spp. The isolates were able to survive in the Optisol-GS medium at the recommended storage temperature. This highlights the need to continue improving protocols for tissue preparation and storage., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Community-genotype methicillin-resistant Staphylococcus aureus (CG-MRSA) emerged in the 1990s as a global community pathogen primarily involved in skin and soft tissue infections (SSTIs) and pneumonia. To date, the CG-MRSA SSTI burden in Latin America (LA) has not been assessed., Objective: The main objective of this study was to report the rate and genotypes of community-genotype methicillin-resistant Staphylococcus aureus (CG-MRSA) causing community-onset skin and soft tissue infections (CO-SSTIs) in LA over the last two decades. In addition, this research determined relevant data related to SSTIs due to CG-MRSA, including risk factors, other invasive diseases, and mortality., Data Sources: Relevant literature was searched and extracted from five major databases: Embase, PubMed, LILACS, SciELO, and Web of Science., Methods: A systematic review was performed, and a narrative review was constructed., Results: An analysis of 11 studies identified epidemiological data across LA, with Argentina presenting the highest percentage of SSTIs caused by CG-MRSA (88%). Other countries had rates of CG-MRSA infection ranging from 0 to 51%. Brazil had one of the lowest rates of CG-MRSA SSTI (4.5-25%). In Argentina, being younger than 50 years of age and having purulent lesions were predictive factors for CG-MRSA CO-SSTIs. In addition, the predominant genetic lineages in LA belonged to sequence types 8, 30, and 5 (ST8, ST30, and ST5)., Conclusion: There are significant regional differences in the rates of CG-MRSA causing CO-SSTIs. It is not possible to conclude whether or not CG-MRSA CO-SSTIs resulted in more severe SSTI presentations or in a higher mortality rate., (Copyright © 2021 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. All rights reserved.)

Purpose: To evaluate the antifungal properties of topical antibiotics (already being used successfully to prevent bacterial endophthalmitis) and some promising antiseptics for antifungal prophylaxis in the setting of artificial corneal implantation., Methods: Several commonly used antibiotics for antimicrobial prophylaxis after artificial corneal implantation, in addition to antiseptics [benzalkonium chloride (BAK), povidone-iodine (PI), and some ionic liquids (ILs)], were tested in vitro against Candida albicans, Fusarium solani, and Aspergillus fumigatus. The time-kill activity was determined. Toxicity was assayed in vitro on human corneal epithelial cultures using trypan blue. Adhesion and tissue invasion experiments were also carried out on porcine corneas and commonly used contact lenses, with or without gamma irradiation, and by analysis with fluorescence microscopy., Results: Polymyxin B (PMB)/trimethoprim/BAK (Polytrim), PMB alone, gatifloxacin with BAK (Zymaxid), and same-concentration BAK alone exhibited antifungal activity in vitro. Moxifloxacin (MOX) or gatifloxacin without BAK-as well as trimethoprim, vancomycin, and chloramphenicol-had no effect. 1% PI and ILs had the highest efficacy/toxicity ratios (>1), and Polytrim was species dependent. Subfungicidal concentrations of Polytrim reduced adhesion of C. albicans to Kontur contact lenses. Gamma-irradiated corneas showed enhanced resistance to fungal invasion., Conclusions: Of antibiotic preparations already in use for bacterial prophylaxis after KPro surgery, Polytrim is a commonly used antibiotic with antifungal effects mediated by both PMB and BAK and may be sufficient for prophylaxis. PI as a 1% solution seems to be promising as a long-term antifungal agent. Choline-undecanoate IL is effective and virtually nontoxic and warrants further development.

