RESUMO
The unprecedented precision and resolution of whole genome sequencing (WGS) can provide definitive identification of infectious agents for epidemiological outbreak tracking. WGS approaches, however, are frequently impeded by low pathogen DNA recovery from available primary specimens or unculturable samples. A cost-effective hybrid capture assay for Legionella pneumophila WGS analysis directly on primary specimens was developed. DNA from a diverse range of sputum and autopsy specimens PCR-positive for L. pneumophila serogroup 1 (LPSG1) was enriched with this method, and WGS was performed. All tested specimens were determined to be enriched for Legionella reads (up to 209,000-fold), significantly improving the discriminatory power to compare relatedness when no clinical isolate was available. We found the WGS data from some enriched specimens to differ by less than five single-nucleotide polymorphisms (SNPs) when compared to the WGS data of a matched culture isolate. This testing and analysis retrospectively provided previously unconfirmed links to environmental sources for clinical specimens of sputum and autopsy lung tissue. The latter provided the additional information needed to identify the source of these culture-negative cases associated with the South Bronx 2015 Legionnaires' disease (LD) investigation in New York City. This new method provides a proof of concept for future direct clinical specimen hybrid capture enrichment combined with WGS and bioinformatic analysis during outbreak investigations.IMPORTANCELegionnaires' disease (LD) is a severe and potentially fatal type of pneumonia primarily caused by inhalation of Legionella-contaminated aerosols from man-made water or cooling systems. LD remains extremely underdiagnosed as it is an uncommon form of pneumonia and relies on clinicians including it in the differential and requesting specialized testing. Additionally, it is challenging to obtain clinical lower respiratory specimens from cases with LD, and when available, culture requires specialized media and growth conditions, which are not available in all microbiology laboratories. In the current study, a method for Legionella pneumophila using hybrid capture by RNA baiting was developed, which allowed us to generate sufficient genome resolution from L. pneumophila serogroup 1 PCR-positive clinical specimens. This new approach offers an additional tool for surveillance of future LD outbreaks where isolation of Legionella is not possible and may help solve previously unanswered questions from past LD investigations.
Assuntos
Legionella pneumophila , Legionella , Doença dos Legionários , Pneumonia , Humanos , Doença dos Legionários/diagnóstico , Estudos Retrospectivos , Legionella pneumophila/genética , Sequenciamento Completo do Genoma , Surtos de Doenças , DNARESUMO
Legionnaires' disease, a severe lung infection caused by the bacterium Legionella pneumophila, occurs as single cases or in outbreaks that are actively tracked by public health departments. To determine the point source of an outbreak, clinical isolates need to be compared to environmental samples to find matching isolates. One confounding factor is the genome plasticity of L. pneumophila, making an exact sequence comparison by whole-genome sequencing (WGS) challenging. Here, we present a WGS analysis pipeline, LegioCluster, that is designed to circumvent this problem by automatically selecting the best matching reference genome prior to mapping and variant calling. This approach reduces the number of false-positive variant calls, maximizes the fraction of all genomes that are being compared, and naturally clusters the isolates according to their reference strain. Isolates that are too distant from any genome in the database are added to the list of candidate references, thereby creating a new cluster. Short insertions or deletions are considered in addition to single-nucleotide polymorphisms for increased discriminatory power. This manuscript describes the use of this automated and "locked down" bioinformatic pipeline deployed at the New York State Department of Health's Wadsworth Center for investigating relatedness between clinical and environmental isolates. A similar pipeline has not been widely available for use to support these critically important public health investigations.
Assuntos
Legionella pneumophila , Doença dos Legionários , Análise por Conglomerados , Biologia Computacional , Surtos de Doenças , Humanos , Legionella pneumophila/genética , Doença dos Legionários/epidemiologia , New YorkRESUMO
We describe a rare instance of donor-derived OXA-23-producing carbapenem-resistant Acinetobacter baumannii transmission during lung transplantation and the subsequent public health response. This investigation highlights how transplantation can introduce rare multidrug-resistant organisms into different healthcare facilities and regions.
