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Find video protocols related to scientific articles indexed in Pubmed.
Efficacy of chemomechanical caries removal in reducing cariogenic microbiota: a randomized clinical trial.
Braz Oral Res
PUBLISHED: 08-18-2014
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The aim of this study was to compare the efficacy of chemochemical methods (Carisolv™ and Papacárie®) versus the manual method (excavators) in reducing the cariogenic microbiota in dentine caries of primary teeth. Forty-six healthy children (5 to 9 years old) having at least one primary tooth with a cavitated dentine carious lesion were included in the study. The teeth presented no clinical or radiographic signs of pulpal involvement. The sample of 74 teeth was randomly divided into three different groups: Papacárie® (n = 25), Carisolv™ (n = 27) and Manual (n = 22). Samples of carious and sound dentine were collected with sterile excavators before and after caries removal in the three groups. The dentine samples were transferred to glass tubes containing a 1mL thioglycollate medium used as a carrier and enriched for microbiological detection of mutans streptococci and Lactobacillus spp, after incubation for 6h at room temperature. The minimum detection value for colony forming units (CFU) was 3.3 x 102 CFU/ml, and the results were converted into scores from 0 to 4. A significant difference was observed in relation to the microbiological scores before and after caries removal for all methods (Wilcoxon test; p < 0.001). The use of chemomechanical methods for caries removal did not improve the reduction of cariogenic microorganisms in dentine caries lesions, in comparison with manual excavation.
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Adherence to abiotic surface induces SOS response in Escherichia coli K-12 strains under aerobic and anaerobic conditions.
Microbiology (Reading, Engl.)
PUBLISHED: 07-10-2014
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During the colonization of surfaces, Escherichia coli bacteria often encounter DNA-damaging agents and these agents can induce several defence mechanisms. Base excision repair (BER) is dedicated to the repair of oxidative DNA damage caused by reactive oxygen species (ROS) generated by chemical and physical agents or by metabolism. In this work, we have evaluated whether the interaction with an abiotic surface by mutants derived from E. coli K-12 deficient in some enzymes that are part of BER causes DNA damage and associated filamentation. Moreover, we studied the role of endonuclease V (nfi gene; 1506 mutant strain) in biofilm formation. Endonuclease V is an enzyme that is involved in DNA repair of nitrosative lesions. We verified that endonuclease V is involved in biofilm formation. Our results showed more filamentation in the xthA mutant (BW9091) and triple xthA nfo nth mutant (BW535) than in the wild-type strain (AB1157). By contrast, the mutant nfi did not present filamentation in biofilm, although its wild-type strain (1466) showed rare filaments in biofilm. The filamentation of bacterial cells attaching to a surface was a consequence of SOS induction measured by the SOS chromotest. However, biofilm formation depended on the ability of the bacteria to induce the SOS response since the mutant lexA Ind(-) did not induce the SOS response and did not form any biofilm. Oxygen tension was an important factor for the interaction of the BER mutants, since these mutants exhibited decreased quantitative adherence under anaerobic conditions. However, our results showed that the presence or absence of oxygen did not affect the viability of BW9091 and BW535 strains. The nfi mutant and its wild-type did not exhibit decreased biofilm formation under anaerobic conditions. Scanning electron microscopy was also performed on the E. coli K-12 strains that had adhered to the glass, and we observed the presence of a structure similar to an extracellular matrix that depended on the oxygen tension. In conclusion, it was proven that bacterial interaction with abiotic surfaces can lead to SOS induction and associated filamentation. Moreover, we verified that endonuclease V is involved in biofilm formation.
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Characterization of the Opp peptide transporter of Corynebacterium pseudotuberculosis and its role in virulence and pathogenicity.
Biomed Res Int
PUBLISHED: 03-07-2014
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Despite the economic importance of caseous lymphadenitis (CLA), a chronic disease caused by Corynebacterium pseudotuberculosis, few genes related to the virulence of its etiologic agent have been characterized. The oligopeptide permease (Opp) transporters are located in the plasma membrane and have functions generally related to the uptake of peptides from the extracellular environment. These peptide transporters, in addition to having an important role in cell nutrition, also participate in the regulation of various processes involving intercellular signaling, including the control of the expression of virulence genes in pathogenic bacteria. To study the role of Opp in C. pseudotuberculosis, an OppD deficient strain was constructed via simple crossover with a nonreplicative plasmid carrying part of the oppD gene sequence. As occurred to the wild-type, the ?oppD strain showed impaired growth when exposed to the toxic glutathione peptide (GSH), indicating two possible scenarios: (i) that this component can be internalized by the bacterium through an Opp-independent pathway or (ii) that there is toxicity while the peptide is extracellular. Additionally, the ?oppD mutant presented a reduced ability to adhere to and infect macrophages compared to the wild-type, although both strains exhibit the same potential to colonize spleens and cause injury and death to infected mice.