Purpose: The aims of this study were to characterize alpha-hemolytic streptococci among isolates from cases of infectious endophthalmitis and keratitis and to determine their distributions., Methods: The sample included 27 and 35 nonduplicated isolates of alpha-hemolytic streptococci recovered from patients with infectious endophthalmitis (2002-2013) and keratitis (2008-2013), respectively. Isolates were identified by the optochin susceptibility and bile solubility tests, using a biochemical identification system. The minimum inhibitory concentration was determined by the broth microdilution method. Molecular identification was performed by analyses of three constitutive genes and the complementary multilocus sequence. The molecular epidemiology of Streptococcus pneumoniae was investigated using multilocus sequence typing, and the presence of the capsular polysaccharide-encoding gene was assessed using conventional polymerase chain reaction. Outcomes were evaluated using the patients' medical records., Results: Phenotypic tests differentiated S. pneumoniae from other alpha-hemolytic streptococci, consistent with later molecular identifications. Streptococcus oralis was significantly prevalent among the endophthalmitis isolates, as was S. pneumoniae in the keratitis isolates. High levels of susceptibility to antibiotics were observed, including vancomycin, cephalosporins, and fluoroquinolones. High genetic variability was detected among the 19 S. pneumoniae strains, with 15 predicted to be encapsulated. The medical records of patients with infectious endophthalmitis were reviewed (n=15/27; 56%), and final visual acuity was assessed in 12 cases (44%). Many patients progressed to a final visual acuity state of "no light perception" (6/12; 50%), "light perception" (3/12; 25%), or "hand motion" (1/12; 8%). The medical records of patients with infectious keratitis were also reviewed (n=24/35; 69%), and final visual acuity was assessed in 18 cases (51%). Similarly, most patients progressed to a final visual acuity state of "no light perception" (6/18; 33%), "light perception" (1/18; 6%), or "hand motion" (6/18; 33%). Overall, the majority of patients progressed to a final visual acuity state of "no light perception" (12/30), "light perception" (4/30), or "hand motion" (7/30)., Conclusions: The distribution of alpha-hemolytic streptococci in ocular infections suggested the presence of a species-specific tissue tropism. The prognoses of patients with ocular streptococcal infections were highly unfavorable, and antibiotic resistance did not contribute to the unfavorable clinical progressions and poor outcomes.

Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of severe and difficult to treat ocular infection. In this study, the population structure of 68 ocular MRSA isolates collected at Massachusetts Eye and Ear between January 2014 and June 2016 was assessed. By using a combination of multilocus sequence typing (MLST) analysis, SCC mec typing and detection of the panton-valentine leukocidin (PVL) gene, we found that the population structure of ocular MRSA is composed of lineages with community and hospital origins. As determined by eBURST analysis of MLST data, the ocular MRSA population consisted of 14 different sequence types (STs) that grouped within two predominant clonal complexes: CC8 (47.0%) and CC5 (41.2%). Most CC8 strains were ST8, harbored type IV SCC mec and were positive for the PVL-toxin (93.7%). The CC5 group was divided between strains carrying SCC mec type II (71.4%) and SCC mec type IV (28.6%). Remaining isolates grouped in 6 different clonal complexes with 3 isolates in CC6 and the other clonal complexes being represented by a single isolate. Interestingly, major MRSA CC5 and CC8 lineages were isolated from discrete ocular niches. Orbital and preseptal abscess/cellulitis were predominantly caused by CC8-SCC mec IV PVL-positive strains. In contrast, infections of the cornea, conjunctiva and lacrimal system were associated with the MDR CC5 lineage, particularly as causes of severe infectious keratitis. This niche specialization of MRSA is consistent with a model where CC8-SCC mec IV PVL-positive strains are better adapted to cause infections of the keratinized and soft adnexal eye tissues, whereas MDR CC5 appear to have greater ability in overcoming innate defense mechanisms of the wet epithelium of the ocular surface., (Copyright © 2020 Bispo, Ung, Chodosh and Gilmore.)

