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1.
Genome Announc ; 4(6)2016 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-28034855

RESUMO

Here, we present the complete genome sequence of Actinomyces naeslundii strain ATCC 27039, isolated from an abdominal wound abscess. This strain is genetically transformable and will thus provide valuable information related to its crucial role in oral multispecies biofilm development.

2.
Genome Announc ; 4(6)2016 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-28034856

RESUMO

Here, we present the complete genome sequence of Rothia aeria type strain JCM 11412, isolated from air in the Russian space laboratory Mir. Recently, there has been an increasing number of reports on infections caused by R. aeria The genomic information will enable researchers to identify the pathogenicity of this organism.

3.
PLoS One ; 11(10): e0165312, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27792773

RESUMO

Pseudomonas aeruginosa infections of wounds in clinical settings are major complications whose outcomes are influenced by host responses that are not completely understood. Herein we evaluated transcriptomic changes of wounds as they counter P. aeruginosa infection-first active infection, and then chronic biofilm infection. We used the dermal full-thickness, rabbit ear excisional wound model. We studied the wound response: towards acute infection at 2, 6, and 24 hrs after inoculating 106 bacteria into day-3 wounds; and, towards more chronic biofilm infection of wounds similarly infected for 24 hrs but then treated with topical antibiotic to coerce biofilm growth and evaluated at day 5 and 9 post-infection. The wounds were analyzed for bacterial counts, expression of P. aeruginosa virulence and biofilm-synthesis genes, biofilm morphology, infiltrating immune cells, re-epithelialization, and genome-wide gene expression (RNA-Seq transcriptome). This analysis revealed that 2 hrs after bacterial inoculation into day-3 wounds, the down-regulated genes (infected vs. non-infected) of the wound edge were nearly all non-coding RNAs (ncRNAs), comprised of snoRNA, miRNA, and RNU6 pseudogenes, and their down-regulation preceded a general down-regulation of skin-enriched coding gene expression. As the active infection intensified, ncRNAs remained overrepresented among down-regulated genes; however, at 6 and 24 hrs they changed to a different set, which overlapped between these times, and excluded RNU6 pseudogenes but included snRNA components of the major and minor spliceosomes. Additionally, the raw counts of multiple types of differentially-expressed ncRNAs increased on post-wounding day 3 in control wounds, but infection suppressed this increase. After 5 and 9 days, these ncRNA counts in control wounds decreased, whereas they increased in the infected, healing-impaired wounds. These data suggest a sequential and coordinated change in the levels of transcripts of multiple major classes of ncRNAs in wound cells transitioning from inflammation to the proliferation phase of healing.


Assuntos
Biofilmes , Infecções por Pseudomonas/fisiopatologia , Pseudomonas aeruginosa , Dermatopatias Bacterianas/fisiopatologia , Transcriptoma/fisiologia , Ferimentos e Lesões/microbiologia , Animais , Feminino , Regulação da Expressão Gênica/fisiologia , RNA/genética , RNA/fisiologia , Coelhos , Dermatopatias Bacterianas/microbiologia , Ferimentos e Lesões/fisiopatologia
4.
FEBS Open Bio ; 6(9): 945-53, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27642558

RESUMO

Macrophages can change their phenotype in response to environmental cues. Polarized macrophages are broadly classified into two groups: classical activated M1 and alternative activated M2. Characterization of human macrophages has been widely studied, but polarized macrophages in rabbits have not been characterized. We characterized rabbit macrophages that were polarized using human recombinant GM-CSF and M-CSF. GM-CSF-treated macrophages had higher mRNA expression of proinflammatory cytokines (M1 phenotype) than did the M-CSF-treated counterpart. By contrast, high levels of TGF-ß and IL-10 expression (M2 phenotype) were found in M-CSF-treated macrophages. The present study may be useful to understand roles of polarized macrophages in rabbit disease models.