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Invasion of endothelial cells and arthritogenic potential of endocarditis associated - Corynebacterium diphtheriae.
Microbiology (Reading, Engl.)
PUBLISHED: 12-16-2013
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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 hematogenic dissemination need further investigation. In this study we evaluated the association and invasion mechanisms to human 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 (AA) pattern and a higher number of internalized viable cells in HUVECs. TEM analysis revealed intracellular bacteria free in the cytoplasm and/or contained in a host-membrane-confined compartment as single microorganisms. Data showed bacterial internalization dependent on microfilament and microtubule stability and involvement of protein phosphorylation in the HUVECs signaling pathway. A high number of affected joints and 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-? was observed for endocarditis-associated strains. In conclusion, higher potential mechanisms by which C. diphtheriae may gain the bloodstream through the endothelial barrier and stimulate the production of IL-6 and TNF-alfa pro-inflammatory cytokines, in addition to the ability to affect the joints and induce arthritis through hematogenic spread are thought to be related to the pathogenesis of endocarditis-associated strains.
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Molecular identification of nocardia isolates from clinical samples and an overview of human nocardiosis in Brazil.
PLoS Negl Trop Dis
PUBLISHED: 12-01-2013
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Nocardia sp. causes a variety of clinical presentations. The incidence of nocardiosis varies geographically according to several factors, such as the prevalence of HIV infections, transplants, neoplastic and rheumatic diseases, as well as climate, socio-economic conditions and laboratory procedures for Nocardia detection and identification. In Brazil the paucity of clinical reports of Nocardia infections suggests that this genus may be underestimated as a cause of human diseases and/or either neglected or misidentified in laboratory specimens. Accurate identification of Nocardia species has become increasingly important for clinical and epidemiological investigations. In this study, seven clinical Nocardia isolates were identified by multilocus sequence analysis (MLSA) and their antimicrobial susceptibility was also determined. Most Nocardia isolates were associated to pulmonary disease.
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Corynebacterium ulcerans isolates from humans and dogs: fibrinogen, fibronectin and collagen-binding, antimicrobial and PFGE profiles.
Antonie Van Leeuwenhoek
PUBLISHED: 09-02-2013
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Corynebacterium ulcerans has been increasingly isolated as an emerging zoonotic agent of diphtheria and other infections from companion animals. Since pets are able to act as symptomless carriers, it is also essential to identify virulence potential for humans of these isolates. In this work the ability of C. ulcerans to bind to fibrinogen (Fbg), fibronectin (Fn) and Type I collagen as well the genetic relationship among strains isolated from human and asymptomatic dogs in Rio de Janeiro (Brazil) were analyzed. Five pulsed-field gel electrophoresis (PFGE) profiles were demonstrated (I, II, III, IV and V). In addition, the IV and V profiles exhibiting ?85 % similarity were expressed by the BR-AD41 and BR-AD61 strains from companion dogs living in the same neighborhood. Independent of the PFGE-types, human and dog isolates showed affinity to Fbg, Fn and collagen. Heterogeneity of PFGE profiles indicated endemicity of C. ulcerans in the Rio de Janeiro metropolitan area. Differences in the expression of adhesins to the human extracellular matrix may contribute to variations in the virulence and zoonotic potential of C. ulcerans strains.
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Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections.
