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1.
Antonie Van Leeuwenhoek ; 107(6): 1387-99, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25828766

RESUMO

Biofilm-related infections are considered a major cause of morbidity and mortality in hospital environments. Biofilms allow microorganisms to exchange genetic material and to become persistent colonizers and/or multiresistant to antibiotics. Corynebacterium pseudodiphtheriticum (CPS), a commensal bacterium that colonizes skin and mucosal sites has become progressively multiresistant and responsible for severe nosocomial infections. However, virulence factors of this emergent pathogen remain unclear. Herein, we report the adhesive properties and biofilm formation on hydrophilic (glass) and hydrophobic (plastic) abiotic surfaces by CPS strains isolated from patients with localized (ATCC10700/Pharyngitis) and systemic (HHC1507/Bacteremia) infections. Adherence to polystyrene attributed to hydrophobic interactions between bacterial cells and this negatively charged surface indicated the involvement of cell surface hydrophobicity in the initial stage of biofilm formation. Attached microorganisms multiplied and formed microcolonies that accumulated as multilayered cell clusters, a step that involved intercellular adhesion and synthesis of extracellular matrix molecules. Further growth led to the formation of dense bacterial aggregates embedded in the exopolymeric matrix surrounded by voids, typical of mature biofilms. Data also showed CPS recognizing human fibrinogen (Fbg) and fibronectin (Fn) and involvement of these sera components in formation of "conditioning films". These findings suggested that biofilm formation may be associated with the expression of different adhesins. CPS may form biofilms in vivo possibly by an adherent biofilm mode of growth in vitro currently demonstrated on hydrophilic and hydrophobic abiotic surfaces. The affinity to Fbg and Fn and the biofilm-forming ability may contribute to the establishment and dissemination of infection caused by CPS.


Assuntos
Aderência Bacteriana , Biofilmes/crescimento & desenvolvimento , Corynebacterium/fisiologia , Microbiologia Ambiental , Fibrinogênio/metabolismo , Fibronectinas/metabolismo , Adesinas Bacterianas/metabolismo , Bacteriemia/microbiologia , Técnicas Bacteriológicas , Corynebacterium/crescimento & desenvolvimento , Corynebacterium/isolamento & purificação , Infecções por Corynebacterium/microbiologia , Vidro , Humanos , Microscopia , Faringite/microbiologia , Plásticos
2.
Microbiology ; 160(Pt 3): 537-546, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24344208

RESUMO

Although infection by Corynebacterium diphtheriae is a model of extracellular mucosal pathogenesis, different clones have been also associated with invasive infections such as sepsis, endocarditis, septic arthritis and osteomyelitis. The mechanisms that promote C. diphtheriae infection and haematogenic dissemination need further investigation. In this study we evaluated the association and invasion mechanisms with human umbilical vein endothelial cells (HUVECs) and experimental arthritis in mice of endocarditis-associated strains and control non-invasive strains. C. diphtheriae strains were able to adhere to and invade HUVECs at different levels. The endocarditis-associated strains displayed an aggregative adherence pattern and a higher number of internalized viable cells in HUVECs. Transmission electron microscopy (TEM) analysis revealed intracellular bacteria free in the cytoplasm and/or contained in a host-membrane-confined compartment as single micro-organisms. Data showed bacterial internalization dependent on microfilament and microtubule stability and involvement of protein phosphorylation in the HUVEC signalling pathway. A high number of affected joints and high arthritis index in addition to the histopathological features indicated a strain-dependent ability of C. diphtheriae to cause severe polyarthritis. A correlation between the arthritis index and increased systemic levels of IL-6 and TNF-α was observed for endocarditis-associated strains. In conclusion, higher incidence of potential mechanisms by which C. diphtheriae may access the bloodstream through the endothelial barrier and stimulate the production of pro-inflammatory cytokines such as IL-6 and TNF-α, in addition to the ability to affect the joints and induce arthritis through haematogenic spread are thought to be related to the pathogenesis of endocarditis-associated strains.