5.
Wound Repair Regen ; 24(1): 26-34, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26562746

RESUMO

Altered inflammation in the early stage has long been assumed to affect subsequent steps of the repair process that could influence proper wound healing and remodeling. However, the lack of explicit experimental data makes the connection between dysregulated wound inflammation and poor wound healing elusive. To bridge this gap, we used the established rabbit ear hypertrophic scar model for studying the causal effect of dysregulated inflammation. We induced an exacerbated and prolonged inflammatory state in these wounds with the combination of trauma-related stimulators of pathogen-associated molecular patterns from heat-killed Pseudomonas aeruginosa and damage-associated molecular patterns from a dermal homogenate. In stimulated wounds, a heightened and lengthened inflammation was observed based on quantitative measurements of IL-6 expression, tissue polymorphonuclear leukocytes infiltration, and tissue myeloperoxidase activity. Along with the high level of inflammation, wound healing parameters (epithelial gap and others) at postoperative day 7 and 16 were significantly altered in stimulated wounds compared to unstimulated controls. By postoperative day 35, scar elevation of stimulated wounds was higher than that of control wounds (scar elevation index: 1.90 vs. 1.39, p < 0.01). Moreover, treatment of these inflamed wounds with Indomethacin (at concentrations of 0.01, 0.1, and 0.4%) reduced scar elevation but with adverse effects of delayed wound closure and increased cartilage hypertrophy. In summary, successful establishment of this inflamed wound model provides a platform to understand these detrimental aspects of unchecked inflammation and to further test agents that can modulate local inflammation to improve wound outcomes.


Assuntos
Cicatriz Hipertrófica/imunologia , Citocinas/imunologia , Inflamação/imunologia , Interleucina-6/imunologia , Neutrófilos/imunologia , Pseudomonas aeruginosa/imunologia , RNA Mensageiro/metabolismo , Cicatrização/imunologia , Animais , Cicatriz Hipertrófica/metabolismo , Cicatriz Hipertrófica/patologia , Citocinas/genética , Modelos Animais de Doenças , Progressão da Doença , Orelha Externa/imunologia , Orelha Externa/lesões , Orelha Externa/metabolismo , Orelha Externa/patologia , Feminino , Inflamação/metabolismo , Inflamação/patologia , Neutrófilos/citologia , Peroxidase/metabolismo , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
6.
Genome Announc ; 3(4)2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26294638

RESUMO

Prevotella intermedia, a Gram-negative black-pigmented anaerobic rod, is frequently isolated from not only periodontal pockets but also purulent infections. We report here the complete genome sequence of P. intermedia strain 17-2, which is a non-exopolysaccharide-producing variant obtained from exopolysaccharide (EPS)-producing P. intermedia strain 17 stock culture.

7.
Arch Oral Biol ; 58(6): 681-9, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23399044

RESUMO

Rothia mucilaginosa is known as a member of commensal bacterial flora in the oral cavity and has received attention as a potential opportunistic pathogen. We previously determined the genomic sequence of R. mucilaginosa DY-18, a clinical strain with biofilm-like structures isolated from an infected root canal of a tooth with persistent apical periodontitis. We found that the DY-18 genome had only two sigma factor genes that encoded the primary and extracytoplasmic function (ECF) sigma factors. Genomic analysis on the available database of R. mucilaginosa ATCC 25296 (a type strain for R. mucilaginosa) revealed that ATCC 25296 has three sigma factors: one primary sigma factor and two ECF sigma factors, one of which was highly homologous to that of DY-18. ECF sigma factors play an important role in the response to environmental stress and to the production of virulence factors. Therefore, we first examined gene-encoding sigma factors on R. mucilaginosa genome in silico. The homologous ECF sigma factors found in strains DY-18 and ATCC 25296 formed a distinct SigH (SigR) clade in a phylogenetic tree and their cognate anti-sigma factor has a HXXXCXXC motif known to respond against disulphide stress. Quantitative reverse transcription polymerase chain reaction (PCR) and microarray analysis showed that the transcriptional levels of sigH were markedly up-regulated under disulphide stress in both strains. Microarray data also demonstrated that several oxidative-stress-related genes (thioredoxin, mycothione reductase, reductase and oxidoreductase) were significantly up-regulated under the diamide stress. On the basis of these results, we conclude that the alternative sigma factor SigH of R. mucilaginosa is a candidate regulator in the redox state.


Assuntos
Actinomycetaceae/metabolismo , Proteínas de Bactérias/isolamento & purificação , Dissulfetos/metabolismo , Estresse Oxidativo/fisiologia , Fator sigma/isolamento & purificação , Actinomycetaceae/genética , Motivos de Aminoácidos/genética , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Diamida , Regulação Bacteriana da Expressão Gênica/genética , Genes Reguladores/genética , Genoma Bacteriano/genética , Humanos , Análise em Microsséries , Oxirredução , Estresse Oxidativo/genética , Oxirredutases/análise , Oxirredutases/genética , Filogenia , Proteínas Repressoras/análise , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fator sigma/análise , Fator sigma/antagonistas & inibidores , Fator sigma/genética , Reagentes de Sulfidrila , Tiorredoxinas/análise , Tiorredoxinas/genética , Transcrição Gênica/genética , Regulação para Cima/genética
8.
J Endod ; 39(1): 44-8, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23228256