Assuntos
Infecções por Acinetobacter/diagnóstico , Infecções por Acinetobacter/transmissão , Surtos de Doenças , Transplante de Pulmão/efeitos adversos , Doadores de Tecidos , Resistência beta-Lactâmica , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/enzimologia , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana Múltipla , Genótipo , Instalações de Saúde , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Fenótipo , Escarro/microbiologia , beta-LactamasesRESUMO
OBJECTIVE: To investigate the source and transmission dynamics of an endoscope-associated New Delhi metallo-ß-lactamase-producing Klebsiella pneumonia (NDM-KP) outbreak. DESIGN: Epidemiological and genomic investigation. SETTING: Academic acute care hospital in New Jersey. PATIENTS: Five patients with active NDM-KP infection identified on clinical isolates, and four NDM-KP colonized patients identified via rectal swab screening. RESULTS: Over a twelve-month period, nine patients were identified with NDM-KP infection or colonization. Whole-genome sequencing (WGS) revealed that all of the identified cases were related by 25 mutational events or less. Seven of the cases were linked to gastrointestinal endoscopic procedures (four clinical cases and three positive screens among patients exposed to endoscopes suspected of transmission). Two cases demonstrated delayed transmission that occurred five months after the initial outbreak, likely through shared usage of a non-therapeutic gastroscope without an elevator channel. CONCLUSIONS: Although all endoscope cultures in our investigation were negative, the epidemiological link to gastrointestinal endoscopes, the high degree of relatedness via WGS, and the identification of asymptomatic NDM-KP colonization among patients exposed to shared endoscopes make the endoscopic mode of transmission most likely. This investigation highlights the probable transmission of NDM-KP via a gastroscope without an elevator channel, observed several months after an initial outbreak. We hypothesize that persistent mechanical defects may have contributed to the delayed device-related transmission of NDM-KP.
RESUMO
Vancomycin-resistant Staphylococcus aureus (VRSA) is a human pathogen of significant public health concern. Although the genome sequences of individual VRSA isolates have been published over the years, very little is known about the genetic changes of VRSA within a patient over time. A total of 11 VRSA, 3 vancomycin-resistant enterococci (VRE), and 4 methicillin-resistant S. aureus (MRSA) isolates, collected over a period of 4.5 months in 2004 from a patient in a long-term-care facility in New York State, were sequenced. A combination of long- and short-read sequencing technologies was used to obtain closed assemblies for chromosomes and plasmids. Our results indicate that a VRSA isolate emerged as the result of the transfer of a multidrug resistance plasmid from a coinfecting VRE to an MRSA isolate. The plasmid then integrated into the chromosome via homologous recombination mediated between two regions derived from remnants of transposon Tn5405. Once integrated, the plasmid underwent further reorganization in one isolate, while two others lost the staphylococcal cassette chromosome mec element (SCCmec) determinant that confers methicillin-resistance. The results presented here explain how a few recombination events can lead to multiple pulsed-field gel electrophoresis (PFGE) patterns that could be mistaken for vastly different strains. A vanA gene cluster that is located on a multidrug resistance plasmid that is integrated into the chromosome could result in the continuous propagation of resistance, even in the absence of selective pressure from antibiotics. The genome comparison presented here sheds light on the emergence and evolution of VRSA within a single patient that will enhance our understanding VRSA genetics. IMPORTANCE High-level vancomycin-resistant Staphylococcus aureus (VRSA) began to emerge in the United States in 2002 and has since then been reported worldwide. Our study reports the closed genome sequences of multiple VRSA isolates obtained in 2004 from a single patient in New York State. Our results show that the vanA resistance locus is located on a mosaic plasmid that confers resistance to multiple antibiotics. In some isolates, this plasmid integrated into the chromosome via homologous recombination between two ant(6)-sat4-aph(3') antibiotic resistance loci. This is, to our knowledge, the first report of a chromosomal vanA locus in VRSA; the effect of this integration event on MIC values and plasmid stability in the absence of antibiotic selection remains poorly understood. These findings highlight the need for a better understanding of the genetics of the vanA locus and plasmid maintenance in S. aureus to address the increase of vancomycin resistance in the health care setting.
RESUMO
In 2017, the New York State Department of Health investigated a large Klebsiella pneumoniae outbreak in a health care facility. A retrospective analysis was conducted to compare the use of multiple molecular typing methods for characterizing the outbreak. Forty-four isolates were characterized using the rapid real-time PCR OpGen Acuitas® AMR Gene Panel. Additionally, short-read whole genome sequencing (WGS) analysis was used to identify antimicrobial resistance (AMR) genes and assess isolate relatedness. Long-read Oxford Nanopore MinION WGS was used to characterize the plasmid content of a subset of isolates. All methods showed overall concordance, identifying four clusters, with a few discrepancies in the clustering of individual isolates. Though short- and long-read WGS results provided a more nuanced understanding of the molecular epidemiology of this outbreak, this study highlights the utility of the Acuitas® PCR-based approach, which can more easily be performed by health care facilities, for rapid clustering of patient isolates.
Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Antibacterianos , Proteínas de Bactérias/genética , Surtos de Doenças , Humanos , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae/genética , New York/epidemiologia , Plasmídeos , Reação em Cadeia da Polimerase , Estudos Retrospectivos , Sequenciamento Completo do Genoma/métodos , beta-Lactamases/genéticaRESUMO
OBJECTIVES: To compare the bactericidal activity of besifloxacin, moxifloxacin and gatifloxacin and determine the contribution of the preservative benzalkonium chloride (BAK) to bactericidal activity. METHODS: Time-kill experiments were performed against four species (n=12) with besifloxacin, moxifloxacin and gatifloxacin, in the presence or absence of BAK, at t=0, 5, 15, 30, 45, 60, 120 and 360 min, according to standard CLSI methods. RESULTS: In the presence of BAK, bactericidal activity was observed within 5 min, regardless of the fluoroquinolone tested. The bactericidal activity of BAK was unaffected by the concurrent presence of besifloxacin and rapid killing (within 5 to 15 min) was not observed at BAK concentrations below 50 mg/L. However, when tested without BAK, besifloxacin was bactericidal in as little as 45 min, while moxifloxacin and gatifloxacin required at least 120 min; besifloxacin kill rates against fluoroquinolone-susceptible and -resistant strains were at least 2- to 4-fold faster than those of gatifloxacin or moxifloxacin. CONCLUSIONS: Besifloxacin was the most rapidly bactericidal fluoroquinolone tested, followed by gatifloxacin and moxifloxacin, both of which had similar activity. Our studies demonstrate that the previously reported rapid in vitro killing by gatifloxacin formulations was probably due to the concurrent presence of 50 mg/L BAK, which is much higher than the 3.2 mg/L BAK observed in human tears 1 min after instillation of ophthalmic gatifloxacin solutions [Friedlaender MH, Breshears D, Amoozgar B et al. The dilution of benzalkonium chloride (BAK) in the tear film. Adv Ther 2006; 23: 835-41].
Assuntos
Antibacterianos/farmacologia , Compostos Aza/farmacologia , Azepinas/farmacologia , Bactérias/efeitos dos fármacos , Fluoroquinolonas/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Quinolinas/farmacologia , Compostos de Benzalcônio/farmacologia , Interações Medicamentosas , Gatifloxacina , Moxifloxacina , Fatores de TempoRESUMO
BACKGROUND: The impact of mutations in DNA gyrase and topoisomerase IV on minimum inhibitory concentrations (MICs) was investigated to better understand why besifloxacin has a higher potency against Staphylococcus aureus when compared to other fluoroquinolones, which was especially pronounced against ciprofloxacin-resistant isolates. METHODS: MICs were determined for 52 clinical isolates against besifloxacin, moxifloxacin, gatifloxacin, ciprofloxacin, and levofloxacin. The genes encoding GyrA, GyrB, ParC, and ParE were sequenced and the potential impact of mutations assessed in light of recent structural data. RESULTS: For all fluoroquinolones tested, the MICs increased with the number of mutations in the quinolone resistance-determining regions. However, this increase was the smallest for besifloxacin and the largest for ciprofloxacin and levofloxacin. In addition to the commonly observed mutations in ParC and GyrA, more unusual mutations in ParE, such as Asp-432âHis or Pro-585âSer, were also detected. CONCLUSIONS: Compared to earlier fluoroquinolones, the higher potency of besifloxacin suggests that the drug's unique combination of a 7-azepinyl ring and an 8-chloro-substituent results in unique interactions with DNA gyrase and topoisomerase IV.