Assuntos
Corynebacterium diphtheriae/fisiologia , Endocardite/microbiologia , Células Endoteliais/microbiologia , Animais , Artrite/microbiologia , Aderência Bacteriana , Linhagem Celular , Citocinas/biossíntese , Endocardite/metabolismo , Células Endoteliais/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/microbiologia , Humanos , Camundongos
3.
Mem Inst Oswaldo Cruz ; 108(1): 23-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23440110

RESUMO

Corynebacterium striatum is a potentially pathogenic microorganism with the ability to produce outbreaks of nosocomial infections. Here, we document a nosocomial outbreak caused by multidrug-resistant (MDR) C. striatum in Rio de Janeiro, Brazil. C. striatum identification was confirmed by 16S rRNA and rpoB gene sequencing. Fifteen C. striatum strains were isolated from adults (half of whom were 50 years of age and older). C. striatum was mostly isolated in pure culture from tracheal aspirates of patients undergoing endotracheal intubation procedures. The analysis by pulsed-field gel electrophoresis (PFGE) indicated the presence of four PFGE profiles, including two related clones of MDR strains (PFGE I and II). The data demonstrated the predominance of PFGE type I, comprising 11 MDR isolates that were mostly isolated from intensive care units and surgical wards. A potential causal link between death and MDR C. striatum (PFGE types I and II) infection was observed in five cases.


Assuntos
Infecções por Corynebacterium/microbiologia , Corynebacterium/efeitos dos fármacos , Infecção Hospitalar/microbiologia , Surtos de Doenças , Farmacorresistência Bacteriana Múltipla/genética , Adulto , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Brasil , Clonagem Molecular , Corynebacterium/genética , Infecções por Corynebacterium/epidemiologia , Infecção Hospitalar/epidemiologia , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Feminino , Genótipo , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Fenótipo , Adulto Jovem
4.
Mem. Inst. Oswaldo Cruz ; 108(1): 23-29, Feb. 2013. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-666039

RESUMO

Corynebacterium striatum is a potentially pathogenic microorganism with the ability to produce outbreaks of nosocomial infections. Here, we document a nosocomial outbreak caused by multidrug-resistant (MDR) C. striatum in Rio de Janeiro, Brazil. C. striatum identification was confirmed by 16S rRNA and rpoB gene sequencing. Fifteen C. striatum strains were isolated from adults (half of whom were 50 years of age and older). C. striatum was mostly isolated in pure culture from tracheal aspirates of patients undergoing endotracheal intubation procedures. The analysis by pulsed-field gel electrophoresis (PFGE) indicated the presence of four PFGE profiles, including two related clones of MDR strains (PFGE I and II). The data demonstrated the predominance of PFGE type I, comprising 11 MDR isolates that were mostly isolated from intensive care units and surgical wards. A potential causal link between death and MDR C. striatum (PFGE types I and II) infection was observed in five cases.


Assuntos
Adulto , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Infecções por Corynebacterium/microbiologia , Corynebacterium/efeitos dos fármacos , Infecção Hospitalar/microbiologia , Surtos de Doenças , Farmacorresistência Bacteriana Múltipla/genética , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Brasil , Clonagem Molecular , Infecções por Corynebacterium/epidemiologia , Corynebacterium/genética , Infecção Hospitalar/epidemiologia , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Genótipo , Testes de Sensibilidade Microbiana , Fenótipo
5.
Mem Inst Oswaldo Cruz ; 107(4): 486-93, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22666859

RESUMO

Corynebacterium pseudodiphtheriticum is a well-known human pathogen that mainly causes respiratory disease and is associated with high mortality in compromised hosts. Little is known about the virulence factors and pathogenesis of C. pseudodiphtheriticum. In this study, cultured human epithelial (HEp-2) cells were used to analyse the adherence pattern, internalisation and intracellular survival of the ATCC 10700 type strain and two additional clinical isolates. These microorganisms exhibited an aggregative adherence-like pattern to HEp-2 cells characterised by clumps of bacteria with a "stacked-brick" appearance. The differences in the ability of these microorganisms to invade and survive within HEp-2 cells and replicate in the extracellular environment up to 24 h post infection were evaluated. The fluorescent actin staining test demonstrated that actin polymerisation is involved in the internalisation of the C. pseudodiphtheriticum strains. The depolymerisation of microfilaments by cytochalasin E significantly reduced the internalisation of C. pseudodiphtheriticum by HEp-2 cells. Bacterial internalisation and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 h. These characteristics may explain how some C. pseudodiphtheriticum strains cause severe infection in human patients.


