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Sample records for aggregatibacter actinomycetemcomitans leukotoxin

  1. Membrane Association and Destabilization by Aggregatibacter actinomycetemcomitans Leukotoxin Requires Changes in Secondary Structures

    PubMed Central

    Walters, Michael J.; Brown, Angela C.; Edrington, Thomas C.; Baranwal, Somesh; Du, Yurong; Lally, Edward T.; Boesze-Battaglia, Kathleen

    2013-01-01

    SUMMARY Aggregatibacter actinomycetemcomitans is a common inhabitant of the upper aerodigestive tract of humans and non-human primates and is associated with disseminated infections, including lung and brain abscesses, pediatric infective endocarditis in children, and localized aggressive periodontitis. A. actinomycetemcomitans secretes a repeats-in-toxin protein, leukotoxin, which exclusively kills lymphocyte function-associated antigen-1-bearing cells. The toxin's pathological mechanism is not fully understood; however, experimental evidence indicates that it involves the association with and subsequent destabilization of the target cell's plasma membrane. We have long hypothesized that leukotoxin secondary structure is strongly correlated with membrane association and/or destabilization. In this study, we tested this hypothesis by analyzing lipid-induced changes in leukotoxin conformation. Upon incubation of leukotoxin with lipids that favor leukotoxin-membrane association, we observed an increase in leukotoxin α-helical content that was not observed with lipids that favor membrane destabilization. The change in leukotoxin conformation after incubation with these lipids suggests that membrane binding and membrane destabilization have distinct secondary structural requirements, suggesting that they are independent events. These studies thus provide insight into the mechanism of cell damage that leads to disease progression by A. actinomycetemcomitans. PMID:23678967

  2. Aggregatibacter actinomycetemcomitans leukotoxin cytotoxicity occurs through bilayer destabilization

    PubMed Central

    Brown, Angela C.; Boesze-Battaglia, Kathleen; Du, Yurong; Stefano, Frank P.; Kieba, Irene R.; Epand, Raquel F.; Kakalis, Lazaros; Yeagle, Philip L.; Epand, Richard M.; Lally, Edward T.

    2012-01-01

    Summary The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA-induced damage while liposomes composed of lipids that do not form non-bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while 31P nuclear magnetic resonance studies showed that the LtxA-induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization. PMID:22309134

  3. Aggregatibacter actinomycetemcomitans leukotoxin: a powerful tool with capacity to cause imbalance in the host inflammatory response.

    PubMed

    Johansson, Anders

    2011-03-01

    Aggregatibacter actinomycetemcomitans has been described as a member of the indigenous oral microbiota of humans, and is involved in the pathology of periodontitis and various non-oral infections. This bacterium selectively kills human leukocytes through expression of leukotoxin, a large pore-forming protein that belongs to the Repeat in Toxin (RTX) family. The specificity of the toxin is related to its prerequisite for a specific target cell receptor, LFA-1, which is solely expressed on leukocytes. The leukotoxin causes death of different leukocyte populations in a variety of ways. It activates a rapid release of lysosomal enzymes and MMPs from neutrophils and causes apoptosis in lymphocytes. In the monocytes/macrophages, the toxin activates caspase-1, a cysteine proteinase, which causes a proinflammatory response by the activation and secretion of IL-1β and IL-18. A specific clone (JP2) of A. actinomycetemcomitans with enhanced leukotoxin expression significantly correlates to disease onset in infected individuals. Taken together, the mechanisms by which this toxin kills leukocytes are closely related to the pathogenic mechanisms of inflammatory disorders, such as periodontitis. Therapeutic strategies targeting the cellular and molecular inflammatory host response in periodontal diseases might be a future treatment alternative.

  4. Profound Effects of Aggregatibacter actinomycetemcomitans Leukotoxin Mutation on Adherence Properties Are Clarified in in vitro Experiments.

    PubMed

    Velusamy, Senthil Kumar; Sampathkumar, Vandana; Godboley, Dipti; Fine, Daniel H

    2016-01-01

    Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans, an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A. actinomycetemcomitans. Unexpectedly, an A. actinomycetemcomitans leukotoxin (ltxA) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A. actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltxA mutation in vitro. Results indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly (P<0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivary-coated hydroxyapatite (SHA) was significantly decreased (P<0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [rcpA (P = 0.018), rcpB (P = 0.02), tadA (P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth (P = 0.008) and exo-polysaccharide production (P<0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltxA restored binding to SHA (P<0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltxA diminished hard tissue binding in vitro, which helps explain the previous in vivo failure of a ltxA knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltxA) could affect other seemingly unrelated genes (such as rcpA, rcpB tadA etc), and 2

  5. Profound Effects of Aggregatibacter actinomycetemcomitans Leukotoxin Mutation on Adherence Properties Are Clarified in in vitro Experiments

    PubMed Central

    Godboley, Dipti; Fine, Daniel H.

    2016-01-01

    Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans, an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A. actinomycetemcomitans. Unexpectedly, an A. actinomycetemcomitans leukotoxin (ltxA) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A. actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltxA mutation in vitro. Results indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly (P<0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivary-coated hydroxyapatite (SHA) was significantly decreased (P<0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [rcpA (P = 0.018), rcpB (P = 0.02), tadA (P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth (P = 0.008) and exo-polysaccharide production (P<0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltxA restored binding to SHA (P<0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltxA diminished hard tissue binding in vitro, which helps explain the previous in vivo failure of a ltxA knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltxA) could affect other seemingly unrelated genes (such as rcpA, rcpB tadA etc), and 2

  6. Profound Effects of Aggregatibacter actinomycetemcomitans Leukotoxin Mutation on Adherence Properties Are Clarified in in vitro Experiments.

    PubMed

    Velusamy, Senthil Kumar; Sampathkumar, Vandana; Godboley, Dipti; Fine, Daniel H

    2016-01-01

    Leukotoxin (Ltx) is a prominent virulence factor produced by Aggregatibacter actinomycetemcomitans, an oral microorganism highly associated with aggressive periodontitis. Ltx compromises host responsiveness by altering the viability of neutrophils, lymphocytes, and macrophages. Previously, we developed a Rhesus (Rh) monkey colonization model designed to determine the effect of virulence gene mutations on colonization of A. actinomycetemcomitans. Unexpectedly, an A. actinomycetemcomitans leukotoxin (ltxA) mutant (RhAa-VS2) failed to colonize in the Rh model. No previous literature suggested that Ltx was associated with A. actinomycetemcomitans binding to tooth surfaces. These results led us to explore the broad effects of the ltxA mutation in vitro. Results indicated that LtxA activity was completely abolished in RhAa-VS2 strain, while complementation significantly (P<0.0001) restored leukotoxicity compared to RhAa-VS2 strain. RT-PCR analysis of ltx gene expression ruled out polar effects. Furthermore, binding of RhAa-VS2 to salivary-coated hydroxyapatite (SHA) was significantly decreased (P<0.0001) compared to wild type RhAa3 strain. Real time RT-PCR analysis of the genes related to SHA binding in RhAa-VS2 showed that genes related to binding were downregulated [rcpA (P = 0.018), rcpB (P = 0.02), tadA (P = 0.002)] as compared to wild type RhAa3. RhAa-VS2 also exhibited decreased biofilm depth (P = 0.008) and exo-polysaccharide production (P<0.0001). Buccal epithelial cell (BEC) binding of RhAa-VS2 was unaffected. Complementation with ltxA restored binding to SHA (P<0.002) but had no effect on biofilm formation when compared to RhAa3. In conclusion, mutation of ltxA diminished hard tissue binding in vitro, which helps explain the previous in vivo failure of a ltxA knockout to colonize the Rh oral cavity. These results suggest that; 1) one specific gene knockout (in this case ltxA) could affect other seemingly unrelated genes (such as rcpA, rcpB tadA etc), and 2

  7. Prophage induction in lysogenic Aggregatibacter actinomycetemcomitans cells co-cultured with human gingival fibroblasts, and its effect on leukotoxin release.

    PubMed

    Stevens, Roy H; de Moura Martins Lobo Dos Santos, Caroline; Zuanazzi, David; de Accioly Mattos, Marcelo Barbosas; Ferreira, Davis Fernandes; Kachlany, Scott C; Tinoco, Eduardo M B

    2013-01-01

    Lysogeny is common among strains of the periodontal pathogen Aggregatibacter actinomycetemcomitans. Since lysogenic induction is known to result in the increased synthesis and release of bacterial toxins from lysogens, it would be important to elucidate the conditions under which induction of these bacteria may occur. Co-cultures of A. actinomycetemcomitans strains (either lysogenic or non-lysogenic) and human cells (either gingival fibroblasts or pharyngeal epithelial cells) were prepared. Following incubation, bacteriophage titers of up to 6.2 × 10(7) pfu/ml were detected in the cell-free, spent culture media from the co-cultures of the lysogenic A. actinomycetemcomitans strains and the fibroblasts. Little (maximum of 2 × 10(0) pfu/ml) or no titers of phage could be detected in the mono-cultures of the lysogenic A. actinomycetemcomitans strains alone. In contrast, no phage were detectable in the cell-free spent culture media of the lysogens cocultured with the epithelial cells. Futhermore, co-culture of the A. actinomycetemcomitans lysogens with the fibroblasts resulted in enhanced release of the A. actinomycetemcomitans leukotoxin into the culture medium, in comparison with the spent culture media from mono-cultures of the lysogens alone. These results are consistent with the concept that interaction with fibroblasts may mediate prophage induction in lysogenic strains of A. actinomycetemcomitans, and that leukotoxin release is greatly augmented following induction of the lysogens. PMID:23022667

  8. Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: interactions with human β2 integrins and erythrocytes.

    PubMed

    Reinholdt, Jesper; Poulsen, Knud; Brinkmann, Christel R; Hoffmann, Søren V; Stapulionis, Romualdas; Enghild, Jan J; Jensen, Uffe B; Boesen, Thomas; Vorup-Jensen, Thomas

    2013-02-01

    Aggregatibacter actinomycetemcomitans is a gram-negative, facultatively anaerobic cocco-bacillus and a frequent member of the human oral flora. It produces a leukotoxin, LtxA, belonging to the repeats-in-toxin (RTX) family of bacterial cytotoxins. LtxA efficiently kills neutrophils and mononuclear phagocytes. The known receptor for LtxA on leukocytes is integrin α(L)β(2) (LFA-1 or CD11a/CD18). However, the molecular mechanisms involved in LtxA-mediated cytotoxicity are poorly understood, partly because LtxA has proven difficult to prepare for experiments as free of contaminants and with its native structure. Here, we describe a protocol for the purification of LtxA from bacterial culture supernatant, which does not involve denaturing procedures. The purified LtxA was monodisperse, well folded as judged by the combined use of synchrotron radiation circular dichroism spectroscopy (SRCD) and in silico prediction of the secondary structure content, and free of bacterial lipopolysaccharide. The analysis by SRCD and similarity to a lipase from Pseudomonas with a known three dimensional structure supports the presence of a so-called beta-ladder domain in the C-terminal part of LtxA. LtxA rapidly killed K562 target cells transfected to express β(2) integrin. Cells expressing α(M)β(2) (CD11b/CD18) or α(X)β(2) (CD11c/CD18) were killed as efficiently as cells expressing α(L)β(2). Erythrocytes, which do not express β(2) integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the β(2) chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins α(M)β(2) and α(X)β(2) as novel receptors for LtxA.

  9. Mlc is a transcriptional activator with a key role in integrating cyclic AMP receptor protein and integration host factor regulation of leukotoxin RNA synthesis in Aggregatibacter actinomycetemcomitans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cAMP receptor protein (CRP) indirectly increases ltxA expression...

  10. Aggregatibacter actinomycetemcomitans leukotoxin (LtxA; Leukothera) induces cofilin dephosphorylation and actin depolymerization during killing of malignant monocytes.

    PubMed

    Kaur, Manpreet; Kachlany, Scott C

    2014-11-01

    Leukotoxin (LtxA; Leukothera), a protein toxin secreted by the oral bacterium Aggregatibacter actinomycetemcomitans, specifically kills white blood cells (WBCs). LtxA binds to the receptor known as lymphocyte function associated antigen-1 (LFA-1), a β2 integrin expressed only on the surface of WBCs. LtxA is being studied as a virulence factor that helps A. actinomycetemcomitans evade host defences and as a potential therapeutic agent for the treatment of WBC diseases. LtxA-mediated cell death in monocytes involves both caspases and lysosomes; however, the signalling proteins that regulate and mediate cell death remain largely unknown. We used a 2D-gel proteomics approach to analyse the global protein expression changes that occur in response to LtxA. This approach identified the protein cofilin, which underwent dephosphorylation upon LtxA treatment. Cofilin is a ubiquitous actin-binding protein known to regulate actin dynamics and is regulated by LIM kinase (LIMK)-mediated phosphorylation. LtxA-mediated cofilin dephosphorylation was dependent on LFA-1 and cofilin dephosphorylation did not occur when LFA-1 bound to its natural ligand, ICAM-1. Treatment of cells with an inhibitor of LIMK (LIMKi) also led to cofilin dephosphorylation and enhanced killing by LtxA. This enhanced sensitivity to LtxA coincided with an increase in lysosomal disruption, and an increase in LFA-1 surface expression and clustering. Both LIMKi and LtxA treatment also induced actin depolymerization, which could play a role in trafficking and surface distribution of LFA-1. We propose a model in which LtxA-mediated cofilin dephosphorylation leads to actin depolymerization, LFA-1 overexpression/clustering, and enhanced lysosomal-mediated cell death.