RESUMO

INTRODUCTION: Although the production of biofilm is thought to be crucial in the pathogenesis of abscess formations caused by oral resident microorganisms, the particular mechanisms are still unknown. The aim of this study was to identify gene(s) responsible for maintaining the cell surface-associated meshwork-like structures, which are found in some biofilm-producing bacteria, in a clinical isolate of Actinomyces oris K20. METHODS: Random insertional mutagenesis by using transposon EZ-Tn5 was performed against the strain K20. Transposon insertion mutants were screened by scanning electron microscopy for the absence of cell surface-associated meshwork-like structures. The disrupted genes by the transposon insertion were determined by direct genome sequencing with the transposon-end primers. RESULTS: Five mutants without the meshwork-like structures were identified from 175 mutants. Sequencing of flanking regions of transposon insertion revealed that 3 mutants had a gene encoded polysaccharide deacetylase, Spo0J containing ParB-like nuclease domain, and hypothetical protein, respectively. The other 2 mutants had an insertion in a noncoding region and an unidentified region, respectively. CONCLUSIONS: Our findings indicated that these genes might be involved in the formation of meshwork-like structures on Actinomyces oris K20.


Assuntos
Actinomyces/genética , Actinomicose/microbiologia , Biofilmes , Genes Bacterianos/genética , Doenças Periapicais/microbiologia , Amidoidrolases/genética , Técnicas Bacteriológicas , Mapeamento Cromossômico , DNA Primase/genética , Elementos de DNA Transponíveis/genética , Humanos , Proteínas de Membrana/genética , Microscopia Eletrônica de Varredura , Mutagênese Insercional/genética
9.
BMC Infect Dis ; 11: 228, 2011 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-21864411

RESUMO

BACKGROUND: Evidence in the literature suggests that exopolysaccharides (EPS) produced by bacterial cells are essential for the expression of virulence in these organisms. Secreted EPSs form the framework in which microbial biofilms are built. METHODS: This study evaluates the role of EPS in Prevotella intermedia for the expression of virulence. This evaluation was accomplished by comparing EPS-producing P. intermedia strains 17 and OD1-16 with non-producing P. intermedia ATCC 25611 and Porphyromonas gingivalis strains ATCC 33277, 381 and W83 for their ability to induce abscess formation in mice and evade phagocytosis. RESULTS: EPS-producing P. intermedia strains 17 and OD1-16 induced highly noticeable abscess lesions in mice at 107 colony-forming units (CFU). In comparison, P. intermedia ATCC 25611 and P. gingivalis ATCC 33277, 381 and W83, which all lacked the ability to produce viscous materials, required 100-fold more bacteria (109 CFU) in order to induce detectable abscess lesions in mice. Regarding antiphagocytic activity, P. intermedia strains 17 and OD1-16 were rarely internalized by human polymorphonuclear leukocytes, but other strains were readily engulfed and detected in the phagosomes of these phagocytes. CONCLUSIONS: These results demonstrate that the production of EPS by P. intermedia strains 17 and OD1-16 could contribute to the pathogenicity of this organism by conferring their ability to evade the host's innate defence response.


Assuntos
Polissacarídeos Bacterianos/metabolismo , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/patogenicidade , Prevotella intermedia/metabolismo , Prevotella intermedia/patogenicidade , Fatores de Virulência/metabolismo , Abscesso/microbiologia , Abscesso/patologia , Animais , Evasão da Resposta Imune , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose , Porphyromonas gingivalis/imunologia , Prevotella intermedia/imunologia , Virulência
10.
FEMS Immunol Med Microbiol ; 59(3): 456-65, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20553325

RESUMO

Escherichia hermannii, formerly classified as enteric group 11 of Escherichia coli, is considered to be nonpathogenic. In this report, we described some of the pathogenic properties of a viscous material-producing E. hermannii strain YS-11, which was clinically isolated from a persistent apical periodontitis lesion. YS-11 possessed cell surface-associated meshwork-like structures that are found in some biofilm-forming bacteria and its viscous materials contained mannose-rich exopolysaccharides. To further examine the biological effect of the extracellular viscous materials and the meshwork structures, we constructed a number of mutants using transposon mutagenesis. Strain 455, which has a transposon inserted into wzt, a gene that encodes an ATP-binding cassette transporter, lacked the expression of the cell surface-associated meshwork structures and the ability to produce extracellular materials. Complementation of the disrupted wzt in strain 455 with an intact wzt resulted in the restoration of these phenotypes. We also compared these strains in terms of their ability to induce abscess formation in mice as an indication of their pathogenicity. Strains with meshwork-like structures induced greater abscesses than those induced by strains that lacked such structures. These results suggest that the ability to produce mannose-rich exopolysaccharides and to form meshwork-like structures on E. hermannii might contribute to its pathogenicity.