Assuntos
Azepinas/farmacologia , DNA Girase/genética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana Múltipla/genética , Fluoroquinolonas/farmacologia , Mutação , Staphylococcus aureus/genética , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacosRESUMO
OBJECTIVES: Besifloxacin is a novel fluoroquinolone that was recently approved for topical treatment of bacterial conjunctivitis. The compound was shown to be active in vitro against a broad spectrum of bacteria, including isolates resistant to other antibacterials. Here, the bactericidal activity of besifloxacin was evaluated against the most common bacterial conjunctivitis pathogens. METHODS: MIC, MBC and time-kill experiments with besifloxacin and comparators were performed according to CLSI guidelines. Quinolone resistance-determining regions (QRDRs) were sequenced using standard PCR-based techniques. RESULTS: MIC and MBC data indicated that besifloxacin was the most potent fluoroquinolone tested against Staphylococcus aureus (n = 30), Staphylococcus epidermidis (n = 15) and Streptococcus pneumoniae (n = 35), while all fluoroquinolones were highly active against Haemophilus influenzae (n = 40). Besifloxacin MBC:MIC ratios were < or = 4 for 97.5% of all isolates tested (n = 120). All fluoroquinolones tested, as well as tobramycin, were bactericidal, while azithromycin was bactericidal against S. pneumoniae and H. influenzae, but bacteriostatic against the staphylococci. Time-kill assays with all four species showed that besifloxacin caused > or = 1000-fold killing within 2 h for 11 of 12 isolates. Only one isolate treated with moxifloxacin and three ciprofloxacin-treated isolates achieved the same level of bactericidal activity under the same conditions. Unlike the comparator fluoroquinolones, besifloxacin maintained a high potency and bactericidal activity even against strains that contained multiple mutations in the genes encoding DNA gyrase and topoisomerase IV. CONCLUSIONS: Overall, besifloxacin demonstrated rapid bactericidal activity against the four major human pathogens tested here, including isolates that showed in vitro resistance to other fluoroquinolones, beta-lactams, macrolides or aminoglycosides.
Assuntos
Antibacterianos/farmacologia , Azepinas/farmacologia , Fluoroquinolonas/farmacologia , Haemophilus influenzae/efeitos dos fármacos , Staphylococcus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Aminoglicosídeos/farmacologia , Conjuntivite/microbiologia , DNA Bacteriano/química , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Genes Bacterianos , Humanos , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Análise de Sequência de DNA , Fatores de Tempo , beta-Lactamas/farmacologiaRESUMO
Carbapenemase-producing Enterobacteriaceae are a major threat to global public health. Klebsiella pneumoniae carbapenemase (KPC) is the most commonly identified carbapenemase in the United States and is frequently found on mobile genetic elements including plasmids, which can be horizontally transmitted between bacteria of the same or different species. Here we describe the results of an epidemiological investigation of KPC-producing bacteria at two healthcare facilities. Using a combination of short-read and long-read whole-genome sequencing, we identified an identical 44 kilobase plasmid carrying the bla KPC-2 gene in four bacterial isolates belonging to three different species (Citrobacter freundii, Klebsiella pneumoniae, and Escherichia coli). The isolates in this investigation were collected from patients who were epidemiologically linked in a region in which KPC was uncommon, suggesting that the antibiotic resistance plasmid was transmitted between these bacterial species. This investigation highlights the importance of long-read sequencing in investigating the relatedness of bacterial plasmids, and in elucidating potential plasmid-mediated outbreaks caused by antibiotic resistant bacteria.
RESUMO
The antibacterial spectrum of besifloxacin, a novel fluoroquinolone recently approved for treatment of ocular infections, was studied using 2,690 clinical isolates representing 40 species. Overall, besifloxacin was the most potent agent tested against gram-positive pathogens and anaerobes and was generally equivalent to comparator fluoroquinolones in activity against most gram-negative pathogens. Besifloxacin demonstrated potent, broad-spectrum activity, which was particularly notable against gram-positive and gram-negative isolates that were resistant to other fluoroquinolones and classes of antibacterial agents.