  11. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles

    PubMed Central

    Kieselbach, Thomas; Zijnge, Vincent; Granström, Elisabeth; Oscarsson, Jan

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. PMID:26381655

  12. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

    PubMed

    Kieselbach, Thomas; Zijnge, Vincent; Granström, Elisabeth; Oscarsson, Jan

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. PMID:26381655

  13. Virulence of Aggregatibacter actinomycetemcomitans serotypes and DGGE subtypes isolated from chronic adult periodontitis in Thailand.

    PubMed

    Pahumunto, Nuntiya; Ruangsri, Praphansri; Wongsuwanlert, Mutita; Piwat, Supatcharin; Dahlen, Gunnar; Teanpaisan, Rawee

    2015-12-01

    A high proportion of non-serotypeable isolates of Aggregatibacter actinomycetemcomitans among Thai periodontitis cases has been previously reported. The aim of this study was to investigate the expression of leukotoxin and toxicity, cytolethal distending toxin (Cdts), and internalization and the killing effect on fibroblasts by A. actinomycetemcomitans subtypes from Thai chronic periodontitis cases. A total of 96 A. actinomycetemcomitans strains from 37 periodontitis cases, previously serotyped with PCR and subtyped with DGGE, were examined for the presence of the ltx gene and cdt genes (cdtBC), and tested for leukotoxin expression, leukotoxicity, internalization, and apoptosis of fibroblast cells. The ltx gene was present in all isolates, while 84.4% showed the cdtBC gene. Two strains with a JP2-like ltx gene with a deletion of 530 bp in the promoter region, serotyped as c, showed virulence of similar magnitude to the JP2 strain. Furthermore, a higher virulence was found in the two non-serotypeable DGGE subtypes, NS1 and NS2, compared with the serotypeable strains (serotype a-f, serotype b and d were absent). Generally, the virulence of strains obtained from deep periodontal pockets was higher than those isolated from shallow non-bleeding pockets. A. actinomycetemcomitans subtypes isolated from adult Thais with chronic periodontitis showed a highly variable virulence, leukotoxin expression, leukotoxicity, internalization and apoptosis of fibroblast, and are regulated both genetically and environmentally. PMID:26529053

  14. The cell envelope proteome of Aggregatibacter actinomycetemcomitans

    PubMed Central

    Smith, Kenneth P.; Fields, Julia G.; Voogt, Richard D.; Deng, Bin; Lam, Ying-Wai; Mintz, Keith P.

    2014-01-01

    Summary The cell envelope of Gram-negative bacteria serves a critical role in maintenance of cellular homeostasis, resistance to external stress, and host-pathogen interactions. Envelope protein composition is influenced by the physiological and environmental demands placed on the bacterium. In this study, we report a comprehensive compilation of cell envelope proteins from the periodontal and systemic pathogen Aggregatibacter actinomycetemcomitans VT1169, an afimbriated serotype b strain. The urea-extracted membrane proteins were identified by mass spectrometry-based shotgun proteomics. The membrane proteome, isolated from actively growing bacteria under normal laboratory conditions, included 648 proteins representing 28% of the predicted ORFs in the genome. Bioinformatic analyses were used to annotate and predict the cellular location and function of the proteins. Surface adhesins, porins, lipoproteins, numerous influx and efflux pumps, multiple sugar, amino acid and iron transporters, and components of the type I, II and V secretion systems were identified. Periplasmic space and cytoplasmic proteins with chaperone function were also identified. 107 proteins with unknown function were associated with the cell envelope. Orthologs of a subset of these uncharacterized proteins are present in other bacterial genomes, while others are found exclusively in A. actinomycetemcomitans. This knowledge will contribute to elucidating the role of cell envelope proteins in bacterial growth and survival in the oral cavity. PMID:25055881

  15. Distribution of biotypes and leukotoxic activity of Aggregatibacter actinomycetemcomitans isolated from Brazilian patients with chronic periodontitis

    PubMed Central

    Gaetti-Jardim Jr., Elerson; Wahasugui, Thais Cristiane; Tomazinho, Paulo Henrique; Marques, Márcia Martins; Nakano, Viviane; Avila-Campos, Mario Julio

    2008-01-01

    Aggregatibacter actinomycetemcomitans is an important etiologic agent of the periodontitis and is associated with extra-oral infections. In this study, the detection of the ltxA gene as well as the ltx promoter region from leukotoxic A. actinomycetemcomitans isolated from 50 Brazilian patients with periodontitis and 50 healthy subjects was performed. The leukotoxic activity on HL-60 cells was also evaluated. Leukotoxic activity was determined using a trypan blue exclusion method. The 530 bp deletion in the promoter region was evaluated by PCR using a PRO primer pair. A. actinomycetemcomitans was detected by culture and directly from crude subgingival biofilm by PCR using specific primers. By culture, A. actinomycetemcomitans was detected in nine (18%) of the periodontal patients and one (2%) healthy subject. However, by PCR, this organism was detected in 44% of the periodontal patients and in 16% of the healthy subjects. It was verified a great discrepancy between PCR detection of the ltx operon promoter directly from crude subgingival biofilm and from bacterial DNA. Only one periodontal sample harbored highly leukotoxic A. actinomycetemcomitans. Moreover, biotype II was the most prevalent and no correlation between biotypes and leukotoxic activity was observed. The diversity of leukotoxin expression by A. actinomycetemcomitans suggests a role of this toxin in the pathogenesis of periodontal disease and other infectious diseases. PMID:24031284

  16. Characterization of leukotoxin from a clinical strain of Actinobacillus actinomycetemcomitans.

    PubMed

    Diaz, Roger; Ghofaily, Lourdes Al; Patel, Jigna; Balashova, Nataliya V; Freitas, Anna C; Labib, Irene; Kachlany, Scott C

    2006-02-01

    Actinobacillus actinomycetemcomitans is a Gram negative pathogen that is the etiologic agent of localized aggressive periodontitis (LAP), a rapidly progressing and severe disease of the oral cavity that affects predominantly adolescents. A. actinomycetemcomitans is also found in extraoral infections including infective endocarditis. As one of its many virulence determinants, A. actinomycetemcomitans produces the RTX (repeats in toxin) exotoxin, leukotoxin (LtxA). LtxA specifically kills leukocytes of humans and Old World Monkeys. All of our current knowledge of A. actinomycetemcomitans LtxA is based on the protein from strain JP2, a nonadherent laboratory isolate. Because laboratory isolates can lose virulence properties, we wished to examine LtxA from a clinical isolate, NJ4500. We show that localization patterns of LtxA do not differ between the strains. Subcellular localization studies with NJ4500 revealed that LtxA localizes to the outer membrane and that the interaction between LtxA and the surface of cells is specific. Surface localized LtxA was not removed with NaCl treatment and protease protection experiments revealed that approximately 10 kDa of LtxA is exposed. We purified secreted LtxA from NJ4500 and found that the specific activity of this toxin was greater than that of secreted LtxA from JP2. For other RTX toxins, fatty acid modification affects toxin activity, and A. actinomycetemcomitans LtxA is predicted to be modified. We show by two-dimensional gel electrophoresis that NJ4500 LtxA is more highly modified than JP2 LtxA, suggesting that the difference in activities could be due to differential modification. Studies of A. actinomycetemcomitans pathogenesis should therefore consider LtxA from clinical isolates.

  17. Stability of the JP2 clone of Aggregatibacter actinomycetemcomitans.

    PubMed

    Haubek, D; Ennibi, O-K; Vaeth, M; Poulsen, S; Poulsen, K

    2009-09-01

    The JP2 clone of Aggregatibacter actinomycetemcomitans is strongly associated with aggressive periodontitis. To obtain information about colonization dynamics of the JP2 clone, we used PCR to examine its presence in 365 Moroccan juveniles from whom periodontal plaque samples were collected at baseline and after one and two years. Periodontal attachment loss was measured at baseline and at the two-year follow-up. At baseline, 43 (12%) carriers of the JP2 clone were found. Nearly half (44 %) of these were persistently colonized with the clone. The relative risk for the development of aggressive periodontitis, adjusted for the concomitant presence of other genotypes of A. actinomycetemcomitans, was highest for individuals continuously infected by the JP2 clone (RR = 13.9; 95% CI, 9.0 to 21.4), indicating a relationship between infectious dose and disease, which further substantiates the evidence for the JP2 clone as a causal factor in aggressive periodontitis.

  18. Pathogenicity of the highly leukotoxic JP2 clone of Aggregatibacter actinomycetemcomitans and its geographic dissemination and role in aggressive periodontitis.

    PubMed

    Haubek, Dorte; Johansson, Anders

    2014-01-01

    For decades, Aggregatibacter actinomycetemcomitans has been associated with aggressive forms of periodontitis in adolescents. In the middle of the 1990s, a specific JP2 clone of A. actinomycetemcomitans, belonging to the cluster of serotype b strains of A. actinomycetemcomitans and having a number of other characteristics, was found to be strongly associated with aggressive forms of periodontitis, particularly in North Africa. Although several longitudinal studies still point to the bacterial species, A. actinomycetemcomitans as a risk factor of aggressive periodontitis, it is now also widely accepted that the highly leukotoxic JP2 clone of A. actinomycetemcomitans is implicated in rapidly progressing forms of aggressive periodontitis. The JP2 clone strains are highly prevalent in human populations living in Northern and Western parts of Africa. These strains are also prevalent in geographically widespread populations that have originated from the Northwest Africa. Only sporadic signs of a dissemination of the JP2 clone strains to non-African populations have been found despite Africans living geographically widespread for hundreds of years. It remains an unanswered question if a particular host tropism exists as a possible explanation for the frequent colonization of the Northwest African population with the JP2 clone. Two exotoxins of A. actinomycetemcomitans are known, leukotoxin (LtxA) and cytolethal distending toxin (Cdt). LtxA is able to kill human immune cells, and Cdt can block cell cycle progression in eukaryotic cells and thus induce cell cycle arrest. Whereas the leukotoxin production is enhanced in JP2 clone strains thus increasing the virulence potential of A. actinomycetemcomitans, it has not been possible so far to demonstrate such a role for Cdt. Lines of evidence have led to the understanding of the highly leukotoxic JP2 clone of A. actinomycetemcomitans as an aetiological factor of aggressive periodontitis. Patients, who are colonized with the JP2

  19. Pathogenicity of the highly leukotoxic JP2 clone of Aggregatibacter actinomycetemcomitans and its geographic dissemination and role in aggressive periodontitis

    PubMed Central

    Haubek, Dorte; Johansson, Anders

    2014-01-01

    For decades, Aggregatibacter actinomycetemcomitans has been associated with aggressive forms of periodontitis in adolescents. In the middle of the 1990s, a specific JP2 clone of A. actinomycetemcomitans, belonging to the cluster of serotype b strains of A. actinomycetemcomitans and having a number of other characteristics, was found to be strongly associated with aggressive forms of periodontitis, particularly in North Africa. Although several longitudinal studies still point to the bacterial species, A. actinomycetemcomitans as a risk factor of aggressive periodontitis, it is now also widely accepted that the highly leukotoxic JP2 clone of A. actinomycetemcomitans is implicated in rapidly progressing forms of aggressive periodontitis. The JP2 clone strains are highly prevalent in human populations living in Northern and Western parts of Africa. These strains are also prevalent in geographically widespread populations that have originated from the Northwest Africa. Only sporadic signs of a dissemination of the JP2 clone strains to non-African populations have been found despite Africans living geographically widespread for hundreds of years. It remains an unanswered question if a particular host tropism exists as a possible explanation for the frequent colonization of the Northwest African population with the JP2 clone. Two exotoxins of A. actinomycetemcomitans are known, leukotoxin (LtxA) and cytolethal distending toxin (Cdt). LtxA is able to kill human immune cells, and Cdt can block cell cycle progression in eukaryotic cells and thus induce cell cycle arrest. Whereas the leukotoxin production is enhanced in JP2 clone strains thus increasing the virulence potential of A. actinomycetemcomitans, it has not been possible so far to demonstrate such a role for Cdt. Lines of evidence have led to the understanding of the highly leukotoxic JP2 clone of A. actinomycetemcomitans as an aetiological factor of aggressive periodontitis. Patients, who are colonized with the JP2

  20. Complete Genome Sequence of Aggregatibacter actinomycetemcomitans Serotype g Strain NUM4039 (JCM 30399)

    PubMed Central

    Saito, Masanori; Hirasawa, Masaaki; Kuwahara, Noriko; Okada, Tamami; Umezawa, Koji; Kobayashi, Taira; Okamoto, Masaaki; Naito, Mariko; Hirasawa, Masatomo