Purpose: To validate a comprehensive clinical algorithm for the assessment and treatment of microbial keratitis (MK)., Design: Retrospective cohort study., Methods: The "1, 2, 3 Rule" for the initial management of MK was conceived by Vital and associates in 2007 to inform the decision as to when to perform corneal cultures. The rule is invoked when any 1 of 3 clinical parameters is met: ≥1+ anterior chamber cells, ≥2 mm infiltrate, or infiltrate ≤3 mm distance from the corneal center. When the rule is met, we added the mandatory use of fortified topical antibiotics after cultures are obtained. We compared outcomes of cases presenting to Massachusetts Eye and Ear 2 years before (Group I, n = 665) and after (Group II, n = 767) algorithm implementation. The primary composite outcome was a vision-threatening complication, such as corneal perforation., Results: At a median follow-up of 67.0 and 60.0 days, respectively, 172 patients experienced a vision-threatening complication (Group I, 12.9% vs Group II, 11.2%, P = .51). While the algorithm codified conventional management practice at either end of disease severity, the effect of algorithm-augmented care was best appreciated in patients with lesions satisfying only 1 criterion. In this group, there was an increase in the proportion of patients undergoing culture at presentation (54.6% vs 67.7%, P = .006), fortified antibiotic prescription (29.7% vs 53.9%, P < .001), and reduction in vision-threatening complications (9.7% vs 1.8%, P = .001). The proportion of patients who were not cultured at presentation but later required culturing decreased (13.4% vs 5.1%, P = .001), as did patients who did not meet any criteria but were nonetheless cultured (23.9% vs 8.5%, P < .001). Multiple logistic regression showed that all algorithm parameters were independently associated with outcome: ≥1+ anterior chamber cells (odds ratio [OR] 1.66, 95% confidence interval 1.09-2.52); ≥2 mm infiltrate (OR 4.74, 2.68-8.40); and ≤3 mm from corneal center (OR 2.82, 1.85-4.31), confirmed with comparison to a bootstrapped sample (n = 10,000)., Conclusions: The implementation of this algorithm reduced vision-threatening complications for patients with lesions satisfying only 1 criterion, arguably the most difficult patients in whom to judge disease severity. Implementation also led to a decrease in patients receiving unnecessary care., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The study of the forces which govern the geographical distributions of life is known as biogeography, a subject which has fascinated zoologists, botanists and ecologists for centuries. Advances in our understanding of community ecology and biogeography-supported by rapid improvements in next generation sequencing technology-have now made it possible to identify and explain where and why life exists as it does, including within the microbial world. In this review, we highlight how a unified model of microbial biogeography, one which incorporates the classic ecological principles of selection, diversification, dispersion and ecological drift, can be used to explain community dynamics in the settings of both health and disease. These concepts operate on a multiplicity of temporal and spatial scales, and together form a powerful lens through which to study microbial population structures even at the finest anatomical resolutions. When applied specifically to curious strains of conjunctivitis-causing, nonencapsulated Streptococcus pneumoniae , we show how this conceptual framework can be used to explain the possible evolutionary and disease-causing mechanisms which allowed these lineages to colonize and invade a separate biogeography. An intimate knowledge of this radical bifurcation in phylogeny, still the only known niche subspecialization for S. pneumoniae to date, is critical to understanding the pathogenesis of ocular surface infections, nature of host-pathogen interactions, and developing strategies to curb disease transmission.