Assuntos
Antibacterianos/farmacologia , Azepinas/farmacologia , Bactérias Anaeróbias/efeitos dos fármacos , Fluoroquinolonas/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Testes de Sensibilidade MicrobianaRESUMO
OBJECTIVE: To compare the clinical and antimicrobial efficacy of besifloxacin ophthalmic suspension 0.6% with that of moxifloxacin ophthalmic solution 0.5% for the treatment of bacterial conjunctivitis. DESIGN: Multicenter, randomized, double-masked, parallel-group, active-controlled, noninferiority study. PARTICIPANTS: Patients 1 year of age or older with clinical manifestations of bacterial conjunctivitis. METHODS: Eligible patients were randomized to either besifloxacin suspension or moxifloxacin solution, instilled in the infected eye(s) 3 times daily for 5 days, and participated in study visits on days 1, 5 (+/-1 day), and 8 (+1 day). Assessments included clinical evaluation of signs and symptoms, visual acuity, biomicroscopy, and culture of the infected eye(s) at each visit, as well as direct ophthalmoscopy on days 1 and 8. MAIN OUTCOME MEASURES: The primary efficacy end points were clinical resolution and microbial eradication of baseline bacterial infection on day 5 in patients with culture-confirmed bacterial conjunctivitis. Secondary end points included clinical resolution and microbial eradication on day 8, individual clinical outcomes, microbial and clinical outcomes by bacterial species, and safety. RESULTS: A total of 1161 patients (533 with culture-confirmed bacterial conjunctivitis) were randomized. Based on the 95% confidence interval (CI) of the difference, besifloxacin was noninferior to moxifloxacin for clinical resolution on day 5 (58.3% vs. 59.4%, respectively; 95% CI, -9.48 to 7.29) and day 8 (84.5% vs. 84.0%, respectively, 95% CI, -5.6% to 6.75%) and for microbial eradication on day 5 (93.3% vs. 91.1%, respectively, 95% CI, -2.44 to 6.74) and day 8 (87.3% vs. 84.7%; 95% CI, -3.32 to 8.53). There was no statistically significant difference between the 2 treatment groups for either efficacy end points on days 5 or 8 (P>0.05). Besifloxacin and moxifloxacin were well tolerated. The cumulative frequency of ocular adverse events was similar between treatments (12% and 14% with besifloxacin and moxifloxacin, respectively). However, eye irritation occurred more often in moxifloxacin-treated eyes (0.3% for besifloxacin vs. 1.4% for moxifloxacin; P = 0.0201). CONCLUSIONS: Besifloxacin ophthalmic suspension was non inferior to moxifloxacin ophthalmic suspension and provided similar safety and efficacy (clinical and microbiological) outcomes when used for the treatment of bacterial conjunctivitis. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.
Assuntos
Antibacterianos/administração & dosagem , Compostos Aza/administração & dosagem , Azepinas/administração & dosagem , Conjuntivite Bacteriana/tratamento farmacológico , Fluoroquinolonas/administração & dosagem , Soluções Oftálmicas/administração & dosagem , Quinolinas/administração & dosagem , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/efeitos adversos , Compostos Aza/efeitos adversos , Azepinas/efeitos adversos , Criança , Pré-Escolar , Túnica Conjuntiva/microbiologia , Conjuntivite Bacteriana/microbiologia , Método Duplo-Cego , Feminino , Fluoroquinolonas/efeitos adversos , Haemophilus influenzae/isolamento & purificação , Humanos , Lactente , Masculino , Microscopia Acústica , Pessoa de Meia-Idade , Moxifloxacina , Soluções Oftálmicas/efeitos adversos , Quinolinas/efeitos adversos , Staphylococcus aureus/isolamento & purificação , Staphylococcus epidermidis/isolamento & purificação , Streptococcus pneumoniae/isolamento & purificação , Resultado do Tratamento , Acuidade VisualRESUMO
The Boston Keratoprosthesis (B-KPro) is a widely accepted modality of corneal restoration in eyes where traditional penetrating keratoplasty has little chance of success. It is the most commonly used keratoprosthesis worldwide. While the introduction of broad-spectrum antibiotic prophylaxis has virtually eliminated cases of bacterial endophthalmitis, fungal colonization and infections are a growing concern. This review of the literature summarizes risk factors for fungal infections in KPro eyes, rates of fungal infection and colonization, clinical presentation, causative organisms, management, and outcomes. We also focus on current recommendations for antifungal prophylaxis, and highlight the role of translational research at the Massachusetts Eye and Ear Infirmary (MEEI, Boston, USA) with its aim of developing novel strategies for reducing rates of fungal infections in KPro patients.