    2016-01-01

    Aggregatibacter actinomycetemcomitans is considered to be a major etiological agent of aggressive periodontitis and includes serotype a to g strains. We herein report the first complete genome sequence of A. actinomycetemcomitans serotype g strain NUM4039. The genome is 2,382,853 bp in length with a G+C content of 44.34%. PMID:26988057

  1. Aggregatibacter actinomycetemcomitans induces Th17 cells in atherosclerotic lesions.

    PubMed

    Jia, Ru; Hashizume-Takizawa, Tomomi; Du, Yuan; Yamamoto, Masafumi; Kurita-Ochiai, Tomoko

    2015-04-01

    Th17 cells have been linked to the pathogenesis of several chronic inflammatory and autoimmune diseases. However, the role of Th17 cells and IL-17 in atherosclerosis remains poorly understood. We previously reported that Aggregatibacter actinomycetemcomitans (Aa) bacteremia accelerated atherosclerosis accompanied by inflammation in apolipoprotein E-deficient spontaneously hyperlipidemic (Apoe(shl)) mice. In this study, we investigated whether Aa promotes the Th17 inducing pathway in Aa-challenged Apoe(shl) mice. Mice were intravenously injected with live Aa HK1651 or vehicles. Time-course analysis of splenic IL-17(+)CD4(+) cell frequencies, the proximal aorta lesion area, serum IL-17, IL-6, TGF-β and IL-1β levels, the mRNA expression of Th17-related molecules such as IL-1β, IL-6, IL17RA, STAT3, IL-21, IL-23, TGF-β and RORγt, Th17-related microRNA levels and the levels of AIM-2, Mincle and NLRP3 were examined. Challenge with Aa time dependently induced tropism of Th17 cells in the spleen and increase in atheromatous lesions in the aortic sinus of Apoe(shl) mice. Serum IL-17, IL-6, TGF-β and IL-1β levels were significantly enhanced by Aa. The gene expression of IL-1β, IL-6, IL-17RA, IL-21, IL-23, TGF-β, STAT3, RORγt, AIM-2, Mincle and NLRP3 was also time dependently stimulated in the aorta of Aa-challenged mice. Furthermore, Aa challenge significantly increased the expression of miR-146b and miR-155 in the aorta. Based on the results, it seems that Aa stimulates Th17 induction that affects the progression of Aa-accelerated atherosclerosis. PMID:25743474

  2. Aggregatibacter actinomycetemcomitans accelerates atherosclerosis with an increase in atherogenic factors in spontaneously hyperlipidemic mice.

    PubMed

    Zhang, Tao; Kurita-Ochiai, Tomoko; Hashizume, Tomomi; Du, Yuan; Oguchi, Sumito; Yamamoto, Masafumi

    2010-07-01

    Cariogenic and periodontal pathogens are thought to be etiological factors in the development of cardiovascular disease. We assessed the involvement of the periodontal pathogen Aggregatibacter actinomycetemcomitans and cariogenic pathogen Streptococcus mutans in the development of atherosclerosis in apolipoprotein E-deficient spontaneously hyperlipidemic (Apoe(shl)) mice. The mice were treated intravenously with A. actinomycetemcomitans HK1651, S. mutans GS-5, or phosphate-buffered saline three times a week for 3 weeks and killed at 15 weeks of age. The areas of the aortic sinus that were covered with atherosclerotic plaque were significantly larger in Apoe(shl) mice challenged with A. actinomycetemcomitans compared with S. mutans- or vehicle-challenged mice. Aggregatibacter actinomycetemcomitans challenge increased serum high-sensitive C-reactive protein and lipopolysaccharide levels. Bacterial DNA was detected in the blood, heart, and spleen, but not in the liver. Furthermore, serum interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha, and MCP-1 levels and Toll-like receptor (TLR)2, TLR4, ICAM-1, E-selectin, P-selectin, LOX-1, HSP60, CCL19, CCL21, CCR7, and MCP-1 expressions in the aorta were significantly increased in mice challenged with A. actinomycetemcomitans. These results suggest that systemic infection with A. actinomycetemcomitans accelerates atherosclerosis in Apoe(shl) mice by exposing the whole microorganisms or their products, followed by initiating inflammation. Increases in proatherogenic factors may explain the aggravation of atherosclerosis by A. actinomycetemcomitans infection. PMID:20482627

  3. LL-37 opsonizes and inhibits biofilm formation of Aggregatibacter actinomycetemcomitans at subbactericidal concentrations.

    PubMed

    Sol, Asaf; Ginesin, Ofir; Chaushu, Stella; Karra, Laila; Coppenhagen-Glazer, Shunit; Ginsburg, Isaac; Bachrach, Gilad

    2013-10-01

    Host defense peptides are immediate responders of the innate immunity that express antimicrobial, immunoregulatory, and wound-healing activities. Neutrophils are a major source for oral host defense peptides, and phagocytosis by neutrophils is a major mechanism for bacterial clearance in the gingival tissue. Dysfunction of or reduction in the numbers of neutrophils or deficiency in the LL-37 host defense peptide was each previously linked with proliferation of oral Aggregatibacter actinomycetemcomitans which resulted in an aggressive periodontal disease. Surprisingly, A. actinomycetemcomitans shows resistance to high concentrations of LL-37. In this study, we demonstrated that submicrocidal concentrations of LL-37 inhibit biofilm formation by A. actinomycetemcomitans and act as opsonins and agglutinins that greatly enhance its clearance by neutrophils and macrophages. Improved uptake of A. actinomycetemcomitans by neutrophils was mediated by their opsonization with LL-37. Enhanced phagocytosis and killing of A. actinomycetemcomitans by murine macrophage-like RAW 264.7 cells were dependent on their preagglutination by LL-37. Although A. actinomycetemcomitans is resistant to the bactericidal effect of LL-37, our results offer a rationale for the epidemiological association between LL-37 deficiency and the expansion of oral A. actinomycetemcomitans and indicate a possible therapeutic use of cationic peptides for host defense.

  4. Mature Biofilm Degradation by Potential Probiotics: Aggregatibacter actinomycetemcomitans versus Lactobacillus spp.

    PubMed Central

    Mizuno, Kouhei; Okinaga, Toshinori

    2016-01-01

    The biofilm degradation of Aggregatibacter actinomycetemcomitans is essential as a complete periodontal disease therapy, and here we show the effects of potential probiotic bacteria such as Lactobacillus spp. for the biofilm of several serotypes of A. actinomycetemcomitans strains. Eight of the 13 species showed the competent biofilm degradation of ≥ 90% reduction in biofilm values in A. actinomycetemcomitans Y4 (serotype b) as well as four of the seven species for the biofilm of A. actinomycetemcomitans OMZ 534 (serotype e). In contrast, the probiotic bacteria did not have a big impact for the degradation of A. actinomycetemcomitans SUNY 75 (serotype a) biofilm. The dispersed A. actinomycetemcomitans Y4 cells through the biofilm detachment were still viable and plausible factors for the biofilm degradation were not due to the lactic acid and low pH conditions. The three enzymes, protease, lipase, and amylase may be responsible for the biofilm degradation; in particular, lipase was the most effective enzyme for the biofilm degradation of A. actinomycetemcomitans Y4 along with the protease activity which should be also important for the other serotypes. Remarkable lipase enzyme activities were detected from some of the potential probiotics and a supporting result using a lipase inhibitor presented corroborating evidence that lipase activity is one of the contributing factors for biofilm degradation outside of the protease which is also another possible factor for the biofilm of the other serotype of A. actinomycetemcomitans strains. On the other hand, the biofilm of A. actinomycetemcomitans SUNY 75 (serotype a) was not powerfully degraded by the lipase enzyme because the lipase inhibitor was slightly functional for only two of potential probiotics. PMID:27438340

  5. Antimicrobial effect of chlorhexidine on Aggregatibacter actinomycetemcomitans biofilms associated with peri-implantitis

    PubMed Central

    Kadkhoda, Zeinab; Amarlu, Zeinab; Eshraghi, Saeed; Samiei, Nazanin

    2016-01-01

    Background. This study aimed to assessthe antimicrobial effect of chlorhexidine (CHX) on Aggregatibacter actinomycetemcomitans biofilms isolated from subgingival plaque of peri-implantitis lesions. Methods. Thirteen patients requiring peri-implantitis treatment were consecutively selected and their subgingival biofilm was collected by inserting fine sterile paper points into peri-implant pockets for 15 seconds. A. actinomycetemcomitans was isolated from the subgingival biofilm and cultured. In this study, the standard strain of A. actinomycetemcomitans served as the positive control group and a blank disc impregnated with water served as the negative control; 0.1 mL of the bacterial suspension was cultured on specific culture medium and blank discs (6 mm in diameter) impregnated with 0.2%CHX mouthrinse (Behsa Pharmaceutical Co.) and negative control discs were placed on two sides of the bacterial culture plate. The size of growth inhibition zone was measured by a blinded independent observer in millimetres. Results. According to the results of disc diffusion test, the mean diameter of growth inhibition zone of A. actinomycetemcomitans around discs impregnated with CHX was larger in both standard (positive control) and biofilm samples of A. actinomycetemcomitans compared to the negative control group (blank disc) (P<0.001). Conclusion. Use of0.2% CHX mouthwash had antibacterial effects on A. actinomycetemcomitans species isolated from peri-implantitis sites.

  6. Antimicrobial effect of chlorhexidine on Aggregatibacter actinomycetemcomitans biofilms associated with peri-implantitis.

    PubMed

    Kadkhoda, Zeinab; Amarlu, Zeinab; Eshraghi, Saeed; Samiei, Nazanin

    2016-01-01

    Background. This study aimed to assessthe antimicrobial effect of chlorhexidine (CHX) on Aggregatibacter actinomycetemcomitans biofilms isolated from subgingival plaque of peri-implantitis lesions. Methods. Thirteen patients requiring peri-implantitis treatment were consecutively selected and their subgingival biofilm was collected by inserting fine sterile paper points into peri-implant pockets for 15 seconds. A. actinomycetemcomitans was isolated from the subgingival biofilm and cultured. In this study, the standard strain of A. actinomycetemcomitans served as the positive control group and a blank disc impregnated with water served as the negative control; 0.1 mL of the bacterial suspension was cultured on specific culture medium and blank discs (6 mm in diameter) impregnated with 0.2%CHX mouthrinse (Behsa Pharmaceutical Co.) and negative control discs were placed on two sides of the bacterial culture plate. The size of growth inhibition zone was measured by a blinded independent observer in millimetres. Results. According to the results of disc diffusion test, the mean diameter of growth inhibition zone of A. actinomycetemcomitans around discs impregnated with CHX was larger in both standard (positive control) and biofilm samples of A. actinomycetemcomitans compared to the negative control group (blank disc) (P<0.001). Conclusion . Use of0.2% CHX mouthwash had antibacterial effects on A. actinomycetemcomitans species isolated from peri-implantitis sites. PMID:27651884

  7. Antimicrobial effect of chlorhexidine on Aggregatibacter actinomycetemcomitans biofilms associated with peri-implantitis

    PubMed Central

    Kadkhoda, Zeinab; Amarlu, Zeinab; Eshraghi, Saeed; Samiei, Nazanin

    2016-01-01

    Background. This study aimed to assessthe antimicrobial effect of chlorhexidine (CHX) on Aggregatibacter actinomycetemcomitans biofilms isolated from subgingival plaque of peri-implantitis lesions. Methods. Thirteen patients requiring peri-implantitis treatment were consecutively selected and their subgingival biofilm was collected by inserting fine sterile paper points into peri-implant pockets for 15 seconds. A. actinomycetemcomitans was isolated from the subgingival biofilm and cultured. In this study, the standard strain of A. actinomycetemcomitans served as the positive control group and a blank disc impregnated with water served as the negative control; 0.1 mL of the bacterial suspension was cultured on specific culture medium and blank discs (6 mm in diameter) impregnated with 0.2%CHX mouthrinse (Behsa Pharmaceutical Co.) and negative control discs were placed on two sides of the bacterial culture plate. The size of growth inhibition zone was measured by a blinded independent observer in millimetres. Results. According to the results of disc diffusion test, the mean diameter of growth inhibition zone of A. actinomycetemcomitans around discs impregnated with CHX was larger in both standard (positive control) and biofilm samples of A. actinomycetemcomitans compared to the negative control group (blank disc) (P<0.001). Conclusion. Use of0.2% CHX mouthwash had antibacterial effects on A. actinomycetemcomitans species isolated from peri-implantitis sites. PMID:27651884

  8. Diverse Toll-like receptors mediate cytokine production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in macrophages.