Assuntos
Biofilmes/crescimento & desenvolvimento , Escherichia/isolamento & purificação , Escherichia/fisiologia , Periodontite Periapical/microbiologia , Transportadores de Cassetes de Ligação de ATP/genética , Abscesso/microbiologia , Abscesso/patologia , Animais , Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , DNA Bacteriano/química , DNA Bacteriano/genética , Modelos Animais de Doenças , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/patologia , Escherichia/genética , Escherichia/patogenicidade , Deleção de Genes , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Insercional , Polissacarídeos Bacterianos/metabolismo , Análise de Sequência de DNA , Virulência
11.
J Endod ; 35(3): 347-52, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19249593

RESUMO

We isolated spore-forming gram-positive aerobic rods from three patients with persistent periapical periodontitis. These cells possessed unique phenotypic characteristics by exhibiting dense meshwork-like structures on their cell surfaces that could be found in a number of biofilm-forming bacteria. We identified these strains as Bacillus subtilis by the API system and 16S ribosomal RNA gene (rRNA) sequencing. Treatment of the meshwork-like structures with protease K and staining with calcofluor for polysaccharides indicated that these structures were polysaccharides in nature and could be essential for biofilm formation by these isolates. Our findings suggest that B. subtilis could form biofilms in periapical periodontitis lesions, and this might contribute to the resistance to treatment resulting in the development of persistent periapical periodontitis observed in these patients. The particular mechanisms for B. subtilis biofilms to develop periapical periodontitis are still unknown. Further studies are needed to clarify the role of biofilms in persistent infections.


Assuntos
Bacillus subtilis/química , Bacillus subtilis/patogenicidade , Periodontite Periapical/microbiologia , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Biofilmes , DNA Bacteriano/análise , Endopeptidase K/metabolismo , Corantes Fluorescentes , Bacilos Gram-Positivos/isolamento & purificação , Humanos , Fosfoglucomutase/genética , Reação em Cadeia da Polimerase , Polissacarídeos Bacterianos/análise , RNA Ribossômico 16S/genética
12.
BMC Microbiol ; 9: 11, 2009 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-19146705

RESUMO

BACKGROUND: Prevotella intermedia (P. intermedia), a gram-negative, black-pigmented anaerobic rod, has been implicated in the development of chronic oral infection. P. intermedia strain 17 was isolated from a chronic periodontitis lesion in our laboratory and described as a viscous material producing strain. The stock cultures of this strain still maintain the ability to produce large amounts of viscous materials in the spent culture media and form biofilm-like structures. Chemical analyses of this viscous material showed that they were mainly composed of neutral sugars with mannose constituting 83% of the polysaccharides. To examine the biological effect of the extracellular viscous materials, we identified and obtained a naturally-occurring variant strain that lacked the ability to produce viscous materials in vitro from our stock culture collections of strain 17, designated as 17-2. We compared these two strains (strains 17 versus 17-2) in terms of their capacities to form biofilms and to induce abscess formation in mice as an indication of their pathogenicity. Further, gene expression profiles between these two strains in planktonic condition and gene expression patterns of strain 17 in solid and liquid cultures were also compared using microarray assays. RESULTS: Strain 17 induced greater abscess formation in mice as compared to that of the variant. Strain 17, but not 17-2 showed an ability to interfere with the phagocytic activity of human neutrophils. Expression of several genes which including those for heat shock proteins (DnaJ, DnaK, ClpB, GroEL and GroES) were up-regulated two to four-fold with statistical significance in biofilm-forming strain 17 as compared to the variant strain 17-2. Strain 17 in solid culture condition exhibited more than eight-fold up-regulated expression levels of several genes which including those for levanase, extracytoplasmic function-subfamily sigma factor (sigmaE; putative) and polysialic acid transport protein (KpsD), as compared to those of strain 17 in liquid culture media. CONCLUSION: These results demonstrate that the capacity to form biofilm in P. intermedia contribute to their resistance against host innate defence responses.


Assuntos
Infecções por Bacteroidaceae/microbiologia , Biofilmes , Periodontite Crônica/microbiologia , Perfilação da Expressão Gênica , Prevotella intermedia/genética , Prevotella intermedia/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Bacteroidaceae/imunologia , Células Cultivadas , Periodontite Crônica/imunologia , Meios de Cultura/química , Regulação Bacteriana da Expressão Gênica , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de Oligonucleotídeos , Fagocitose , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/metabolismo , Prevotella intermedia/química , Prevotella intermedia/fisiologia , Virulência
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