Mem. Inst. Oswaldo Cruz
PUBLISHED: 06-20-2013
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Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis constitute a group of potentially toxigenic microorganisms that are related to different infectious processes in animal and human hosts. Currently, there is a lack of information on the prevalence of disease caused by these pathogens, which is partially due to a reduction in the frequency of routine laboratory testing. In this study, a multiplex polymerase chain reaction (mPCR) assay that can simultaneously identify and determine the toxigenicity of these corynebacterial species with zoonotic potential was developed. This assay uses five primer pairs targeting the following genes: rpoB (Corynebacterium spp), 16S rRNA (C. ulcerans and C. pseudotuberculosis), pld (C. pseudotuberculosis), dtxR (C. diphtheriae) and tox [diphtheria toxin (DT) ]. In addition to describing this assay, we review the literature regarding the diseases caused by these pathogens. Of the 213 coryneform strains tested, the mPCR results for all toxigenic and non-toxigenic strains of C . diphtheriae, C. ulcerans and C. pseudotuberculosis were in 100% agreement with the results of standard biochemical tests and PCR-DT. As an alternative to conventional methods, due to its advantages of specificity and speed, the mPCR assay used in this study may successfully be applied for the diagnosis of human and/or animal diseases caused by potentially toxigenic corynebacterial species.
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Antimicrobial resistance and virulence traits of Enterococcus faecalis from primary endodontic infections.
J Dent
PUBLISHED: 05-10-2013
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To determine the phenotypic and molecular characteristics of Enterococcus faecalis recovered from primary endodontic infections in Brazilian patients.
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Staphylococcus haemolyticus disseminated among neonates with bacteremia in a neonatal intensive care unit in Rio de Janeiro, Brazil.
Diagn. Microbiol. Infect. Dis.
PUBLISHED: 03-11-2013
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Oxacillin-resistant Staphylococcus haemolyticus (ORSH) was found as the most prevalent (77.5%) species of coagulase-negative staphylococci associated with bacteremia in neonates making use of intravenous catheters in an intensive care unit of a Brazilian teaching hospital. Thirty-one blood isolates were confirmed as S. haemolyticus by sequencing of the 16S and clustered in 6 pulsed-field gel electrophoresis types (with 58% of the strains belonging to 2 predominant types B and D). S. haemolyticus was mostly oxacillin-resistant (90.3%) displaying multiresistance profiles (70.4%). However, the mecA gene was undetected in 22.6% strains. ORSH exhibited slime production on Congo-Red agar (67.7%), adherence to polystyrene (96.7%), and glass (87%) surfaces. Interestingly, ica-operon was detected in 58% strains, mostly belonging to the B, D, and F genotypes, which is a significantly higher percentage when compared to other studies conducted at different parts of the globe. Data indicated that ica operon and biofilm-forming ORSH are endemic in Brazilian nosocomial environment.
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Clonal multidrug-resistant Corynebacterium striatum within a nosocomial environment, Rio de Janeiro, Brazil.
Mem. Inst. Oswaldo Cruz
PUBLISHED: 02-27-2013
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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.
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The role of DNA base excision repair in filamentation in Escherichia coli K-12 adhered to epithelial HEp-2 cells.
Antonie Van Leeuwenhoek
PUBLISHED: 07-14-2011
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Base excision repair (BER) is dedicated to the repair of oxidative DNA damage caused by reactive oxygen species generated by chemical and physical agents or by metabolism which can react with DNA and cause a variety of mutations. Epithelial cells are typically the first type of host cell to come into contact with potential microbial invaders. In this work, we have evaluated whether the adherence to human epithelial cells causes DNA damage and associated filamentation. Experiments concerning adherence to HEp-2 cells were carried out with mutants deficient in BER that were derived from Escherichia coli K-12. Since the removal of mannose during bacterial interaction with HEp-2 cells allows adhesion through mannose-sensitive adhesins, the experiments were also performed in the presence and the absence of mannose. Our results showed enhanced filamentation for the single xth (BW9091) and triple xth nfo nth (BW535) mutants in adherence assays with HEp-2 cells performed without D: -mannose. The increased filamentation growth was inhibited by complementation of BER mutants with a wild type xth gene. Moreover, we measured SOS induction of bacteria adhered to HEp-2 cells in the presence and absence of D: -mannose through of SOS-chromotest assay and we observed a higher ?-galactosidase expression in the absence of mannose. In this context, data showed evidence that bacterial attachment to HEp-2 epithelial surfaces can generate DNA lesions and SOS induction.
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Comparative analysis of two complete Corynebacterium ulcerans genomes and detection of candidate virulence factors.