    PubMed

    Park, Se-Ra; Kim, Dong-Jae; Han, Seung-Hyun; Kang, Min-Jung; Lee, Jun-Young; Jeong, Yu-Jin; Lee, Sang-Jin; Kim, Tae-Hyoun; Ahn, Sang-Gun; Yoon, Jung-Hoon; Park, Jong-Hwan

    2014-05-01

    Toll-like receptors (TLRs) orchestrate a repertoire of immune responses in macrophages against various pathogens. Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans are two important periodontal pathogens. In the present study, we investigated TLR signaling regulating cytokine production of macrophages in response to F. nucleatum and A. actinomycetemcomitans. TLR2 and TLR4 are redundant in the production of cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) in F. nucleatum- and A. actinomycetemcomitans-infected macrophages. The production of cytokines by macrophages in response to F. nucleatum and A. actinomycetemcomitans infection was impaired in MyD88-deficient macrophages. Moreover, cytokine concentrations were lower in MyD88-deficient macrophages than in TLR2/TLR4 (TLR2/4) double-deficient cells. An endosomal TLR inhibitor, chloroquine, reduced cytokine production in TLR2/4-deficient macrophages in response to F. nucleatum and A. actinomycetemcomitans, and DNA from F. nucleatum or A. actinomycetemcomitans induced IL-6 production in bone marrow-derived macrophages (BMDMs), which was abolished by chloroquine. Western blot analysis revealed that TLR2/4 and MyD88 were required for optimal activation of NF-κB and mitogen-activated protein kinases (MAPKs) in macrophages in response to F. nucleatum and A. actinomycetemcomitans, with different kinetics. An inhibitor assay showed that NF-κB and all MAPKs (p38, extracellular signal-regulated kinase [ERK], and Jun N-terminal protein kinase [JNK]) mediate F. nucleatum-induced production of cytokines in macrophages, whereas NF-κB and p38, but not ERK and JNK, are involved in A. actinomycetemcomitans-mediated cytokine production. These findings suggest that multiple TLRs may participate in the cytokine production of macrophages against periodontal bacteria.

  9. Trimeric Form of Intracellular ATP Synthase Subunit β of Aggregatibacter actinomycetemcomitans Binds Human Interleukin-1β

    PubMed Central

    Paino, Annamari; Tuominen, Heidi; Jääskeläinen, Mari; Alanko, Jonna; Nuutila, Jari; Asikainen, Sirkka E.; Pelliniemi, Lauri J.; Pöllänen, Marja T.; Chen, Casey; Ihalin, Riikka

    2011-01-01

    Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1β, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1β receptor has been identified, current knowledge of the bacterial IL-1β sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1β than inactive ones. The effect of IL-1β on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1β to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1β, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1β and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1β slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1β. Our results suggest that IL-1β might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit β interacted with IL-1β, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1β during inflammation. PMID:21533109

  10. Human Serum-Specific Activation of Alternative Sigma Factors, the Stress Responders in Aggregatibacter actinomycetemcomitans

    PubMed Central

    Tang-Siegel, Gaoyan; Bumgarner, Roger; Ruiz, Teresa; Kittichotirat, Weerayuth; Chen, Weizhen; Chen, Casey

    2016-01-01

    Aggregatibacter actinomycetemcomitans, a known pathogen causing periodontal disease and infective endocarditis, is a survivor in the periodontal pocket and blood stream; both environments contain serum as a nutrient source. To screen for unknown virulence factors associated with this microorganism, A. actinomycetemcomitans was grown in serum-based media to simulate its in vivo environment. Different strains of A. actinomycetemcomitans showed distinct growth phenotypes only in the presence of human serum, and they were grouped into high- and low-responder groups. High-responders comprised mainly serotype c strains, and showed an unusual growth phenomenon, featuring a second, rapid increase in turbidity after 9-h incubation that reached a final optical density 2- to 7-fold higher than low-responders. Upon further investigation, the second increase in turbidity was not caused by cell multiplication, but by cell death. Whole transcriptomic analysis via RNA-seq identified 35 genes that were up-regulated by human serum, but not horse serum, in high-responders but not in low-responders, including prominently an alternative sigma factor rpoE (σE). A lacZ reporter construct driven by the 132-bp rpoE promoter sequence of A. actinomycetemcomitans responded dramatically to human serum within 90 min of incubation only when the construct was carried by a high responder strain. The rpoE promoter is 100% identical among high- and low-responder strains. Proteomic investigation showed potential interactions between human serum protein, e.g. apolipoprotein A1 (ApoA1) and A. actinomycetemcomitans. The data clearly indicated a different activation process for rpoE in high- versus low-responder strains. This differential human serum-specific activation of rpoE, a putative extra-cytoplasmic stress responder and global regulator, suggests distinct in vivo adaptations among different strains of A. actinomycetemcomitans. PMID:27490177

  11. Human Serum-Specific Activation of Alternative Sigma Factors, the Stress Responders in Aggregatibacter actinomycetemcomitans.

    PubMed

    Tang-Siegel, Gaoyan; Bumgarner, Roger; Ruiz, Teresa; Kittichotirat, Weerayuth; Chen, Weizhen; Chen, Casey

    2016-01-01

    Aggregatibacter actinomycetemcomitans, a known pathogen causing periodontal disease and infective endocarditis, is a survivor in the periodontal pocket and blood stream; both environments contain serum as a nutrient source. To screen for unknown virulence factors associated with this microorganism, A. actinomycetemcomitans was grown in serum-based media to simulate its in vivo environment. Different strains of A. actinomycetemcomitans showed distinct growth phenotypes only in the presence of human serum, and they were grouped into high- and low-responder groups. High-responders comprised mainly serotype c strains, and showed an unusual growth phenomenon, featuring a second, rapid increase in turbidity after 9-h incubation that reached a final optical density 2- to 7-fold higher than low-responders. Upon further investigation, the second increase in turbidity was not caused by cell multiplication, but by cell death. Whole transcriptomic analysis via RNA-seq identified 35 genes that were up-regulated by human serum, but not horse serum, in high-responders but not in low-responders, including prominently an alternative sigma factor rpoE (σE). A lacZ reporter construct driven by the 132-bp rpoE promoter sequence of A. actinomycetemcomitans responded dramatically to human serum within 90 min of incubation only when the construct was carried by a high responder strain. The rpoE promoter is 100% identical among high- and low-responder strains. Proteomic investigation showed potential interactions between human serum protein, e.g. apolipoprotein A1 (ApoA1) and A. actinomycetemcomitans. The data clearly indicated a different activation process for rpoE in high- versus low-responder strains. This differential human serum-specific activation of rpoE, a putative extra-cytoplasmic stress responder and global regulator, suggests distinct in vivo adaptations among different strains of A. actinomycetemcomitans. PMID:27490177

  12. Aggregatibacter actinomycetemcomitans arcB influences hydrophobic properties, biofilm formation and adhesion to hydroxyapatite

    PubMed Central

    Longo, PL; Ota-Tsuzuki, C; Nunes, ACR; Fernandes, BL; Mintz, K; Fives-Taylor, P; Mayer, MPA

    2009-01-01

    The regulation of gene expression in the oral pathogen Aggregatibacter actinomycetemcomitans is still not fully elucidated. ArcAB is a two-component system which allows facultative anaerobic bacteria to sense various respiratory growth conditions and adapt their gene expression accordingly.This study investigated in A. actinomycetemcomitans the role of ArcB on the regulation of biofilm formation, adhesion to saliva coated hydroxyapatite (SHA) and the hydrophobic properties of the cell. These phenotypic traits were determined for an A. actinomycetemcomitans arcB deficient type and a wild type strain. Differences in hydrophobic properties were shown at early and late exponential growth phases under microaerobic incubation and at late exponential phase under anaerobiosis.The arcB mutant formed less biofilm than the wild type strain when grown under anaerobic incubation, but displayed higher biofilm formation activity under microaerobic conditions. The adherence to SHA was significantly lower in the mutant when compared with the wild type strain. These results suggest that the transmembrane sensor kinase ArcB, in A. actinomycetemcomitans, senses redox growth conditions and regulates the expression of surface components of the bacterial cell related to biofilm formation and adhesion to saliva coated surfaces. PMID:24031399

  13. Azithromycin Enhances Phagocytic Killing of Aggregatibacter actinomycetemcomitans Y4 by Human Neutrophils

    PubMed Central

    Lai, Pin-Chuang; Schibler, Mark R.; Walters, John D.

    2015-01-01

    Background Aggregatibacter actinomycetemcomitans resists killing by neutrophils and is inhibited by azithromycin (AZM) and amoxicillin (AMX). AZM actively concentrates inside host cells, whereas AMX enters by diffusion. The present study is conducted to determine whether AZM is more effective than AMX at enhancing phagocytic killing of A. actinomycetemcomitans by neutrophils. Methods Killing assays were conducted in the presence of either 2 μg/mL AZM or 16 μg/mL AMX (equipotent against A. actinomycetemcomitans). Neutrophils were loaded by incubation with the appropriate antibiotic. Opsonized A. actinomycetemcomitans strain Y4 was incubated with the indicated antibiotic alone, with loaded neutrophils and antibiotic, or with control neutrophils (without antibiotic) at multiplicities of infection (MOIs) of 30 and 90 bacteria per neutrophil. Results Neutrophil incubation with 2 μg/mL AZM yielded an intracellular concentration of 10 μg/mL. At an MOI of 30, neutrophils loaded with AZM failed to kill significantly more bacteria than control neutrophils during the 60- and 90-minute assay periods. At an MOI of 90, neutrophils loaded with AZM killed significantly more bacteria than either AZM alone or control neutrophils during 60- and 90-minute incubations (P <0.05), and killed significantly more bacteria after 90 minutes than the sum of the killing produced by AZM alone or neutrophils alone. Neutrophils incubated with AMX under identical conditions also killed significantly more bacteria than either AMX alone or control neutrophils, but there was no evidence of synergism between AMX and neutrophils. Conclusions Neutrophils possess a concentrative transport system for AZM that may enhance killing of A. actinomycetemcomitans. Its effects are most pronounced when neutrophils are greatly outnumbered by bacteria. PMID:25186779

  14. [Microbiological approach to a possible infective endocarditis case caused by Aggregatibacter actinomycetemcomitans].

    PubMed

    Gürcan, Şaban; Ünlü, Selahattin; Kuloğlu, Figen; Karadenizli, Aynur; Kuşkucu, Mert Ahmet

    2016-04-01

    Aggregatibacter (Actinobacillus) actinomycetemcomitans, a small, gram-negative coccobacillus that grows slow and fastidious, is generally colonized in the oral cavity. It is a rarely seen bacterium because of the difficulty of isolation but it can be a causative agent for dental infections and infective endocarditis (IE) particularly in the persons having prosthetic heart valves. In this report, a possible IE case caused by A.actinomycetemcomitans in a patient with aortic valve replacement has been presented. A 36-year-old man has admitted to Trakya University Hospital, Health Center for Medical Research and Practice, with the complaints of chills, malaise, intermittent fever, severe arthralgia and weight loss (20 kg). During his follow-up period, the blood cultures that were obtained three week intervals yielded the identical gram-negative coccobacilli morphology. The patient was then diagnosed as possible IE on the basis of having one major (growth of the typical microorganisms that may cause IE in two different blood cultures) and two minor (presence of prosthetic valve and high fever) criterias. The isolate could not be identified with conventional methods, while it was identified as Francisella tularensis with VITEK 2 (bioMerieux, France) system. Hence this identification was not confirmed by real-time Taqman polymerase chain reaction, so MALDI-TOF mass spectrometry was used to identify this bacteria. In the first run of the study, the isolate was named as Shigella dysenteriae initially, however when it was retested the next day it was identified as A.actinomycetemcomitans. In order to enlighten these conflicting results, 16S and 23S ribosomal DNA sequence analysis was performed, and consequently the bacterium was identified as A.actinomycetemcomitans. Doxycycline (2 x 100 mg po, 20 days) and streptomycin (2 x 10 mg/kg im, 10 days) therapy were initiated, considering the initial suspicious identification (F.tularensis), and on the fifth day of therapy the

  15. Photosensitization of Aggregatibacter actinomycetemcomitans with methylene blue: a microbiological and spectroscopic study

    NASA Astrophysics Data System (ADS)

    Yamada Júnior, Aécio M.; Prates, Renato A.; Cai, Silvana; Ribeiro, Martha S.

    2008-03-01

    The aim of this study was to determinate the efficiency of methylene blue (MB) to kill cultures of Aggregatibacter actinomycetemcomitans under red light and to investigate MB photobleaching by optical absorption spectroscopy. Bacteria were diluted in aqueous solution, putted in glass tubes and distributed in 5 groups: (L-MB-) control group; (L+MB-) laser alone by 5min; (L-MB+) MB alone through 5min; (3L+MB+) MB+laser 3min; (5L+MB+) MB+laser 5min. Laser parameters were P=30mW, λ=660nm, E=9J in 5min and E=5.4J in 3min. The samples were diluted and bacterial colonies were counted and converted into colony forming units (CFU). Absorption spectra of the MB-stained bacterial suspension and photosensitized bacterial suspension were obtained. Groups L-MB-, L+MB-, and L-MB+ did not show a decrease in CFU/mL. L+MB+ groups showed a significant decrease in CFU/mL but no statistically significant differences were observed between 3min and 5min. Spectroscopy showed that MB is photodegraded after irradiation and that dimer species are more notably consumed than monomeric species. These results suggest that MB is a suitable photosensitizer to reduce A. actinomycetemcomitans, and that 3min of irradiation are enough to produce a significant effect. Due to the spectral changes observed on MB solution after irradiation a type I mechanism may be involved.

