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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.
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Anti-Infecciosos , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Humanos , Antibacterianos/farmacologia , Staphylococcus aureus , Azitromicina , Farmacorresistência Bacteriana/genética , Macrolídeos , Infecções Estafilocócicas/microbiologia , Staphylococcus/genética , Fluoroquinolonas , Streptococcus , Testes de Sensibilidade MicrobianaRESUMO
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.
Candida species are the most common pathogen responsible for ocular infections in the temperature regions of the world. Here, we sequenced and molecularly characterized the Candida species seen in patients who present to our hospital with infection to understand the species' distribution over time.
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Endoftalmite , Infecções Oculares Fúngicas , Ceratite , Animais , Antifúngicos/farmacologia , Leveduras , Infecções Oculares Fúngicas/epidemiologia , Infecções Oculares Fúngicas/microbiologia , Infecções Oculares Fúngicas/veterinária , Ceratite/epidemiologia , Ceratite/microbiologia , Ceratite/veterinária , Candida albicans , Endoftalmite/epidemiologia , Endoftalmite/microbiologia , Endoftalmite/veterinária , Candida tropicalis , Candida parapsilosis , Testes de Sensibilidade Microbiana/veterináriaRESUMO
Staphylococcus epidermidis is an abundant member of the microbiota of the human skin and wet mucosa, which is commonly associated with sight-threatening infections in eyes with predisposing factors. Ocular S. epidermidis has become notorious because of its capability to form biofilms on different ocular devices and due to the evolving rates of antimicrobial resistance. In this study, the molecular epidemiology of 30 ocular methicillin-resistant S. epidermidis (MRSE) isolates was assessed using multilocus sequence typing (MLST). Antimicrobial resistance, accessory gene-regulator and staphylococcal cassette chromosome mec (SCCmec) types, biofilm formation, and the occurrence of biofilm-associated genes were correlated with MLST clonal complexes. Sequence types (STs) frequently found in the hospital setting were rarely found in our collection. Overall, 12 different STs were detected with a predominance of ST59 (30%), ST5 and ST6 (13.3% each). Most of the isolates (93.3%) belonged to the clonal complex 2 (CC2) and grouped mainly within subcluster CC2-II (92.9%). Isolates grouped within this subcluster were frequently biofilm producers (92.3%) with a higher occurrence of the aap (84.5%) and bhp (46.1%) genes compared to icaA (19.2%). SCCmec type IV (53.8%) was predominant within CC2-II strains, while 38.4% were nontypeable. In addition, CC2-II strains were frequently multidrug resistant (80.7%) and demonstrated to be particularly resistant to ciprofloxacin (80.8%), ofloxacin (77%), azithromycin (61.5%), and gentamicin (57.7%). Our findings demonstrate the predominance of a particular MRSE cluster causing ocular infections, which was associated with high rates of antimicrobial resistance and particularly the carriage of biofilm-related genes coding for proteinaceous factors implicated in biofilm accumulation.
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Infecções Oculares/microbiologia , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/isolamento & purificação , Anti-Infecciosos/farmacologia , Biofilmes , Farmacorresistência Bacteriana Múltipla/genética , Infecções Oculares/epidemiologia , Genes Bacterianos/genética , Hospitais , Humanos , Resistência a Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Epidemiologia Molecular/métodos , Tipagem de Sequências Multilocus/métodos , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/efeitos dos fármacosRESUMO
Fluoroquinolones remain the most commonly used antimicrobials for the prevention and management of bacterial endophthalmitis. Coagulase-negative staphylococci are the most frequently recovered pathogens. Increasing resistance among this group has paralleled the presence of methicillin resistance. From 2005 to 2010, we recovered 38 methicillin-susceptible Staphylococcus epidermidis (MSSE) isolates from endophthalmitis patients at our institute, including 15 (39.5%) isolates resistant to gatifloxacin and moxifloxacin, members of the C-8-methoxyfluoroquinolones family. Mutations in the quinolone resistance-determining regions (QRDR) of gyrA and parC were determined and correlated with fluoroquinolone MICs based on Etests of these 15 MSSE isolates. High-level resistance (MIC, >32 µg/ml) to gatifloxacin and moxifloxacin was documented for 46.7% of the MSSE isolates, and low-level resistance (MIC, 2 to 4 µg/ml) was determined for 53.3%. The MICs for ciprofloxacin, levofloxacin, and ofloxacin were >32 µg/ml for all isolates. The amino acid substitution Ser84Phe in gyrA was found among all isolates. A second mutation in gyrA (Glu88Lys) resulted in high-level resistance to moxifloxacin and gatifloxacin. Almost all (92.8%) isolates presented double point mutations in the parC gene at codons 80 and 84 with different combinations. Eighty-seven percent of the patients had prior exposure to topical 8-methoxyfluoroquinolones. Prior exposure to the 8-methoxyfluoroquinolones may contribute to the selection of MSSE strains containing multiple mutations in the QRDRs of gyrA and parC that results in low- and high-level resistance to these agents.