Microbial keratitis is a potentially blinding condition that must be treated emergently to preserve vision. Although long recognized as a significant cause of corneal blindness, our understanding of its true global scale, associated burden of disease, and etiological patterns remains somewhat limited. Current epidemiological data suggest that microbial keratitis may be epidemic in parts of the world-particularly within South, South-East, and East Asia-and may exceed 2 million cases per year worldwide. Etiological patterns vary between economically developed and developing countries, with bacterial predominance in the former and fungal predominance in the latter. The key to effective management lies in timely diagnosis; however, the current gold standard of stain and culture remains time consuming and often yields no clinically useful results. For this reason, there are attempts to develop highly sensitive and accurate molecular diagnostic tools to provide rapid diagnosis, inform treatment decision making, and minimize the threat of antimicrobial resistance. We provide an overview of these key areas and of avenues for further research toward the goal of more effectively addressing the problem of microbial keratitis on both an individual and public health level., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Introduction : Infectious uveitis is a serious inflammatory condition that often causes grave ocular morbidity including permanent vision loss and damage to the structures of the eye. The most common causes of infectious uveitis include herpesviruses and Toxoplasma gondii . Traditionally, these infections have been identified and differentiated based on characteristic clinical examination findings; however, there is often overlap between these presentations and the unique cause of a given patient's infection is not always clear. Therefore, a reliable and fast method for definitively diagnosing infectious uveitis would be helpful and potentially sight-saving. Several groups have recently found experimental success with real-time multiplex polymerase chain reaction (PCR) techniques. Methods : A comprehensive review of the literature was undertaken to further understand the current state of real-time multiplex PCR and its clinical use. Search terms including "real time multiplex PCR", "infectious uveitis", and "uveitis diagnosis" were used. Appropriate English-language articles were included in this review. Results : Publications from four main groups (two from the United States, one from Japan, and one from India) citing success with real-time multiplex PCR were compared and contrasted. All four groups used the same technique to develop a highly sensitive and specific multiplex PCR analysis and found that their tests maintained high sensitivity and specificity during validation testing. These tests confirmed clinical suspicions in the majority of cases of infectious uveitis, but there were also cases of clinical misdiagnosis that were corrected based on molecular pathogen detection. These patients were then initiated on appropriate antimicrobial therapy with subsequent clinical improvement. Discussion : Real-time multiplex PCR is a highly sensitive and specific laboratory assay that allows for rapid and reliable molecular diagnosis of causative agents in infectious uveitis. This in turn facilitates swift initiation of effective therapy and prevents long-term ocular damage and vision loss.

Staphylococcus aureus is an important human pathogen that causes serious antibiotic-resistant infections. Its population structure is marked by the appearance and dissemination of successful lineages across different settings. To begin understanding the population structure of S. aureus causing ocular and otolaryngology infections, we characterized 262 isolates by antimicrobial sensitivity testing and multilocus sequence typing (MLST). Methicillin-resistant S. aureus were subjected to SCCmec typing and Panton-Valentine leukocidin (PVL) screening. Although we detected a high level of genetic diversity among methicillin-sensitive (MSSA) isolates, (63 sequence types-STs), the population was dominated by five lineages: ST30, ST5, ST8, ST15 and ST97. Resistance to penicillin, erythromycin and clindamycin was common among the major MSSA lineages, with fluctuations markedly impacted by genetic background. Isolates belonging to the predominant lineage, ST30, displayed high rates of resistance to penicillin (100%), erythromycin (71%), and clindamycin (63%). Overall, 21% of the isolates were methicillin-resistant (MRSA), with an apparent enrichment among otitis and orbital cellulitis isolates (>40%). MRSA isolates belonged to 14 STs grouped in 5 clonal complexes (CC), however, CC5 (56.1%) and CC8 (38.6%) dominated the population. Most CC5 strains were SCCmec type II, and resembled the hospital-adapted USA100 clone. CC8 strains were SCCmec type IV, and 86% were positive for the PVL toxin, common features of the community-acquired clone USA300. CC5 strains harboring a SCCmec type IV, typical for the USA800 clone, comprised 15.5% of the population. USA100 strains were highly resistant to clindamycin, erythromycin and levofloxacin (100%), while USA300 strains were frequently resistant to erythromycin (89%) but displayed lower rates of resistance to levofloxacin (39%) and clindamycin (17%). Our data demonstrate that the ocular and otolaryngology S. aureus populations are composed of strains that are commonly resistant to clinically relevant antibiotics, and are associated with the major epidemic clonal complexes of both community and hospital origins., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Infectious uveitis is a serious sight-threatening infection commonly caused by herpesviruses and Toxoplasma gondii. Etiologic diagnosis based on the clinical evaluation is often challenging. We developed and validated a multiplex real-time PCR assay coupled with high-resolution melting (HRM) for rapid detection and identification of herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), and T. gondii., Methods: The assay was designed to target pathogen genome regions that yield products with distinct melting temperatures. Analytical specificity, sensitivity, and precision of HRM identification were determined. Clinical validation was performed by testing 108 intraocular fluids collected from eyes suffering with infectious uveitis (n = 30) and controls (n = 78)., Results: A nonoverlapping high-precision profile for each pathogen was generated following HRM (coefficient of variation 0%). The assay was highly sensitive, with a limit of detection of 20 genome copies for herpesviruses and 200 genome copies for T. gondii. The intra- and interassay variability of cycle threshold (Ct) measurement was ≤4% and ≤6%, respectively. Thirteen intraocular specimens collected from suspected cases of infectious uveitis were positive (mean Ct values varied from 19.4 to 27.7). Melting profiles of positive cases were consistent with HSV-2 (n = 5), VZV (n = 5), CMV (n = 2), and T. gondii (n = 1). Amplicon identities were confirmed by sequencing. Control intraocular samples from patients without a clinical diagnosis of infectious uveitis were all negative., Conclusions: This assay allows rapid, sensitive, and reliable detection and identification of the most common known causes of infectious uveitis, making early pathogen information-based intervention possible.