  16. Serotype-dependent expression patterns of stabilized lipopolysaccharide aggregates in Aggregatibacter actinomycetemcomitans strains.

    PubMed

    Kikuchi, Haruko; Fujise, Osamu; Miura, Mayumi; Tanaka, Ayako; Hisano, Kyoko; Haraguchi, Akira; Hamachi, Takafumi; Maeda, Katsumasa

    2012-10-01

    Above a critical concentration, amphiphilic lipopolysaccharide (LPS) molecules in an aqueous environment form aggregate structures, probably because of interactions involving hydrophobic bonds. Ionic bonds involving divalent cations stabilize these aggregate structures, making them resistant to breakdown by detergents. The aim of this study was to examine expression patterns of stabilized LPS aggregates in Aggregatibacter actinomycetemcomitans, a microorganism that causes periodontitis. A. actinomycetemcomitans strains of various serotypes and truncated LPS mutants were prepared for this study. Following treatment with a two-phase separation system using the detergent Triton X-114, crude LPS extracts of the study strains were separated into detergent-phase LPS (DP-LPS) and aqueous-phase LPS (AP-LPS). Repeated treatment of the aqueous phase with the two-phase separation system produced only a slight decrease in AP-LPS, suggesting that AP-LPS was resistant to the detergent and thus distinguishable from DP-LPS. The presence of divalent cations increased the yield of AP-LPS. AP-LPS expression patterns were serotype-dependent; serotypes b and f showing early expression, and serotypes a and c late expression. In addition, highly truncated LPS from a waaD (rfaD) mutant were unable to generate AP-LPS, suggesting involvement of the LPS structure in the generation of AP-LPS. The two-phase separation was able to distinguish two types of LPS with different physical states at the supramolecular structure level. Hence, AP-LPS likely represents stabilized LPS aggregates, whereas DP-LPS might be derived from non-stabilized aggregates. Furthermore, time-dependent expression of stabilized LPS aggregates was found to be serotype-dependent in A. actinomycetemcomitans.

  17. Detection of antimicrobial activity of banana peel (Musa paradisiaca L.) on Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans: An in vitro study

    PubMed Central

    Kapadia, Suraj Premal; Pudakalkatti, Pushpa S.; Shivanaikar, Sachin

    2015-01-01

    Introduction and Aim: Banana is used widely because of its nutritional values. In past, there are studies that show banana plant parts, and their fruits can be used to treat the human diseases. Banana peel is a part of banana fruit that also has the antibacterial activity against microorganisms but has not been studied extensively. Since, there are no studies that relate the antibacterial activity of banana peel against periodontal pathogens. Hence, the aim of this study is to determine the antimicrobial activity of banana peel extract on Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans). Material and Methods: Standard strains of P. gingivalis and A. actinomycetemcomitans were used in this study which was obtained from the in-house bacterial bank of Department of Molecular Biology and Immunology at Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre. The banana peel extract was prepared, and the antibacterial activity was assessed using well agar diffusion method and minimum inhibitory concentration was assessed using serial broth dilution method. Results: In the current study, both the tested microorganisms showed antibacterial activity. In well diffusion method, P. gingivalis and A. actinomycetemcomitans showed 15 mm and 12 mm inhibition zone against an alcoholic extract of banana peel, respectively. In serial broth dilution method P. gingivalis and A. actinomycetemcomitans were sensitive until 31.25 μg/ml dilutions. Conclusion: From results of the study, it is suggested that an alcoholic extract of banana peel has antimicrobial activity against P. gingivalis and A. actinomycetemcomitans. PMID:26681854

  18. Construction of new cloning, lacZ reporter and scarless-markerless suicide vectors for genetic studies in Aggregatibacter actinomycetemcomitans.

    PubMed

    Juárez-Rodríguez, María Dolores; Torres-Escobar, Ascención; Demuth, Donald R

    2013-05-01

    To elucidate the putative function of a gene, effective tools are required for genetic characterization that facilitate its inactivation, deletion or modification on the bacterial chromosome. In the present study, the nucleotide sequence of the Escherichia coli/Aggregatibacter actinomycetemcomitans shuttle vector pYGK was determined, allowing us to redesign and construct a new shuttle cloning vector, pJT4, and promoterless lacZ transcriptional/translational fusion plasmids, pJT3 and pJT5. Plasmids pJT4 and pJT5 contain the origin of replication necessary to maintain shuttle vector replication. In addition, a new suicide vector, pJT1, was constructed for the generation of scarless and markerless deletion mutations of genes in the oral pathogen A. actinomycetemcomitans. Plasmid pJT1 is a pUC-based suicide vector that is counter-selectable for sucrose sensitivity. This vector does not leave antibiotic markers or scars on the chromosome after gene deletion and thus provides the option to combine several mutations in the same genetic background. The effectiveness of pJT1 was demonstrated by the construction of A. actinomycetemcomitans isogenic qseB single deletion (ΔqseB) mutant and lsrRK double deletion mutants (ΔlsrRK). These new vectors may offer alternatives for genetic studies in A. actinomycetemcomitans and other members of the HACEK (Haemophilus spp., A. actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) group of Gram-negative bacteria.

  19. Development of an Animal Model for Aggregatibacter Actinomycetemcomitans Biofilm-Mediated Oral Osteolytic Infection: A Preliminary Study

    PubMed Central

    Freire, Marcelo O.; Sedghizadeh, Parish P.; Schaudinn, Christoph; Gorur, Amita; Downey, Jennifer S.; Choi, Jeong-Ho; Chen, Weizhen; Kook, Joong-Ki; Chen, Casey; Goodman, Steven D.; Zadeh, Homayoun H.

    2012-01-01

    Background Biofilm-induced inflammatory osteolytic oral infections, such as periodontitis and peri-implantitis, have complex etiology and pathogenesis. A significant obstacle to research has been the lack of appropriate animal models where the inflammatory response to biofilms can be investigated. The aim of this study is to develop a novel animal model to study the host response to Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans)–biofilm colonizing titanium implants. Methods Titanium implants were inoculated in vitro with A. actinomycetemcomitans, establishing a biofilm for 1 to 3 days. Biofilm-inoculated and control implants were transmucosally placed into rat hard palate or alveolar ridge. Analysis included documentation of clinical inflammation, polymerase chain reaction, and culture detection of A. actinomycetemcomitans and microcomputed tomography quantitation of peri-implant bone volume. Results Viable A. actinomycetemcomitans biofilm was successfully established on titanium implants in vitro, detected by confocal laser scanning microscopy. An inflammatory response characterized by clinical inflammation, bleeding, ulceration, hyperplasia, and necrosis was observed around biofilm-inoculated implants. A. actinomycetemcomitans was detected by polymerase chain reaction and culture analysis on 100%of biofilm-inoculated implants for up to 3 weeks and 25%for up to 6 weeks. Microcomputed tomography analysis demonstrated significantly lower bone volume (P <0.05) around biofilm-inoculated implants (29.6% ± 7.6%) compared to non-inoculated implants (50.5% ± 9.6%) after 6 weeks. Conclusions These results describe a novel animal model where A. actinomycetemcomitans biofilm was established in vitro on titanium implants before placement in rat oral cavity, leading to an inflammatory response, osteolysis, and tissue destruction. This model may have potential use for investigation of host responses to biofilm pathogens and

  20. A Consortium of Aggregatibacter actinomycetemcomitans, Streptococcus parasanguinis, and Filifactor alocis Is Present in Sites Prior to Bone Loss in a Longitudinal Study of Localized Aggressive Periodontitis

    PubMed Central

    Markowitz, Kenneth; Fairlie, Karen; Tischio-Bereski, Debbie; Ferrendiz, Javier; Furgang, David; Paster, Bruce J.; Dewhirst, Floyd E.

    2013-01-01

    Aggregatibacter actinomycetemcomitans-induced localized aggressive periodontitis (LAP) in African-American adolescents has been documented but is poorly understood. Two thousand fifty-eight adolescents aged 11 to 17 years were screened for their periodontal status and the presence of A. actinomycetemcomitans in their oral cavity. Seventy-one A. actinomycetemcomitans-negative and 63 A. actinomycetemcomitans-positive periodontally healthy subjects were enrolled, sampled, examined, and radiographed yearly for 3 years. Gingival and periodontal pocket depth and attachment levels were recorded. Disease presentation was characterized by bone loss (BL). Subgingival sites were sampled every 6 months to assess (i) the role of A. actinomycetemcomitans in BL and (ii) the association of A. actinomycetemcomitans and other microbes in their relationships to BL. Sixteen of 63 subjects with A. actinomycetemcomitans developed BL (the other 47 subjects with A. actinomycetemcomitans had no BL). No A. actinomycetemcomitans-negative subjects developed BL. Human oral microbe identification microarray (HOMIM) was used for subgingival microbial assessment. On a subject level, pooled data from A. actinomycetemcomitans-positive subjects who remained healthy had higher prevalences of Streptococcus and Actinomyces species, while A. actinomycetemcomitans-positive subjects with BL had higher prevalences of Parvimonas micra, Filifactor alocis, A. actinomycetemcomitans, and Peptostreptococcus sp. human oral taxon 113 (HOT-113). At vulnerable sites, A. actinomycetemcomitans, Streptococcus parasanguinis, and F. alocis levels were elevated prior to BL. In cases where the three-organism consortium (versus A. actinomycetemcomitans alone) was detected, the specificity for detecting sites of future BL increased from 62% to 99%, with a sensitivity of 89%. We conclude that detecting the presence of A. actinomycetemcomitans, S. parasanguinis, and F. alocis together indicates sites of future BL in LAP. A

  1. Transcriptome Profiling of Wild-Type and pga-Knockout Mutant Strains Reveal the Role of Exopolysaccharide in Aggregatibacter actinomycetemcomitans

    PubMed Central

    Shanmugam, Mayilvahanan; El Abbar, Faiha; Ramasubbu, Narayanan

    2015-01-01

    Exopolysaccharides have a diverse set of functions in most bacteria including a mechanistic role in protecting bacteria against environmental stresses. Among the many functions attributed to the exopolysaccharides, biofilm formation, antibiotic resistance, immune evasion and colonization have been studied most extensively. The exopolysaccharide produced by many Gram positive as well as Gram negative bacteria including the oral pathogen Aggregatibacter actinomycetemcomitans is the homopolymer of β(1,6)-linked N-acetylglucosamine. Recently, we reported that the PGA-deficient mutant of A. actinomycetemcomitans failed to colonize or induce bone resorption in a rat model of periodontal disease, and the colonization genes, apiA and aae, were significantly down regulated in the mutant strain. To understand the role of exopolysaccharide and the pga locus in the global expression of A. actinomycetemcomitans, we have used comparative transcriptome profiling to identify differentially expressed genes in the wild-type strain in relation to the PGA-deficient strain. Transcriptome analysis revealed that about 50% of the genes are differently expressed (P < 0.05 and fold change >1.5). Our study demonstrated that the absence of the pga locus affects the genes involved in peptidoglycan recycling, glycogen storage, and virulence. Further, using confocal microscopy and plating assays, we show that the viability of pga mutant strain is significantly reduced during biofilm growth. Thus, this study highlights the importance of pga genes and the exopolysaccharide in the virulence of A. actinomycetemcomitans. PMID:26221956

  2. Serotypes of Aggregatibacter actinomycetemcomitans in relation to periodontal status and geographic origin of individuals-a review of the literature

    PubMed Central

    da Silveira, Virginia R S.; Rego, Rodrigo O.; Nogueira, Nádia A P.

    2014-01-01

    Objectives: Several studies have focused on the relationship among serotype distribution, ethnical status and geographic populations, and periodontal conditions. Studies that have investigated the prevalence and the distribution of A. actinomycetemcomitans serotypes and the relation between the different serotypes of the bacterium and periodontal status were reviewed. Material and Methods: A systematic literature search for publications regarding the distribution of A. actinomycetemcomitans serotypes in subgingival samples of periodontitis patients and periodontally healthy subjects by employing polymerase chain reaction (PCR) was conducted. Results: From the 85 studies identified in the first analysis, only 12 met all inclusion and exclusion criteria. Clinical isolates from diverse geographic populations with different periodontal conditions were evaluated. Serotypes a, b and c were largely found, and serotype c was the most prevalent. They were isolated from various periodontal conditions, including aggressive periodontitis. Conclusions: The available literature suggests that serotypes a, b, and c are globally dominant, serotypes d and e are rare, and the prevalence of the most recently identified serotype fis still unknown. It is widely accepted that distribution patterns of A. actinomycetemcomitans vary among subjects of different ethnicity and geographic regions. The correlation of different serotypes with various periodontal conditions remains unclear. Key words:Aggregatibacter actinomycetemcomitans, serotypes, periodontal disease, prevalence. PMID:24316700

  3. Surface display of Aggregatibacter actinomycetemcomitans autotransporter Aae and dispersin B hybrid act as antibiofilm agents.