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Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Endoftalmite/microbiologia , Fluoroquinolonas/farmacologia , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/isolamento & purificação , Idoso , Idoso de 80 Anos ou mais , DNA Girase/genética , DNA Topoisomerase IV/genética , Feminino , Genótipo , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Staphylococcus epidermidis/genéticaRESUMO
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.
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Epitélio Corneano , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Humanos , Vancomicina/toxicidade , Vancomicina/uso terapêutico , Lipoglicopeptídeos/toxicidade , Lipoglicopeptídeos/uso terapêutico , Staphylococcus , Antibacterianos/toxicidade , Antibacterianos/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Testes de Sensibilidade MicrobianaRESUMO
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.
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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.
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Endoftalmite , Infecções Oculares , Humanos , Sensibilidade e Especificidade , Endoftalmite/diagnóstico , Bactérias/genética , Reação em Cadeia da PolimeraseRESUMO
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.
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We present a rare case of recalcitrant fungal keratitis caused by Coniochaeta mutabilis, successfully managed with a course of oral, topical, intrastromal, and intracameral antifungals. A 57-year-old male on their fourth week of treatment for presumed left herpes simplex keratitis presented to clinic with severe left-sided foreign body sensation after gardening in his yard. On examination, a white corneal plaque was observed at 8 o'clock, shown to be a dense collection of fungal hyphae on confocal microscopy. Corneal cultures revealed yeast-like cells, initially identified as Kabatiella zeae by matching 100% identity with K. zeae strains CBS 767.71 and CBS 265.32 in BLASTn search using the internal transcribed spacer (ITS) sequence. Treated for over four months with topical amphotericin B and oral voriconazole without improvement, recourse to intrastromal and intracameral amphotericin B injections, coupled with the application of cyanoacrylate glue to the lesion and a bandage contact lens, led to eventual resolution. The patient subsequently underwent cataract surgery, achieving a BCVA of 20/20 in the eye. Surprisingly, upon further sequence analyses of combined ITS and large subunit ribosomal ribonucleic acid (LSU) and investigation of the K. zeae German strain CBS 767.71, the organism was revealed to be Coniochaeta mutabilis (formerly Lecythospora mutabilis). This means that the correct name for CBS 767.71 and CBS 265.32 is C. mutabilis and should be corrected in the GenBank record to avoid misleading identification in the future. This case also underscores the urgent unmet need for improved molecular diagnostic modalities in the care of corneal infections.
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Úlcera da Córnea , Infecções Oculares Fúngicas , Ceratite , Masculino , Humanos , Pessoa de Meia-Idade , Anfotericina B/uso terapêutico , Antifúngicos/uso terapêutico , Voriconazol/uso terapêutico , Ceratite/diagnóstico , Ceratite/tratamento farmacológico , Ceratite/microbiologia , Infecções Oculares Fúngicas/diagnóstico , Infecções Oculares Fúngicas/tratamento farmacológico , Infecções Oculares Fúngicas/microbiologiaRESUMO
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-1ß. Cultured cells were incubated with siRNA to knock down the different TLRs. After stimulation with toxigenic S. aureus RN6390, pro-IL-1ß synthesis, caspase-1 activity, and mature IL-1ß 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-1ß. 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 Ca2+. We conclude that α toxin by these two different mechanisms triggers the synthesis of pro-IL-1ß and NLRP3 components, activation of capase-1, and secretion of mature IL-1ß to defend against bacterial infection.
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Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Inflamassomos/metabolismo , Receptor 2 Toll-Like/metabolismo , Staphylococcus aureus/metabolismo , Células Caliciformes , Receptor 1 Toll-Like , Caspase 1/metabolismo , Interleucina-1beta/metabolismoRESUMO
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.
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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.