Assuntos
Bioprótese/microbiologia , Úlcera da Córnea/microbiologia , Endoftalmite/microbiologia , Infecções Oculares Fúngicas/microbiologia , Micoses/microbiologia , Infecções Relacionadas à Prótese/microbiologia , Antifúngicos/uso terapêutico , Úlcera da Córnea/prevenção & controle , Endoftalmite/prevenção & controle , Infecções Oculares Fúngicas/prevenção & controle , Fungos/isolamento & purificação , Humanos , Micoses/prevenção & controle , Infecções Relacionadas à Prótese/prevenção & controle , Fatores de RiscoRESUMO
PURPOSE: To review the current literature describing cases of fungal keratitis and endophthalmitis after Boston keratoprosthesis (KPro) implantation and to characterize the antifungal activity of 0.01% hypochlorous acid against medically relevant fungi. METHODS: A literature review of fungal keratitis or endophthalmitis in KPro patients from January 2001 to April 2015, and an in vitro time kill assay characterizing the fungicidal activity of 0.01% hypochlorous acid against fungi causing ocular infections. RESULTS: Fifteen publications, predominantly retrospective case series, were identified. Infection rates after KPro implantation ranged from 0.009 to 0.02 fungal infections per patient-year of follow-up. The largest single-surgeon series reported an incidence of 2.4% for fungal endophthalmitis during a 10-year period. Causative organisms included both yeasts and molds. Outcomes were favorable if infections were caught early and treated appropriately; less favorable outcomes were reported in developing countries where fungal species are endemic and resources are limited. 0.01% hypochlorous acid is rapidly fungicidal, reducing the number of viable yeast cells or mold conidia by at least 99.99% within 60 seconds. The antifungal activity extended to all molds (Acremonium kiliense, Aspergillus flavus, Aspergillus fumigatus, Fusarium solani, and Mucor indicus) and yeast species (Candida albicans and Candida parapsilosis) tested. CONCLUSIONS: Fungal infections remain a lifelong concern in patients after KPro implantation. There is a growing need for a standard antifungal prophylaxis regimen, especially in the developing world. The rapid broad-spectrum in vitro fungicidal activity of 0.01% hypochlorous acid against all fungi tested makes it an attractive candidate as an antifungal prophylaxis in KPro patients.
Assuntos
Antifúngicos/farmacologia , Bioprótese , Úlcera da Córnea/tratamento farmacológico , Endoftalmite/tratamento farmacológico , Infecções Oculares Fúngicas/tratamento farmacológico , Ácido Hipocloroso/farmacologia , Implantação de Prótese , Doenças da Córnea/cirurgia , Úlcera da Córnea/microbiologia , Endoftalmite/microbiologia , Infecções Oculares Fúngicas/microbiologia , Fungos/efeitos dos fármacos , Humanos , Oxidantes/farmacologia , Estudos RetrospectivosRESUMO
The ability to form biofilms in a variety of environments is a common trait of bacteria, and may represent one of the earliest defenses against predation. Biofilms are multicellular communities usually held together by a polymeric matrix, ranging from capsular material to cell lysate. In a structure that imposes diffusion limits, environmental microgradients arise to which individual bacteria adapt their physiologies, resulting in the gamut of physiological diversity. Additionally, the proximity of cells within the biofilm creates the opportunity for coordinated behaviors through cell-cell communication using diffusible signals, the most well documented being quorum sensing. Biofilms form on abiotic or biotic surfaces, and because of that are associated with a large proportion of human infections. Biofilm formation imposes a limitation on the uses and design of ocular devices, such as intraocular lenses, posterior contact lenses, scleral buckles, conjunctival plugs, lacrimal intubation devices and orbital implants. In the absence of abiotic materials, biofilms have been observed on the capsule, and in the corneal stroma. As the evidence for the involvement of microbial biofilms in many ocular infections has become compelling, developing new strategies to prevent their formation or to eradicate them at the site of infection, has become a priority.
RESUMO
Streptococcus pneumoniae, an inhabitant of the upper respiratory mucosa, causes respiratory and invasive infections as well as conjunctivitis. Strains that lack the capsule, a main virulence factor and the target of current vaccines, are often isolated from conjunctivitis cases. Here we perform a comparative genomic analysis of 271 strains of conjunctivitis-causing S. pneumoniae from 72 postal codes in the United States. We find that the vast majority of conjunctivitis strains are members of a distinct cluster of closely related unencapsulated strains. These strains possess divergent forms of pneumococcal virulence factors (such as CbpA and neuraminidases) that are not shared with other unencapsulated nasopharyngeal S. pneumoniae. They also possess putative adhesins that have not been described in encapsulated pneumococci. These findings suggest that the unencapsulated strains capable of causing conjunctivitis utilize a pathogenesis strategy substantially different from that described for S. pneumoniae at other infection sites.