Objective: Otologic methicillin-resistant Staphylococcus aureus (MRSA) infection has historically been rare, but given the rise in community-acquired MRSA carriage and infection at other body sites, prevalence rates may be changing. The goal of this study was to determine the prevalence of MRSA in recent otologic cultures from patients with acute otitis externa (AOE)., Study Design: Retrospective review of an institutional microbiologic database., Methods: A retrospective analysis was performed on serial culture isolates taken from the ear at a quaternary care hospital from January 2014 to April 2016. The causative pathogen and antibiotic sensitivity was determined by culture isolation and end point mean inhibitory concentration (MIC) testing. Medical records were reviewed to document patient characteristics, chronicity of infection, symptomatology, and previous treatments., Results: Over the study period, 173 patients were diagnosed with AOE and underwent otologic cultures of the ear. Fifty-three (30.6%) of cultures grew S . aureus (SA). Of SA infections, 15 (28.3%) were identified as MRSA. MRSA patients were typically older than patients with methicillin-sensitive SA (MSSA) (mean age 46.7 ± 17.9 vs 29 ± 19.4, P  = 0.003) and had more medical comorbidities (4 vs 1.7, P  = 0.001). Compared to patients with MSSA, patients with MRSA were significantly more likely to have had prior ototopical antibiotic exposure (37% vs 73%, P  = 0.019)., Conclusion: Contemporary ear culture isolates at quaternary care center show higher rates of MRSA compared to historical reports in the literature. Clinicians should consider ear cultures to identify MRSA AOE., Level of Evidence: IV.

Objective:: Bacterial keratitis occurs worldwide, and despite recent developments, it remains a potentially blinding condition. This study assesses the presence of herpes simplex virus (HSV-1 and -2) and varicella zoster virus (VZV) by quantitative real-time polymerase chain reaction (qPCR) in corneal scrapings from patients with bacterial keratitis., Methods:: A total of 65 patients with clinical diagnoses of infectious corneal ulcers prospectively underwent clinical eye examinations. Corneal scrapings were investigated by Gram staining, Giemsa staining, culture, and qPCR (the study group). Risk factors and epidemiological data were recorded. The control group comprising 25 eyes with typical herpes dendritic keratitis was also analyzed by qPCR., Results:: From the study group (n=65), nine patients (13.8%) had negative smears, cultures, and qPCR findings. Fifty-six (86.2%) patients had positive cultures: 51 for bacteria, 4 for fungi, and 1 for amoebae. Of the patients who had positive bacterial cultures, qPCR identified 10 patients who were also positive for virus: one for VZV and nine for HSV-1. Of the 25 patients in the control group, 21 tested positive for HSV-1 by qPCR analysis., Conclusions:: Herpes may be present in patients with bacterial corneal ulcers, and qPCR may be useful in its detection.