BMC Genomics
PUBLISHED: 06-01-2011
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Corynebacterium ulcerans has been detected as a commensal in domestic and wild animals that may serve as reservoirs for zoonotic infections. During the last decade, the frequency and severity of human infections associated with C. ulcerans appear to be increasing in various countries. As the knowledge of genes contributing to the virulence of this bacterium was very limited, the complete genome sequences of two C. ulcerans strains detected in the metropolitan area of Rio de Janeiro were determined and characterized by comparative genomics: C. ulcerans 809 was initially isolated from an elderly woman with fatal pulmonary infection and C. ulcerans BR-AD22 was recovered from a nasal sample of an asymptomatic dog.
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Corynebacterium ulcerans diphtheria: an emerging zoonosis in Brazil and worldwide.
Rev Saude Publica
PUBLISHED: 05-27-2011
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The article is a literature review on the emergence of human infections caused by Corynebacterium ulcerans in many countries including Brazil. Articles in Medline/PubMed and SciELO databases published between 1926 and 2011 were reviewed, as well as articles and reports of the Brazilian Ministry of Health. It is presented a fast, cost-effective and easy to perform screening test for the presumptive diagnosis of C. ulcerans and C. diphtheriae infections in most Brazilian public and private laboratories. C. ulcerans spread in many countries and recent isolation of this pathogen in Rio de Janeiro, southeastern Brazil, is a warning to clinicians, veterinarians, and microbiologists on the occurrence of zoonotic diphtheria and C. ulcerans dissemination in urban and rural areas of Brazil and/or Latin America.
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Group B Streptococcus serotypes III and V induce apoptosis and necrosis of human epithelial A549 cells.
Int. J. Mol. Med.
PUBLISHED: 01-21-2011
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Although group B Streptococcus (GBS) has been classically described as an exclusively extracellular pathogen, growing evidence suggests that it may be internalized by epithelial cells. However, the fates of intracellular GBS and of infected respiratory epithelial cells remain unclear. Little is known about the bacterial components involved in these processes. The present study investigated the bacterial internalization by A549 cells and the apoptosis/necrosis of the infected human epithelial cells. The morphological changes in A549 cells observed from 2 h post-infection with GBS included vacuolization and the formation of apoptotic bodies. Flow cytometry revealed that 81.2% of apoptotic A549 cells were infected with GBS serotype III 90356-liquor. Moreover, a double-staining assay using propidium iodide (PI)/Annexin V (AV) gave information about the numbers of viable (PI-/AV-) (18.27%) vs. early apoptotic (PI-/AV+) (73.83%) and late apoptotic cells (PI+/AV+) (7.37%) during infection of A549 cells with GBS III 90356-liquor. In addition, 37% necrotic cells were observed in A549 cells infected with GBS serotype V 90186-blood. In conclusion, GBS serotypes III and V induce apoptosis of epithelial cells in the early stages of GBS infection, resulting in tissue destruction, bacterial spreading and, in consequence, invasive disease or systemic infection.
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Fever temperature enhances mechanisms of survival of Streptococcus agalactiae within human endothelial cells.
Int. J. Mol. Med.
PUBLISHED: 09-07-2010
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Group B streptococci (GBS) are the most common cause of pneumonia and sepsis during the neonatal period. However, the pathogenesis of invasive infection is poorly understood. We investigated the ability of GBS grown at 37 degrees C and 40 degrees C to adhere and invade human umbilical vein endothelial cells (HUVECs) at different periods of incubation (0, 0.5, 1, 2, 18 and 24 h). All strains tested, except strain 88641-vagina survived for 24 h in the intracellular environment at 40 degrees C. For serotype III grown at 40 degrees C, both strains (80340-vagina and 90356-liquor) showed increased adherence and intracellular survival when compared to bacteria grown at 37 degrees C (P<0.01). GBS serotype V strains (88641-vagina and 90186-blood) showed ability to survive inside HUVECs until 2 and 24 h post-infection at 40 degrees C and 37 degrees C, respectively (P<0.01). Influence of growth temperature in bacterial interaction with endothelial cells was partially dependent of serotypes and the clinical origin of strains. Serotypes III and V strains grown at both temperatures remained viable within acidic endothelial vacuoles which acquired Rab7 and LAMP-1 endosomal markers. The data emphasize the influence of temperature on cellular events of phagocytosis and pathogenesis of GBS diseases.
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The antimicrobial activity of gray-colored mineral trioxide aggregate (GMTA) and white-colored MTA (WMTA) under aerobic and anaerobic conditions.