Assuntos
Conjuntivite Bacteriana/microbiologia , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/genética , Adesinas Bacterianas/genética , Infecções Assintomáticas , Cápsulas Bacterianas/genética , Western Blotting , Conjuntivite Bacteriana/epidemiologia , Genoma Bacteriano/genética , Humanos , Família Multigênica/genética , Tipagem de Sequências Multilocus , Filogenia , Filogeografia , Infecções Pneumocócicas/epidemiologia , Streptococcus pneumoniae/patogenicidade , Estados Unidos/epidemiologia , Fatores de Virulência/genéticaRESUMO
INTRODUCTION: Previous work has shown that besifloxacin, an 8-chloro-fluoroquinolone, has more potent activity against gram-positive pathogens than moxifloxacin and gatifloxacin, which carry an 8-methoxy group. This study was conducted to determine the contribution of the R7 and R8 substituent to fluoroquinolone antibacterial activity. MATERIALS AND METHODS: Besifloxacin, moxifloxacin, gatifloxacin, their R8 structural analogs, and ciprofloxacin were tested against representative isolates of various gram-positive and gram-negative species and previously characterized fluoroquinolone-resistant mutants of Staphylococcus aureus. Minimum inhibitory and minimum bactericidal concentrations were determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Reserpine was used to determine the effect of efflux pumps on antibacterial activity. RESULTS: In general, exchanging the R8 residue in besifloxacin slightly reduced the molecule's potency, while introducing an 8-chloro group in moxifloxacin increased its potency. A similar change in gatifloxacin had little to no effect. Substituting the R8 residues did not increase the susceptibility to the efflux pump inhibitor reserpine or result in a loss of bactericidal activity. In contrast, the positive control, ciprofloxacin, was shown to be a substrate for reserpine and lost bactericidal activity against some fluoroquinolone-resistant isolates of S. aureus. CONCLUSION: The data presented here show that, depending on the R7 substituent, replacing an 8-methoxy group with an 8-chloro substituent can improve potency or can have little-to-no effect. These findings highlight the importance of the interplay between the R7 and R8 substituents in determining antibacterial potency.
RESUMO
INTRODUCTION: Bacterial conjunctivitis is a contagious infection of the surface of the eye usually treated empirically with topical antibiotics. Since the etiologic agent is rarely identified, it is important to monitor which bacteria cause conjunctivitis and determine their antibacterial resistance profiles. METHODS: A total of 496 bacterial samples were isolated during a randomized, double-masked, vehicle-controlled, parallel-group study conducted in the United States with besifloxacin ophthalmic suspension 0.6% dosed twice daily. Species were determined by standard biochemical and/or molecular identification methods. Minimum inhibitory concentrations were determined according to Clinical and Laboratory Standards Institute standards. RESULTS: The most prevalent species was Haemophilus influenzae, followed by Staphylococcus epidermidis, Staphylococcus aureus, the Streptococcus mitis group, and Streptococcus pneumoniae. One species identified in this study, which was not previously noted as a common cause of bacterial conjunctivitis, was Dolosigranulum pigrum. Ampicillin resistance was common among H. influenzae isolates, while macrolide resistance was high among S. pneumoniae, S. epidermidis, and S. aureus. The latter two species also included a number of isolates resistant to methicillin and ciprofloxacin. CONCLUSION: Antibiotic resistance among isolates remains a concern and the appearance of an emerging ocular pathogen, D. pigrum, suggests the need for continued observation. The topical ophthalmic fluoroquinolones continue to provide a good balance of low to moderate (i.e., manageable) levels of resistance plus broad-spectrum coverage for empiric treatment of ocular infections.