    PubMed

    Ragunath, C; DiFranco, K; Shanmugam, M; Gopal, P; Vyas, V; Fine, D H; Cugini, C; Ramasubbu, N

    2016-08-01

    Among the various proteins expressed by the periodontopathogen Aggregatibacter actinomycetemcomitans, two proteins play important roles for survival in the oral cavity. The autotransporter Aae facilitates the attachment of the pathogen to oral epithelial cells, which act as a reservoir, while the biofilm-degrading glycoside hydrolase dispersin B facilitates the movement of daughter cells from the mature biofilm to a new site. The objective of this study was to use the potential of these two proteins to control biofilms. To this end, we generated a hybrid construct between the Aae C-terminal translocating domain and dispersin B, and mobilized it into Escherichia coli Rosetta (DE3) pLysS cells. Immunofluorescence analysis of the modified E. coli cells confirmed the presence of dispersin B on the surface. Further, the membrane localization of the displayed dispersin B was confirmed with Western blot analysis. The integrity of the E. coli cells displaying the dispersin B was confirmed through FACS analysis. The hydrolytic activity of the surface-displayed dispersin B was confirmed by using 4-methylumbelliferyl-β-d-glucopyranoside as the substrate. The detachment ability of the dispersin B surface-displaying E. coli cells was shown using Staphylococcus epidermidis and Actinobacillus pleuropneumoniae biofilms in a microtiter assay. We concluded that the Aae β-domain is sufficient to translocate foreign enzymes in the native folded form and that the method of Aae-mediated translocation of surface displayed enzymes might be useful for control of biofilms. PMID:26280561

  4. Alteration of Homeostasis in Pre-osteoclasts Induced by Aggregatibacter actinomycetemcomitans CDT

    PubMed Central

    Kawamoto, Dione; Ando-Suguimoto, Ellen S.; Bueno-Silva, Bruno; DiRienzo, Joseph M.; Mayer, Marcia P. A.

    2016-01-01

    The dysbiotic microbiota associated with aggressive periodontitis includes Aggregatibacter actinomycetemcomitans, the only oral species known to produce a cytolethal distending toxin (AaCDT). Give that CDT alters the cytokine profile in monocytic cells, we aimed to test the hypothesis that CDT plays a role in bone homeostasis by affecting the differentiation of precursor cells into osteoclasts. Recombinant AaCDT was added to murine bone marrow monocytes (BMMC) in the presence or absence of RANKL and the cell viability and cytokine profile of osteoclast precursor cells were determined. Multinucleated TRAP+ cell numbers, and relative transcription of genes related to osteoclastogenesis were also evaluated. The addition of AaCDT did not lead to loss in cell viability but promoted an increase in the average number of TRAP+ cells with 1-2 nuclei in the absence or presence of RANKL (Tukey, p < 0.05). This increase was also observed for TRAP+ cells with ≥3nuclei, although this difference was not significant. Levels of TGF-β, TNF-α, and IL-6, in the supernatant fraction of cells, were higher when in AaCDT exposed cells, whereas levels of IL-1β and IL-10 were lower than controls under the same conditions. After interaction with AaCDT, transcription of the rank (encoding the receptor RANK), nfatc1 (transcription factor), and ctpK (encoding cathepsin K) genes was downregulated in pre-osteoclastic cells. The data indicated that despite the presence of RANKL and M-CSF, AaCDT may inhibit osteoclast differentiation by altering cytokine profiles and repressing transcription of genes involved in osteoclastogenesis. Therefore, the CDT may impair host defense mechanisms in periodontitis. PMID:27064424

  5. Antibacterial Effect of an Herbal Product Persica on Porphyromonas Gingivalis and Aggregatibacter Actinomycetemcomitans: An In-Vitro Study

    PubMed Central

    Jelvehgaran Esfahani, Zahra; Kadkhoda, Zeinab; Eshraghi, Seyed Saeed; Salehi Surmaghi, Mohammad Hossein

    2014-01-01

    Objective: The plant Salvadora persica is used for oral hygiene in many parts of the world. It has been suggested that it has antibacterial properties, in addition to its ability to mechanically remove plaques. The aim of this study was to assess the antimicrobial activity of the herbal product Persica containing Salvadora persica against periodontopathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in vitro. Materials and Methods: Fifty patients with moderate and severe periodontitis were recruited. Using paper points, subgingival plaque samples were taken from pockets with attachment loss ≥ 3mm. The samples were subjected to microbial culture to yield P. gingivalis and A. actinomycetemcomitans. The ditch plate method was used for antimicrobial susceptibility testing of the bacteria to Persica compared to chlorhexidine and distilled water. The growth inhibition zones of microorganisms around the ditches were measured in millimeters. The data were analyzed using SPSS 16. Freidman test and Wilcoxon signed ranks test with Bonferroni adjustment were used for analysis of variance with 5% significance level. P<0.05 for main comparisons and P< 0.017 for multiple comparisons were considered statistically significant. Results: P. gingivalis was sensitive to chlorhexidine and persica. There was a significant difference (P=0.001) between antimicrobial activity of chlorhexidine (mean 28.733mm, SD 5.216) and Persica (mean 16.333mm, SD 5.259) compared to water against P. gingivalis. There was a significant difference (P< 0.001) between the antimicrobial activity of chlorhexidine (24.045mm, SD 3.897) and Persica (0.545mm, SD 2.558) with respect to A. actinomycetemcomitans. There was no significant difference (P=0.317) between the antimicrobial activity of Persica and water against A. actinomycetemcomitans. Conclusion: The herbal product Persica had significant antimicrobial activity against P. gingivalis and negligible antimicrobial activity against A

  6. Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes

    PubMed Central

    ALVAREZ, Carla; BENÍTEZ, Alvaro; ROJAS, Leticia; PUJOL, Myriam; CARVAJAL, Paola; DÍAZ-ZÚÑIGA, Jaime; VERNAL, Rolando

    2015-01-01

    In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production. Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the different A. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analysed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation. Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the different A. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels. Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b. Conclusion A T-lymphocyte response biased towards a

  7. Differential expression of CC chemokines (CCLs) and receptors (CCRs) by human T lymphocytes in response to different Aggregatibacter actinomycetemcomitans serotypes

    PubMed Central

    ALVAREZ, Carla; BENÍTEZ, Alvaro; ROJAS, Leticia; PUJOL, Myriam; CARVAJAL, Paola; DÍAZ-ZÚÑIGA, Jaime; VERNAL, Rolando

    2015-01-01

    ABSTRACT In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Recently, our research group has reported a differential immunogenicity when T lymphocytes were stimulated with these different serotypes. In particular, it was demonstrated that the serotype b of A. actinomycetemcomitans has a stronger capacity to trigger Th1- and Th17-type cytokine production. Objective This study aimed to quantify the expression of different CC chemokines (CCLs) and receptors (CCRs) in T lymphocytes stimulated with the different A. actinomycetemcomitans serotypes. In addition, the expression of the transcription factors T-bet, GATA-3, RORC2, and Foxp3, master-switch genes implied in the Th1, Th2, Th17, and T-regulatory differentiation, respectively, was analyzed in order to determine T-cell phenotype-specific patterns of CCL and CCR expression upon A. actinomycetemcomitans stimulation. Material and Methods Human naïve CD4+ T lymphocytes were obtained from healthy subjects and stimulated with autologous dendritic cells primed with the different A. actinomycetemcomitans serotypes. The expression levels for the chemokines CCL1, CCL2, CCL3, CCL5, CCL11, CCL17, CCL20, CCL21, CCL25, and CCL28, as well as the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10 were quantified by qPCR. Similarly, the expression levels for the transcription factors T-bet, GATA-3, RORC2, and Foxp3 were quantified and correlated with the CCL and CCR expression levels. Results Higher expression levels of CCL2, CCL3, CCL5, CCL20, CCL21, CCL28, CCR1, CCR2, CCR5, CCR6, CCR7, and CCR9 were detected in T lymphocytes stimulated with the serotype b of A. actinomycetemcomitans compared with the other serotypes. In addition, these higher expression levels of CCLs and CCRs positively correlated with the increased levels of T-bet and RORC2 when T lymphocytes were stimulated with the serotype b. Conclusion A T-lymphocyte response biased

  8. Inflammatory bone loss in experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in interleukin-1 receptor antagonist knockout mice.

    PubMed

    Izawa, A; Ishihara, Y; Mizutani, H; Kobayashi, S; Goto, H; Okabe, E; Takeda, H; Ozawa, Y; Kamiya, Y; Sugita, Y; Kubo, K; Kamei, H; Kikuchi, T; Mitani, A; Hayashi, J; Nishihara, T; Maeda, H; Noguchi, T

    2014-05-01

    The interleukin-1 receptor antagonist (IL-1Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is not clear whether IL-1Ra plays a protective role in periodontal disease. This study was undertaken to compare experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in IL-1Ra knockout (KO) mice and wild-type (WT) mice. Computed tomography (CT) analysis and hematoxylin-and-eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining were performed. In addition, osteoblasts were isolated; the mRNA expression of relevant genes was assessed by real-time quantitative PCR (qPCR); and calcification was detected by Alizarin Red staining. Infected IL-1Ra KO mice exhibited elevated (P, <0.05) levels of antibody against A. actinomycetemcomitans, bone loss in furcation areas, and alveolar fenestrations. Moreover, protein for tumor necrosis factor alpha (TNF-α) and IL-6, mRNA for macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL) in IL-1Ra KO mouse osteoblasts stimulated with A. actinomycetemcomitans were increased (P, <0.05) compared to in WT mice. Alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN)/bone gla protein (BGP), and runt-related gene 2 (Runx2) mRNA levels were decreased (P, <0.05). IL-1α mRNA expression was increased, and calcification was not observed, in IL-1 Ra KO mouse osteoblasts. In brief, IL-1Ra deficiency promoted the expression of inflammatory cytokines beyond IL-1 and altered the expression of genes involved in bone resorption in A. actinomycetemcomitans-infected osteoblasts. Alterations consistent with rapid bone loss in infected IL-Ra KO mice were also observed for genes expressed in bone formation and calcification. In short, these data suggest that IL-1Ra may serve as a potential therapeutic drug for periodontal disease.

  9. Inflammatory bone loss in experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in interleukin-1 receptor antagonist knockout mice.

    PubMed

    Izawa, A; Ishihara, Y; Mizutani, H; Kobayashi, S; Goto, H; Okabe, E; Takeda, H; Ozawa, Y; Kamiya, Y; Sugita, Y; Kubo, K; Kamei, H; Kikuchi, T; Mitani, A; Hayashi, J; Nishihara, T; Maeda, H; Noguchi, T

    2014-05-01

    The interleukin-1 receptor antagonist (IL-1Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is not clear whether IL-1Ra plays a protective role in periodontal disease. This study was undertaken to compare experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in IL-1Ra knockout (KO) mice and wild-type (WT) mice. Computed tomography (CT) analysis and hematoxylin-and-eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining were performed. In addition, osteoblasts were isolated; the mRNA expression of relevant genes was assessed by real-time quantitative PCR (qPCR); and calcification was detected by Alizarin Red staining. Infected IL-1Ra KO mice exhibited elevated (P, <0.05) levels of antibody against A. actinomycetemcomitans, bone loss in furcation areas, and alveolar fenestrations. Moreover, protein for tumor necrosis factor alpha (TNF-α) and IL-6, mRNA for macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL) in IL-1Ra KO mouse osteoblasts stimulated with A. actinomycetemcomitans were increased (P, <0.05) compared to in WT mice. Alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN)/bone gla protein (BGP), and runt-related gene 2 (Runx2) mRNA levels were decreased (P, <0.05). IL-1α mRNA expression was increased, and calcification was not observed, in IL-1 Ra KO mouse osteoblasts. In brief, IL-1Ra deficiency promoted the expression of inflammatory cytokines beyond IL-1 and altered the expression of genes involved in bone resorption in A. actinomycetemcomitans-infected osteoblasts. Alterations consistent with rapid bone loss in infected IL-Ra KO mice were also observed for genes expressed in bone formation and calcification. In short, these data suggest that IL-1Ra may serve as a potential therapeutic drug for periodontal disease. PMID:24566623

  10. Photocatalytical Antibacterial Activity of Mixed-Phase TiO2 Nanocomposite Thin Films against Aggregatibacter actinomycetemcomitans.