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Otite Média com Derrame , Humanos , Otite Média com Derrame/diagnóstico , Ventilação da Orelha Média/métodos , Orelha Média/patologia , Próteses e Implantes , AntibacterianosRESUMO
Polymyxins have been the only alternative therapeutic option for the treatment of serious infections caused by multidrug-resistant Acinetobacter baumannii or Pseudomonas aeruginosa isolates. For this reason, it is of crucial importance that susceptibility tests provide accurate results when testing these drug-pathogen combinations. In this study, the effect of cation concentration variability found on different commercial brands of Mueller-Hinton agar (MHA) for testing polymyxin B susceptibility was evaluated. The polymyxin B susceptibilities determined using Etest and disk diffusion were compared to those determined by the CLSI reference broth microdilution method. In general, the polymyxin B MIC values were higher when determined by Etest than when determined by broth microdilution against both A. baumannii and P. aeruginosa isolates. A high very major error rate (10%) was observed, as well as a trend toward lower MICs, compared to those determined by broth microdilution when the Merck MHA was tested by Etest. Poor essential agreement rates (10 to 70%) were observed for P. aeruginosa when all MHA brands were tested by Etest. Although an excellent categorical agreement rate (100%) was seen between the disk diffusion and broth microdilution methods for P. aeruginosa, larger zones of inhibition were shown obtained using the Merck MHA. The high cation concentration variability found for the MHA brands tested correlated to the low accuracy, and discrepancies in the polymyxin B MICs were determined by Etest method, particularly for P. aeruginosa isolates.
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Antibacterianos/farmacologia , Cátions/análise , Meios de Cultura/química , Polimixina B/farmacologia , Acinetobacter baumannii/efeitos dos fármacos , Ágar , Erros de Diagnóstico/estatística & dados numéricos , Testes de Sensibilidade Microbiana/métodos , Pseudomonas aeruginosa/efeitos dos fármacosRESUMO
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.
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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 105 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.
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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 (60â%), and ST448 (53.3â%) 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 95.5â% of the isolates were non-PCV-13 vaccine types. Most of the conjunctivitis isolates (80â%) were unencapsulated, with the remaining belonging to serotypes 15B, 3 and 23B. On the other hand, S. pneumoniae causing keratitis were predominantly encapsulated (95.2â%) with 13 different serotypes identified, mostly being non-vaccine types. Carriage of macrolide resistance genes was common in our ocular S. pneumoniae population (42.2â%), 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 tetM 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.
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Conjuntivite , Infecções Oculares , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Humanos , Macrolídeos , Vacinas Pneumocócicas , Streptococcus pneumoniae/genética , Vacinas ConjugadasRESUMO
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.
Assuntos
Úlcera da Córnea/diagnóstico , Infecções por Corynebacterium/diagnóstico , Infecções Oculares Bacterianas/diagnóstico , Síndrome de Stevens-Johnson/diagnóstico , Adulto , Idoso , Antibacterianos/uso terapêutico , Doença Crônica , Úlcera da Córnea/tratamento farmacológico , Úlcera da Córnea/microbiologia , Corynebacterium/isolamento & purificação , Infecções por Corynebacterium/tratamento farmacológico , Infecções por Corynebacterium/microbiologia , Combinação de Medicamentos , Infecções Oculares Bacterianas/tratamento farmacológico , Infecções Oculares Bacterianas/microbiologia , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Polimixinas/uso terapêutico , Síndrome de Stevens-Johnson/tratamento farmacológico , Síndrome de Stevens-Johnson/microbiologia , Trimetoprima/uso terapêutico , Vancomicina/uso terapêuticoRESUMO
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.
Assuntos
Achromobacter/isolamento & purificação , Contaminação de Medicamentos , Limbo da Córnea/microbiologia , Soluções Oftálmicas , Soluções para Preservação de Órgãos , Achromobacter/genética , Técnicas Bacteriológicas , DNA Ribossômico/genética , Bancos de Olhos/métodos , Humanos , Ceratoplastia Penetrante , Testes de Sensibilidade Microbiana , RNA Ribossômico 16S/genética , Doadores de Tecidos , Coleta de Tecidos e ÓrgãosRESUMO
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, SCCmec 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 SCCmec and were positive for the PVL-toxin (93.7%). The CC5 group was divided between strains carrying SCCmec type II (71.4%) and SCCmec 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-SCCmec 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-SCCmec 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.
Assuntos
Infecção Hospitalar/microbiologia , Ceratite , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas/microbiologia , Adulto , Idoso , Feminino , Hospitais , Humanos , Ceratite/epidemiologia , Ceratite/microbiologia , Masculino , Massachusetts , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/genética , Pessoa de Meia-Idade , Tipagem de Sequências MultilocusRESUMO
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.