Oral Surg Oral Med Oral Pathol Oral Radiol Endod
PUBLISHED: 02-03-2010
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The objective of this study was to test the hypothesis that the antimicrobial activity of gray-colored MTA (GMTA) and white-colored MTA (WMTA) is related to the induction of reactive oxygen species (ROS).
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Fibrinogen binds to nontoxigenic and toxigenic Corynebacterium diphtheriae strains.
Mem. Inst. Oswaldo Cruz
PUBLISHED: 01-25-2010
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The production of fibrinous exudates may play an important role in determining the outcome of bacterial infection. Although pseudomembrane formation is a characteristic feature of diphtheria, little is known about the fibrinogen (Fbn)-binding properties of Corynebacterium diphtheriae strains and the influence of the gene that codes for diphtheria toxin (tox gene) in this process. In this study we demonstrated the ability of C. diphtheriae strains to bind to Fbn and to convert Fbn to fibrin. Bacterial interaction with rabbit plasma was evaluated by both slide and tube tests. Interaction of microorganisms with human Fbn was evaluated by both enzyme linked immunosorbent assay (ELISA) and fluorescein isothiocyanate-conjugated (FITC) Fbn binding assays. Nontoxigenic and toxigenic strains formed bacterial aggregates in the presence of plasma in the slide tests. The ability to convert Fbn to a loose web of fibrin in the plasma solution in the tube tests appeared to be a common characteristic of the species, including strains that do not carry the tox gene. Fbn binding to C. diphtheriae strains occurred at varying intensities, as demonstrated by the FITC-Fbn and ELISA binding assays. Our data suggest that the capacity to bind to Fbn and to convert Fbn to fibrin may play a role in pseudomembrane formation and act as virulence determinants of both nontoxigenic and toxigenic strains.
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Non-opsonic phagocytosis of homologous non-toxigenic and toxigenic Corynebacterium diphtheriae strains by human U-937 macrophages.
Microbiol. Immunol.
PUBLISHED: 01-09-2010
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As interactions between bacteria and macrophages dictate the outcome of most infectious diseases, analyses of molecular mechanisms of non-opsonic phagocytosis should lead to new approaches for the prevention of diphtheria and systemic Corynebacterium diphtheriae infections. The present study aimed to evaluate human macrophage-bacteria interactions in the absence of opsonin antibodies and the influence of the tox gene on this process. Homologous C. diphtheriae tox+ and tox- strains were evaluated for adhesion, entering and survival within U-937 human macrophages at different incubation periods. Higher numbers of viable bacteria associated with and internalized by macrophages were demonstrated for the tox+ strain. However, viable intracellular bacteria were detected at T-24 hr only for the tox- strain. Cytoskeletal inhibitors, cytochalasin E, genistein and colchicine, inhibited intracellular viability of both strains at different levels. Bacterial replication was evidenced at T-24 hr in supernatants of monolayers infected with the tox- strain. Host cell death and nuclear alterations were evidenced by the Trypan blue exclusion assay and DAPI fluorescence microscopy. ELISA of histone-associated DNA fragments allowed detection of apoptosis and necrosis induced by tox+ and tox- strains at T-1 hr and T-3 hr. In conclusion, human macrophages in the absence of opsonins may not be promptly effective at killing diphtheria bacilli. The presence of the tox gene influences the susceptibility of C. diphtheriae to human macrophages and the outcome of non-opsonic phagocytosis. C. diphtheriae strains exhibit strategies to survive within macrophages and to exert apoptosis and necrosis in human phagocytic cells, independent of the tox gene.
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Corynebacterium ulcerans isolated from an asymptomatic dog kept in an animal shelter in the metropolitan area of Rio de Janeiro, Brazil.
Vector Borne Zoonotic Dis.
PUBLISHED: 01-07-2010
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Corynebacterium ulcerans was isolated from nares of one asymptomatic dog kept in an animal shelter in the metropolitan area of Rio de Janeiro, Brazil. The RNA polymerase beta subunit-encoding gene was sequenced to confirm the species identity. C. ulcerans strains producing phospholipase D, but not diphtheria toxin, are able to cause severe disease in humans, such as pneumonia and granulomatous nodules in pulmonary tissues. The infection rate varies really widely by region, probably because of the variations in the reported infection rates. Dogs with unapparent C. ulcerans infections may be considered as potentially capable of infecting other animals and humans, including pet owners. Medical and veterinary staff should be aware that asymptomatic animals can carry C. ulcerans and cooperate in eliminating infections and monitoring animals also in the developing countries.