    PubMed

    Yeniyol, Sinem; Mutlu, Ilven; He, Zhiming; Yüksel, Behiye; Boylan, Robert Joseph; Ürgen, Mustafa; Karabuda, Zihni Cüneyt; Basegmez, Cansu; Ricci, John Lawrence

    2015-01-01

    Mixed-phase TiO2 nanocomposite thin films consisting of anatase and rutile prepared on commercially pure Ti sheets via the electrochemical anodization and annealing treatments were investigated in terms of their photocatalytic activity for antibacterial use around dental implants. The resulting films were characterized by scanning electron microscopy (SEM), and X-ray diffraction (XRD). The topology was assessed by White Light Optical Profiling (WLOP) in the Vertical Scanning Interferometer (VSI) mode. Representative height descriptive parameters of roughness R a and R z were calculated. The photocatalytic activity of the resulting TiO2 films was evaluated by the photodegradation of Rhodamine B (RhB) dye solution. The antibacterial ability of the photocatalyst was examined by Aggregatibacter actinomycetemcomitans suspensions in a colony-forming assay. XRD showed that anatase/rutile mixed-phase TiO2 thin films were predominantly in anatase and rutile that were 54.6 wt% and 41.9 wt%, respectively. Craters (2-5 µm) and protruding hills (10-50 µm) on Ti substrates were produced after electrochemical anodization with higher R a and R z surface roughness values. Anatase/rutile mixed-phase TiO2 thin films showed 26% photocatalytic decolorization toward RhB dye solution. The number of colonizing bacteria on anatase/rutile mixed-phase TiO2 thin films was decreased significantly in vitro. The photocatalyst was effective against A. actinomycetemcomitans colonization. PMID:26576430

  11. Photocatalytical Antibacterial Activity of Mixed-Phase TiO2 Nanocomposite Thin Films against Aggregatibacter actinomycetemcomitans

    PubMed Central

    Yeniyol, Sinem; Mutlu, Ilven; He, Zhiming; Yüksel, Behiye; Boylan, Robert Joseph; Ürgen, Mustafa; Karabuda, Zihni Cüneyt; Basegmez, Cansu; Ricci, John Lawrence

    2015-01-01

    Mixed-phase TiO2 nanocomposite thin films consisting of anatase and rutile prepared on commercially pure Ti sheets via the electrochemical anodization and annealing treatments were investigated in terms of their photocatalytic activity for antibacterial use around dental implants. The resulting films were characterized by scanning electron microscopy (SEM), and X-ray diffraction (XRD). The topology was assessed by White Light Optical Profiling (WLOP) in the Vertical Scanning Interferometer (VSI) mode. Representative height descriptive parameters of roughness Ra and Rz were calculated. The photocatalytic activity of the resulting TiO2 films was evaluated by the photodegradation of Rhodamine B (RhB) dye solution. The antibacterial ability of the photocatalyst was examined by  Aggregatibacter actinomycetemcomitans suspensions in a colony-forming assay. XRD showed that anatase/rutile mixed-phase TiO2 thin films were predominantly in anatase and rutile that were 54.6 wt% and 41.9 wt%, respectively. Craters (2–5 µm) and protruding hills (10–50 µm) on Ti substrates were produced after electrochemical anodization with higher Ra and Rz surface roughness values. Anatase/rutile mixed-phase TiO2 thin films showed 26% photocatalytic decolorization toward RhB dye solution. The number of colonizing bacteria on anatase/rutile mixed-phase TiO2 thin films was decreased significantly in vitro. The photocatalyst was effective against A. actinomycetemcomitans colonization. PMID:26576430

  12. Enterococcus faecalis lipoteichoic acid suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced IL-8 expression in human periodontal ligament cells.

    PubMed

    Im, Jintaek; Baik, Jung Eun; Kim, Kyoung Whun; Kang, Seok-Seong; Jeon, Jun Ho; Park, Ok-Jin; Kim, Hyun Young; Kum, Kee-Yeon; Yun, Cheol-Heui; Han, Seung Hyun

    2015-08-01

    Periodontitis is caused by multi-bacterial infection and Aggregatibacter actinomycetemcomitans and Enterococcus faecalis are closely associated with inflammatory periodontal diseases. Although lipopolysaccharide (LPS) of A. actinomycetemcomitans (Aa.LPS) and lipoteichoic acid of E. faecalis (Ef.LTA) are considered to be major virulence factors evoking inflammatory responses, their combinatorial effect on the induction of chemokines has not been investigated. In this study, we investigated the interaction between Aa.LPS and Ef.LTA on IL-8 expression in human periodontal ligament (PDL) cells. Aa.LPS, but not Ef.LTA, substantially induced IL-8 expression at the protein and mRNA levels. Interestingly, Ef.LTA suppressed Aa.LPS-induced IL-8 expression without affecting the binding of Aa.LPS to Toll-like receptor (TLR) 4. Ef.LTA reduced Aa.LPS-induced phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38 kinase. Furthermore, Ef.LTA inhibited the Aa.LPS-induced transcriptional activities of the activating protein 1, CCAAT/enhancer-binding protein and nuclear factor-kappa B transcription factors, all of which are known to regulate IL-8 gene expression. Ef.LTA augmented the expression of IL-1 receptor-associated kinase-M (IRAK-M), a negative regulator of TLR intracellular signaling pathways, in the presence of Aa.LPS at both the mRNA and protein levels. Small interfering RNA silencing IRAK-M reversed the attenuation of Aa.LPS-induced IL-8 expression by Ef.LTA. Collectively, these results suggest that Ef.LTA down-regulates Aa.LPS-induced IL-8 expression in human PDL cells through up-regulation of the negative regulator IRAK-M.

  13. A Comparison of Aggregatibacter actinomycetemcomitans (Aa) Virulence Traits in a Rat Model for Periodontal Disease

    PubMed Central

    Schreiner, Helen; Li, Yu; Cline, Joshua; Tsiagbe, Vincent K.; Fine, Daniel H.

    2013-01-01

    Our aim was to explore the effects of Cytolethal Distending toxin (Cdt) in a well established rat model of periodontal disease where leukotoxin (LtxA) was thought to have no known effect. In vitro studies, were used to assess CdtB activity using Aa Leukotoxin as a negative control. These studies showed that both CdtB and LtxA (unexpectedly) exerted significant effects on CD4+ T cells. As a result we decided to compare the effects of these two prominent Aa virulence factors on bone loss using our rat model of Aa-induced periodontitis. In this model, Aa strains, mutant in cdtB and ltxA, were compared to their parent non-mutant strains and evaluated for colonization, antibody response to Aa, bone loss and disease. We found that bone loss/disease caused by the ltxA mutant strain, in which cdtB was expressed, was significantly less (p<0.05) than that due to the wild type strain. On the other hand, the disease caused by cdtB mutant strain, in which ltxA was expressed, was not significantly different from the wild type strain. This data indicates that Aa LtxA exerts a greater effect on bone loss than Cdt in this rat model of periodontal disease and supports the utility of this model to dissect specific virulence factors as they relate to immunopathology in studies of Aa-induced disease. PMID:23936002

  14. ygiW and qseBC are co-expressed in Aggregatibacter actinomycetemcomitans and regulate biofilm growth.

    PubMed

    Juárez-Rodríguez, María Dolores; Torres-Escobar, Ascención; Demuth, Donald R

    2013-06-01

    The quorum-sensing Escherichia coli regulators B and C (QseBC) two-component system were previously shown to regulate biofilm growth of the oral pathogen Aggregatibacter actinomycetemcomitans and to be essential for virulence. In this study, we use RT-PCR to show that an open reading frame, ygiW, residing upstream of qseBC and encoding a hypothetical protein is co-expressed with qseBC. In addition, using a series of lacZ transcriptional fusion constructs and 5'-rapid amplification of cDNA Ends (RACE), the promoter that drives expression of the ygiW-qseBC operon and the transcriptional start site was mapped to the 372 bp intergenic region upstream from ygiW. No internal promoters drive qseBC expression independently from ygiW. However, qseBC expression is attenuated by approximately ninefold by a putative attenuator stem-loop (ΔG = -77.0 KJ/mol) that resides in the 137 bp intergenic region between ygiW and qseB. The QseB response regulator activates expression of the ygiW-qseBC operon and transcription from the ygiW promoter is drastically reduced in ΔqseB and ΔqseBC mutants of A. actinomycetemcomitans. In addition, transcriptional activity of the ygiW promoter is significantly reduced in a mutant expressing an in-frame deletion of qseC that lacks the sensor domain of QseC, suggesting that a periplasmic signal is required for QseB activation. Finally, a non-polar in-frame deletion in ygiW had little effect on biofilm depth but caused a significant increase in surface coverage relative to wild-type. Complementation of the mutant with a plasmid-borne copy of ygiW reduced surface coverage back to wild-type levels. Interestingly, deletion of the sensor domain of QseC or of the entire qseC open reading frame resulted in significant reductions in biofilm depth, biomass and surface coverage, indicating that the sensor domain is essential for optimal biofilm formation by A. actinomycetemcomitans. Thus, although ygiW and qseBC are co-expressed, they regulate biofilm

  15. Identification of a Novel Bacterial Outer Membrane Interleukin-1Β-Binding Protein from Aggregatibacter actinomycetemcomitans

    PubMed Central

    Paino, Annamari; Ahlstrand, Tuuli; Nuutila, Jari; Navickaite, Indre; Lahti, Maria; Tuominen, Heidi; Välimaa, Hannamari; Lamminmäki, Urpo; Pöllänen, Marja T.; Ihalin, Riikka

    2013-01-01

    Aggregatibacteractinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control protein for IL-1

  16. A Cytolethal Distending Toxin Variant from Aggregatibacter actinomycetemcomitans with an Aberrant CdtB That Lacks the Conserved Catalytic Histidine 160.

    PubMed

    Obradović, Davor; Gašperšič, Rok; Caserman, Simon; Leonardi, Adrijana; Jamnik, Maja; Podlesek, Zdravko; Seme, Katja; Anderluh, Gregor; Križaj, Igor; Maček, Peter; Butala, Matej

    2016-01-01

    The periodontopathogen Aggregatibacter actinomycetemcomitans synthesizes several virulence factors, including cytolethal distending toxin (CDT). The active CDT holoenzyme is an AB-type tripartite genotoxin that affects eukaryotic cells. Subunits CdtA and CdtC (B-components) allow binding and intracellular translocation of the active CdtB (A-component), which elicits nuclear DNA damage. Different strains of A. actinomycetemcomitans have diverse virulence genotypes, which results in varied pathogenic potential and disease progression. Here, we identified an A. actinomycetemcomitans strain isolated from two patients with advance chronic periodontitis that has a regular cdtABC operon, which, however, codes for a unique, shorter, variant of the CdtB subunit. We describe the characteristics of this CdtBΔ116-188, which lacks the intact nuclear localisation signal and the catalytic histidine 160. We show that the A. actinomycetemcomitans DO15 isolate secretes CdtBΔ116-188, and that this subunit cannot form a holotoxin and is also not genotoxic if expressed ectopically in HeLa cells. Furthermore, the A. actinomycetemcomitans DO15 isolate is not toxic, nor does it induce cellular distention upon infection of co-cultivated HeLa cells. Biological significance of this deletion in the cdtB remains to be explained. PMID:27414641

  17. A Cytolethal Distending Toxin Variant from Aggregatibacter actinomycetemcomitans with an Aberrant CdtB That Lacks the Conserved Catalytic Histidine 160

    PubMed Central

    Obradović, Davor; Gašperšič, Rok; Caserman, Simon; Leonardi, Adrijana; Jamnik, Maja; Podlesek, Zdravko; Seme, Katja; Anderluh, Gregor; Križaj, Igor; Maček, Peter; Butala, Matej

    2016-01-01

    The periodontopathogen Aggregatibacter actinomycetemcomitans synthesizes several virulence factors, including cytolethal distending toxin (CDT). The active CDT holoenzyme is an AB-type tripartite genotoxin that affects eukaryotic cells. Subunits CdtA and CdtC (B-components) allow binding and intracellular translocation of the active CdtB (A-component), which elicits nuclear DNA damage. Different strains of A. actinomycetemcomitans have diverse virulence genotypes, which results in varied pathogenic potential and disease progression. Here, we identified an A. actinomycetemcomitans strain isolated from two patients with advance chronic periodontitis that has a regular cdtABC operon, which, however, codes for a unique, shorter, variant of the CdtB subunit. We describe the characteristics of this CdtBΔ116–188, which lacks the intact nuclear localisation signal and the catalytic histidine 160. We show that the A. actinomycetemcomitans DO15 isolate secretes CdtBΔ116–188, and that this subunit cannot form a holotoxin and is also not genotoxic if expressed ectopically in HeLa cells. Furthermore, the A. actinomycetemcomitans DO15 isolate is not toxic, nor does it induce cellular distention upon infection of co-cultivated HeLa cells. Biological significance of this deletion in the cdtB remains to be explained. PMID:27414641

  18. Evaluation of chemical composition and efficacy of Chinese propolis extract on Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans: An in vitro study

    PubMed Central

    Agarwal, Garima; Vemanaradhya, Gayathri G.; Mehta, Dhoom S.