Assuntos
Antibacterianos/uso terapêutico , Azepinas/uso terapêutico , Conjuntivite Bacteriana/tratamento farmacológico , Conjuntivite Bacteriana/microbiologia , Farmacorresistência Bacteriana , Fluoroquinolonas/uso terapêutico , Administração Oftálmica , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/administração & dosagem , Azepinas/administração & dosagem , Bactérias/classificação , Bactérias/isolamento & purificação , Técnicas Bacteriológicas , Criança , Pré-Escolar , Ensaios Clínicos como Assunto , Conjuntivite Bacteriana/epidemiologia , Esquema de Medicação , Feminino , Fluoroquinolonas/administração & dosagem , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
PURPOSE: Methicillin-resistant Staphylococcus aureus (MRSA) strains are commonly classified as hospital-acquired (HA) or community-acquired (CA). Typical HA-MRSA isolates are characterized by multidrug resistance and the SCCmec type II cassette, while CA-MRSA isolates are generally susceptible to more drug classes, are often of SCCmec type IV, and frequently carry the Panton-Valentine leukocidin (PVL) genes. This study determined the presence of traits characteristic for CA and HA strains in ocular MRSA isolates. MATERIALS AND METHODS: Fifty-six recent ocular isolates, consisting of 40 MRSA and 16 methicillin-susceptible Staphylococcus aureus (MSSA) comparator strains, were characterized. Minimum inhibitory concentration (MIC) testing was done according to current Clinical and Laboratory Standards Institute guidelines. Detection of the PVL encoding genes and determination of the SCCmec type was done by polymerase chain reaction (PCR), while spa typing and cluster analysis was performed following DNA sequencing. RESULTS: Of the 38 typeable MRSA isolates, 22 were of SCCmec type II and 16 were of SCCmec type IV. All SCCmec type II isolates were multidrug-resistant, lacked the PVL genes, and were of spa type t002 or closely related spa types. In contrast, the SCCmec type IV isolates were resistant to fewer classes of antimicrobial agents, often possessed the PVL genes (75.0%), and were of spa type t008 or closely related spa types. CONCLUSIONS: While the majority of ocular MRSA strains in this study fit the classical profile of HA- and CA-MRSA, some CA-MRSA isolates exhibited higher levels of antimicrobial resistance, which should be of particular concern to eye-care professionals. Furthermore, the apparent association of spa types and SCCmec types observed here warrants further investigation and suggests that spa typing may be useful in future HA- and CA-MRSA characterization studies.
Assuntos
Antibacterianos/farmacologia , Infecções Oculares Bacterianas/microbiologia , Staphylococcus aureus Resistente à Meticilina/genética , Meticilina/farmacologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Toxinas Bacterianas/genética , Técnicas de Tipagem Bacteriana , Infecções Comunitárias Adquiridas/epidemiologia , Infecções Comunitárias Adquiridas/microbiologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Impressões Digitais de DNA , DNA Bacteriano/genética , Exotoxinas/genética , Infecções Oculares Bacterianas/epidemiologia , Genótipo , Humanos , Leucocidinas/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Reação em Cadeia da Polimerase , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificaçãoRESUMO
PURPOSE: To determine the antibacterial susceptibility profile of bacterial pathogens from ocular infections against relevant aminoglycoside, ß-lactam, cephalosporin, chloramphenicol, fluoroquinolone, glycopeptide, lincosamide, and macrolide antibacterial agents. DESIGN: Laboratory investigation. METHODS: Isolates from patients with bacterial eye infections were collected prospectively by 34 institutions across the United States and were submitted to a central laboratory for inclusion in the Antibiotic Resistance Monitoring in Ocular micRorganisms (ARMOR) study. Minimum inhibitory concentrations were determined by microbroth dilution for 200 Staphylococcus aureus (S. aureus), 144 coagulase-negative staphylococci, 75 Streptococcus pneumoniae (S. pneumoniae), 73 Haemophilus influenzae (H. influenzae), and 100 Pseudomonas aeruginosa (P. aeruginosa) isolates. RESULTS: A large proportion of S. aureus and coagulase-negative staphylococci isolates were resistant to oxacillin/methicillin, azithromycin, or fluoroquinolones; 46.5% of S. aureus, 58.3% of coagulase-negative staphylococci, 9.0% of P. aeruginosa, and 9.3% of pneumococcal isolates were nonsusceptible to 2 or more antibacterial drug classes. Only 2.7% of H. influenzae isolates were nonsusceptible to 1 of the agents tested. Methicillin-resistant staphylococci were statistically more likely (all P < .0038) also to be resistant to fluoroquinolones, aminoglycosides, and macrolides. CONCLUSIONS: Resistance to 1 or more antibiotics is prevalent among ocular bacterial pathogens. Current resistance trends should be considered before initiating empiric treatment of common eye infections.