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Effect of a glaze/composite sealant on the 3-D surface roughness of esthetic restorative materials.
Oper Dent
PUBLISHED: 12-04-2009
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The main goal of the current study was to evaluate the surface roughness of tooth-colored restorative materials after different finishing/polishing protocols, including a liquid polisher (BisCover, BISCO, Schaumburg, IL, USA). The restorative materials tested included two nanofilled resin composites (Filtek Supreme, 3M Dental Products, St Paul, MN, USA and Grandio, Voco, Cuxhaven, Germany), one resin-modified glass ionomer cement (Vitremer, 3M Dental Products) and one conventional glass ionomer cement (Meron Molar ART, Voco). The finishing/polishing methods were divided into five groups: G1 (compression with Mylar matrix), G2 (finishing with diamond burs), G3 (Sof-Lex, 3M Dental Products), G4 (BisCover, BISCO, after diamond burs) and G5 (BisCover after Sof-Lex). Five cylindrical specimens of each material were prepared for each group according to the manufacturers instructions. The finishing/polishing methods were performed by a single operator in one direction to avoid variations at low speed (15,000 RPM). The surface roughness was evaluated using a 3-D scanning instrument with two parameters considered (Ra and Rz). The data was analyzed using one-way ANOVA followed by a multiple comparison Tukeys test. The results showed that BisCover (BISCO) was capable of reducing surface roughness and provided polished surfaces for all materials, enhancing smoothness over already polished surfaces (Sof-Lex, 3M Dental Products) and achieving polishing after finishing with diamond burs.
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Cytotoxicity evaluation of two root canal sealers and a commercial calcium hydroxide paste on THP1 cell line by Trypan Blue assay.
J Appl Oral Sci
PUBLISHED: 11-26-2009
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The aim of this investigation was to evaluate the cytotoxicity of two brands of root canal sealers, epoxy-resin based and zinc oxide-eugenol based, and one commercial calcium hydroxide paste on a monocyte cell line THP-1.
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Higher levels of salivary MUC5B and MUC7 in individuals with gastric diseases who harbor Helicobacter pylori.
Arch. Oral Biol.
PUBLISHED: 08-01-2009
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The aim of the present study was to assess the salivary levels of MUC5B and MUC7 in individuals with dyspeptic disease and Helicobacter pylori (H. pylori) in the stomach, compared to individuals without dyspeptic disease.
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Corynebacterium diphtheriae as an emerging pathogen in nephrostomy catheter-related infection: evaluation of traits associated with bacterial virulence.
J. Med. Microbiol.
PUBLISHED: 07-23-2009
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Corynebacterium diphtheriae still represents a global medical challenge, particularly due to the significant number of individuals susceptible to diphtheria and the emergence of non-toxigenic strains as the causative agents of invasive infections. In this study, we characterized the clinical and microbiological features of what we believe to be the first case of C. diphtheriae infection of a percutaneous nephrostomy catheter insertion site in an elderly patient with a fatal bladder cancer. Moreover, we demonstrated the potential role of adherence, biofilm formation and fibrin deposition traits in C. diphtheriae from the catheter-related infection. Non-toxigenic C. diphtheriae isolated from the purulent discharge (named strain BR-CAT5003748) was identified by the API Coryne system (code 1 010 324) and a multiplex PCR for detection of dtxR and tox genes. Strain BR-CAT5003748 showed resistance to oxacillin, ceftazidime and ciprofloxacin. In experiments performed in vitro, the catheter isolate was classified as moderately hydrophobic and as moderately adherent to polystyrene surfaces. Glass provided a more effective surface for biofilm formation than polystyrene. Micro-organisms adhered to (>1.5 x 10(6) c.f.u.) and multiplied on surfaces of polyurethane catheters. Microcolony formation (a hallmark of biofilm formation) and amorphous accretions were observed by scanning electron microscopy on both external and luminal catheter surfaces. Micro-organisms yielded simultaneous expression of localized adherence-like and aggregative-like (LAL/AAL) adherence patterns to HEp-2 cells. Interestingly, the coagulase tube test resulted in the formation of a thin layer of fibrin embedded in rabbit plasma by the non-toxigenic BR-CAT5003748 strain. In conclusion, C. diphtheriae should be recognized as a potential cause of catheter-related infections in at-risk populations such as elderly and cancer patients. LAL/AAL strains may be associated with virulence traits that enable C. diphtheriae to effectively produce biofilms on catheter surfaces. Biofilm formation and fibrin deposition could have contributed to the persistence of C. diphtheriae at the infected insertion site and the obstruction of the nephrostomy catheter.