Assuntos
Aderência Bacteriana/fisiologia , Corynebacterium/patogenicidade , Células Epiteliais/microbiologia , Corynebacterium/fisiologia , Células Hep G2 , Humanos , Virulência
6.
Mem. Inst. Oswaldo Cruz ; 107(4): 486-493, June 2012. ilus, graf
Artigo em Inglês | LILACS | ID: lil-626442

RESUMO

Corynebacterium pseudodiphtheriticum is a well-known human pathogen that mainly causes respiratory disease and is associated with high mortality in compromised hosts. Little is known about the virulence factors and pathogenesis of C. pseudodiphtheriticum. In this study, cultured human epithelial (HEp-2) cells were used to analyse the adherence pattern, internalisation and intracellular survival of the ATCC 10700 type strain and two additional clinical isolates. These microorganisms exhibited an aggregative adherence-like pattern to HEp-2 cells characterised by clumps of bacteria with a "stacked-brick" appearance. The differences in the ability of these microorganisms to invade and survive within HEp-2 cells and replicate in the extracellular environment up to 24 h post infection were evaluated. The fluorescent actin staining test demonstrated that actin polymerisation is involved in the internalisation of the C. pseudodiphtheriticum strains. The depolymerisation of microfilaments by cytochalasin E significantly reduced the internalisation of C. pseudodiphtheriticum by HEp-2 cells. Bacterial internalisation and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 h. These characteristics may explain how some C. pseudodiphtheriticum strains cause severe infection in human patients.


Assuntos
Humanos , Aderência Bacteriana/fisiologia , Corynebacterium/patogenicidade , Células Epiteliais/microbiologia , Corynebacterium/fisiologia , Virulência
7.
Microb Pathog ; 52(3): 165-76, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22239957

RESUMO

Although Corynebacterium diphtheriae has been classically described as an exclusively extracellular pathogen, there is growing evidence that it may be internalized by epithelial cells. The aim of the present report was to investigate the nature and involvement of the surface-exposed non-fimbrial 67-72 kDa proteins (67-72p), previously characterized as adhesin/hemagglutinin, in C. diphtheriae internalization by HEp-2 cells. Transmission electron microscopy and bacterial internalization inhibition assays indicated the role of 67-72p as invasin for strains of varied sources. Cytoskeletal changes with accumulation of polymerized actin in HEp-2 cells beneath adherent 67-72p-adsorbed microspheres were observed by the Fluorescent actin staining test. Trypan blue staining method and Methylthiazole tetrazolium reduction assay showed a significant decrease in viability of HEp-2 cells treated with 67-72p. Morphological changes in HEp-2 cells observed after treatment with 67-72p included vacuolization, nuclear fragmentation and the formation of apoptotic bodies. Flow cytometry revealed an apoptotic volume decrease in HEp-2 cells treated with 67-72p. Moreover, a double-staining assay using Propidium Iodide/Annexin V gave information about the numbers of vital vs. early apoptotic cells and late apoptotic or secondary necrotic cells. The comparative analysis of MALDI-TOF MS experiments with the probes provided for 67-72p CDC-E8392 with an in silico proteome deduced from the complete genome sequence of C. diphtheriae identified with significant scores 67-72p as the protein DIP0733. In conclusion, DIP0733 (67-72p) may be directly implicated in bacterial invasion and apoptosis of epithelial cells in the early stages of diphtheria and C. diphtheriae invasive infection.


Assuntos
Apoptose , Corynebacterium diphtheriae/patogenicidade , Endocitose , Hemaglutininas/metabolismo , Hepatócitos/microbiologia , Hepatócitos/fisiologia , Fatores de Virulência/metabolismo , Actinas/metabolismo , Linhagem Celular , Sobrevivência Celular , Corynebacterium diphtheriae/genética , Hemaglutininas/genética , Humanos , Multimerização Proteica , Fatores de Virulência/genética
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