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Antimicrobial activity of calcium hydroxide pastes on Enterococcus faecalis cultivated in root canal systems.
Braz Dent J
PUBLISHED: 05-26-2009
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The effectiveness of calcium hydroxide pastes: Calen and PMCC-Calen associated to chemo-mechanical preparation was assessed on Enterococcus faecalis grown within root canals. Seventy incisors were inserted into TSB medium, sterilized and contaminated with E. faecalis. Culture medium was replaced each 24 h and incubated at 37 degrees C for 72 h. After chemo-mechanical preparation, root canals were filled with Calen or PMCC-Calen (7 or 14 days). Pastes were removed and teeth were inserted into test tubes containing Enterococcosel broth. Calen paste (maintained for 7 and 14 days) induced 70% elimination of enterococci and PMCC-Calen 100% elimination only after maintenance for 14 days. These medications were significantly more effective (p<0.001) than chemo-mechanical protocol alone and PMCC-Calen maintained for 7 days, both incapable to eliminate the viability of enterococci. Calcium hydroxide pastes demonstrated important adjuvant effects in the elimination of enterococci during chemo-mechanical preparation of root canal systems. When associated with PMCC, calcium hydroxide pastes should be maintained for at least 14 days.
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Diphtheria Antibodies and T lymphocyte Counts in Patients Infected With HIV-1.
Braz. J. Microbiol.
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We assessed the IgG levels anti-diphtheria (D-Ab) and T cell counts (CD4+ and CD8+) in HIV-1 infected subjects undergoing or not highly active antiretroviral therapy (HAART). Approximately 70% of all HIV-1 patients were unprotected against diphtheria. There were no differences in D-Ab according to CD4 counts. Untreated patients had higher D-Ab (geometric mean of 0.62 IU/ml) than HAART-patients (geometric mean of 0.39 IU/ml). The data indicated the necessity of keeping all HIV-1 patients up-to-date with their vaccination.
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Aggregative adherent strains of Corynebacterium pseudodiphtheriticum enter and survive within HEp-2 epithelial cells.
Mem. Inst. Oswaldo Cruz
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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.
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Pangenomic study of Corynebacterium diphtheriae that provides insights into the genomic diversity of pathogenic isolates from cases of classical diphtheria, endocarditis, and pneumonia.
J. Bacteriol.
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Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae. Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ?(tox)(+) phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox(+) corynephage. DNA binding sites of the tox-controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.
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PIPS: pathogenicity island prediction software.
PLoS ONE
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The adaptability of pathogenic bacteria to hosts is influenced by the genomic plasticity of the bacteria, which can be increased by such mechanisms as horizontal gene transfer. Pathogenicity islands play a major role in this type of gene transfer because they are large, horizontally acquired regions that harbor clusters of virulence genes that mediate the adhesion, colonization, invasion, immune system evasion, and toxigenic properties of the acceptor organism. Currently, pathogenicity islands are mainly identified in silico based on various characteristic features: (1) deviations in codon usage, G+C content or dinucleotide frequency and (2) insertion sequences and/or tRNA genetic flanking regions together with transposase coding genes. Several computational techniques for identifying pathogenicity islands exist. However, most of these techniques are only directed at the detection of horizontally transferred genes and/or the absence of certain genomic regions of the pathogenic bacterium in closely related non-pathogenic species. Here, we present a novel software suite designed for the prediction of pathogenicity islands (pathogenicity island prediction software, or PIPS). In contrast to other existing tools, our approach is capable of utilizing multiple features for pathogenicity island detection in an integrative manner. We show that PIPS provides better accuracy than other available software packages. As an example, we used PIPS to study the veterinary pathogen Corynebacterium pseudotuberculosis, in which we identified seven putative pathogenicity islands.
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Corynebacterium diphtheriae 67-72p hemagglutinin, characterized as the protein DIP0733, contributes to invasion and induction of apoptosis in HEp-2 cells.
Microb. Pathog.
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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.
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