    2012-01-01

    Background: Propolis as a natural remedy has maintained its popularity over long periods of time. The aim of this study was to determine the chemical composition in terms of total phenolic compounds and flavonoids present in Chinese propolis and to carry out an in vitro evaluation of its antimicrobial activity and the minimal inhibitory concentrations for Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa). Materials and Methods: From the ethanolic extract of propolis (EEP), total phenol content was determined by the Folin–Ciocalteau method, flavones and flavonols by the modified aluminum chloride colorimetric method, and flavanones by the 2.4-dinitrophenylhydrazine (2,4-DNP) method. Agar well diffusion assay was used to evaluate the antimicrobial potential of propolis against Pg and Aa. The minimum inhibitory concentration of propolis against the two bacteria was determined using serial tube dilution technique. Results: The total concentration of phenol in the EEP was 19.44%, flavones and flavonols 2.616%, and flavanones 16.176%. The inhibitory zone depicting antimicrobial activity ranged from 18 to 25 mm for Pg and from 12 to 14 mm for Aa. The concentration range of Chinese propolis that is sensitive to inhibit the growth of Pg was 0.1–0.0125 μg/ml and for Aa it was 0.1–0.025 μg/ml. Conclusion: These data suggest that Chinese propolis has potent antimicrobial activity against the two periodontopathogens, suggesting its possible use as a natural alternative to the widely used synthetic antibiotics for periodontal therapy. PMID:23293477

  19. Aggregatibacter actinomycetemcomitans QseBC is activated by catecholamines and iron and regulates genes encoding proteins associated with anaerobic respiration and metabolism.

    PubMed

    Weigel, W A; Demuth, D R; Torres-Escobar, A; Juárez-Rodríguez, M D

    2015-10-01

    Aggregatibacter actinomycetemcomitans QseBC regulates its own expression and is essential for biofilm growth and virulence. However, the signal that activates the QseC sensor has not been identified and the qseBC regulon has not been defined. In this study, we show that QseC is activated by catecholamine hormones and iron but not by either component alone. Activation of QseC requires an EYRDD motif in the periplasmic domain of the sensor and site-specific mutations in EYRDD or the deletion of the periplasmic domain inhibits catecholamine/iron-dependent induction of the ygiW-qseBC operon. Catecholamine/iron-dependent induction of transcription also requires interaction of the QseB response regulator with its binding site in the ygiW-qseBC promoter. Whole genome microarrays were used to compare gene expression profiles of A. actinomycetemcomitans grown in a chemically defined medium with and without catecholamine and iron supplementation. Approximately 11.5% of the A. actinomycetemcomitans genome was differentially expressed by at least two-fold upon exposure to catecholamines and iron. The expression of ferritin was strongly induced, suggesting that intracellular iron storage capacity is increased upon QseBC activation. Consistent with this, genes encoding iron binding and transport proteins were down-regulated by QseBC. Strikingly, 57% of the QseBC up-regulated genes (56/99) encode proteins associated with anaerobic metabolism and respiration. Most of these up-regulated genes were recently reported to be induced during in vivo growth of A. actinomycetemcomitans. These results suggest that detection of catecholamines and iron by QseBC may alter the cellular metabolism of A. actinomycetemcomitans for increased fitness and growth in an anaerobic host environment.

  20. Al(III), Pd(II), and Zn(II) phthalocyanines for inactivation of dental pathogen Aggregatibacter actinomycetemcomitans as planktonic and biofilm-cultures

    NASA Astrophysics Data System (ADS)

    Kussovski, V.; Mantareva, V.; Angelov, I.; Avramov, L.; Popova, E.; Dimitrov, S.

    2012-06-01

    The Gram-negative, oral bacterium Aggregatibacter actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. The new periodontal disease treatments are emergence in order to prevent infection progression. Antimicrobial photodynamic therapy (a-PDT) can be a useful tool for this purpose. It involves the use of light of specific wavelength to activate a nontoxic photosensitizing agent in the presence of oxygen for eradication of target cells, and appears effective in photoinactivation of microorganisms. The phthalocyanine metal complexes of Pd(II)- (PdPcC) and Al(III)- (AlPc1) were evaluated as photodynamic sensitizers towards a dental pathogen A. actinomycetemcomitans in comparison to the known methylpyridyloxy-substituted Zn(II) phthalocyanine (ZnPcMe). The planktonic and biofilm-cultivated species of A. actinomycetemcomitans were treated. The photophysical results showed intensive and far-red absorbance with high tendency of aggregation for Pd(II)-phthalocyanine. The dark toxicities of both photosensitizers were negligible at concentrations used (< 0.5 log decrease of viable cells). The photodynamic response for planktonic cultured bacteria was full photoinactivation after a-PDT with ZnPcMe. In case of the newly studied complexes, the effect was lower for PdPcC (4 log) as well as for AlPc1 (1.5-2 log). As it is known the bacterial biofilms were more resistant to a-PDT, which was confirmed for A. actinomycetemcomitans biofilms with 3 log reductions of viable cells after treatment with ZnPcMe and approximately 1 log reduction of biofilms after PdPcC and AlPc1. The initial results suggest that a-PDT can be useful for effective inactivation of dental pathogen A. actinomycetemcomitans.

  1. Aggregatibacter actinomycetemcomitans QseBC is activated by catecholamines and iron and regulates genes encoding proteins associated with anaerobic respiration and metabolism

    PubMed Central

    Weigel, WA; Demuth, DR; Torres-Escobar, A; Juárez-Rodríguez, MD

    2015-01-01

    Aggregatibacter actinomycetemcomitans QseBC regulates its own expression and is essential for biofilm growth and virulence. However, the signal that activates the QseC sensor has not been identified and the qseBC regulon has not been defined. In this study, we show that QseC is activated by catecholamine hormones and iron but not by either component alone. Activation of QseC requires an EYRDD motif in the periplasmic domain of the sensor and site-specific mutations in EYRDD or the deletion of the periplasmic domain inhibits catecholamine/iron-dependent induction of the ygiW-qseBC operon. Catecholamine/iron-dependent induction of transcription also requires interaction of the QseB response regulator with its binding site in the ygiW-qseBC promoter. Whole genome microarrays were used to compare gene expression profiles of A. actinomycetemcomitans grown in a chemically defined medium with and without catecholamine and iron supplementation. Approximately 11.5% of the A. actinomycetemcomitans genome was differentially expressed by at least two-fold upon exposure to catecholamines and iron. The expression of ferritin was strongly induced, suggesting that intracellular iron storage capacity is increased upon QseBC activation. Consistent with this, genes encoding iron binding and transport proteins were down-regulated by QseBC. Strikingly, 57% of the QseBC up-regulated genes (56/99) encode proteins associated with anaerobic metabolism and respiration. Most of these up-regulated genes were recently reported to be induced during in vivo growth of A. actinomycetemcomitans. These results suggest that detection of catecholamines and iron by QseBC may alter the cellular metabolism of A. actinomycetemcomitans for increased fitness and growth in an anaerobic host environment. PMID:25923132

  2. Inverse Association of Plasma IgG Antibody to Aggregatibacter actinomycetemcomitans and High C-Reactive Protein Levels in Patients with Metabolic Syndrome and Periodontitis.

    PubMed

    Thanakun, Supanee; Pornprasertsuk-Damrongsri, Suchaya; Gokyu, Misa; Kobayashi, Hiroaki; Izumi, Yuichi

    2016-01-01

    The association between clinically diagnosed periodontitis, a common chronic oral infection, and metabolic syndrome has been previously reported. The aim of this study was to investigate the association of plasma IgG levels against Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia, C-reactive protein, and periodontal status with metabolic syndrome. Plasma IgG levels and C-reactive protein were measured by enzyme-linked immunosorbent assay, and salivary levels of A. actinomycetemcomitans and P. gingivalis were determined by quantitative real-time polymerase chain reaction. Among 127 individuals aged 35-76 years, 57 participants had metabolic syndrome and severe periodontitis, 25 had metabolic syndrome and an absence of severe periodontitis, 17 healthy individuals had severe periodontitis, and 28 healthy individuals were without severe periodontitis. Patients with metabolic syndrome had reduced humoral immune response to A. actinomycetemcomitans (p = 0.008), regardless of their salivary levels or periodontitis status compared with healthy participants. The IgG antibody response to P. gingivalis, regardless of their salivary levels or participants' health condition, was significantly higher in severe periodontitis patients (p<0.001). Plasma IgG titers for P. intermedia were inconsistent among metabolic syndrome or periodontal participants. Our results indicate that the presence of lower levels of IgG antibodies to A. actinomycetemcomitans (OR = 0.1; 95%CI 0.0-0.7), but not P. gingivalis, a severe periodontitis status (OR = 7.8; 95%CI 1.1-57.0), high C-reactive protein levels (OR = 9.4; 95%CI 1.0-88.2) and body mass index (OR = 3.0; 95%CI 1.7-5.2), are associated with the presence of metabolic syndrome. The role of the decreased IgG antibody response to A. actinomycetemcomitans, increased C-reactive protein levels on the association between periodontal disease and metabolic syndrome in a group of Thai patients is suggested. PMID

  3. Inactivation of Aggregatibacter actinomycetemcomitans by two different modalities of photodynamic therapy using Toluidine blue O or Radachlorin as photosensitizers: an in vitro study.

    PubMed

    Moslemi, Neda; Soleiman-Zadeh Azar, Pardis; Bahador, Abbas; Rouzmeh, Nina; Chiniforush, Nasim; Paknejad, Mojgan; Fekrazad, Reza

    2015-01-01

    Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is one of the periodontopathogens strongly associated with aggressive periodontitis. The aim of this investigation was to compare the effect of laser and light-emitting diode on the photodynamic inactivation of A. actinomycetemcomitans. Eighty-four samples of bacterial suspensions (200 μL) were prepared and divided in seven groups: control group (no treatment), laser group (indium-gallium-aluminum-phosphate laser with wavelength of 662 ± 0.1 nm, energy density of 6 j/cm(2), and irradiation time of 34 s), light-emitting diode (LED) group (wavelength 625-635 nm, energy density 6 j/cm(2), time of irradiation 30 s), Toluidine blue O (TBO) group (0.1 mg/mL), Radachlorin group (0.1 %), Radachlorin + laser group (after pre-irradiation time of 10 min, laser was irradiated), and TBO + LED group (after preirradiation time of 10 min, LED was irradiated). Then, 100 μL of each sample was cultured in brain heart infusion (BHI) plates and incubated for 48-72 h in microaerophilic atmosphere for colony counting. Application of Radachlorin + laser resulted in a significant decrease in the concentration of A. actinomycetemcomitans (P values <0.05). Photodynamic therapy with laser + Radachlorin was more effective than that of LED + TBO in suppression of this microorganism (P value <0.05). Within the limits of this study, it can be concluded that photodynamic inactivation using laser and Radachlorin was more effective than that of LED and TBO in eradication of A. actinomycetemcomitans. PMID:24981641

  4. Colonization and Persistence of Labeled and “Foreign” Strains of Aggregatibacter actinomycetemcomitans Inoculated into the Mouths of Rhesus Monkeys

    PubMed Central

    Fine, Daniel H.; Karched, Maribasappa; Furgang, David; Sampathkumar, Vandana; Velusamy, Senthil; Godboley, Dipti

    2015-01-01

    Aggregatibacter actinomycetemcomitans (Aa) is a pathobiont and part of a consortium of bacteria that can lead to periodontitis in humans. Our aim was to develop a model for oral inoculation of labeled Aa into a suitable host in order to study Aa traits and ecological factors that either enhance or repress its persistence. Primate species were screened for Aa to select a host for colonization studies. Macaca mulatta (Rhesus/Rh) was selected. Rh Aa strains were isolated, subjected to sequencing and functional analysis for comparison to human strains. “Best” methods for microbial decontamination prior to inoculation were assessed. Three groups were studied; Group 1 (N=5) was inoculated with Aa Spectinomycin resistant (SpecR) Rh strain 4.35, Group 2 (N=5) inoculated with Aa SpecR human strain IDH 781, and Group 3 (N=5) the un-inoculated control. Repeated feeding with pancakes spiked with SpecRAa followed high dose oral inoculation. Cheek, tongue, and plaque samples collected at baseline 1, 2, 3, and 4 weeks after inoculation were plated on agar; 1) selective for Aa, 2) enriched for total counts, and 3) containing 50 µg/ml of Spec. Aa was identified by colonial morphology and DNA analysis. Rh and human Aa had > 93–98 % genome identity. Rh Aa attached to tissues better than IDH 781 in vitro (p < 0.05). SpecR IDH 781 was not recovered from any tissue at any time; whereas, RhSpecR 4.35 was detected in plaque, but never tongue or cheek, in all monkeys at all times (> 1 × 105 colonies/ml; p < 0.001). In conclusion, the primate model provides a useful platform for studying integration of Aa strains into a reduced but established oral habitat. Primate derived SpecRAa was consistently detected in plaque at all collection periods; however, human derived Aa was never detected. The model demonstrated both microbial as well as tissue specificity. PMID:26213715

  5. Determination of antibacterial activity of green coffee bean extract on periodontogenic bacteria like Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans: An in vitro study

    PubMed Central

    Bharath, Nagaraj; Sowmya, Nagur Karibasappa; Mehta, Dhoom Singh

    2015-01-01

    Background: The aim of this study was to evaluate the antibacterial activity of pure green coffee bean extract on periodonto pathogenic bacteria Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Fusobacterium nucleatum (Fn) and Aggregatibacter actinomycetemcomitans (Aa). Materials and Methods: Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBC) were used to assess the antibacterial effect of pure green coffee bean extract against periodonto pathogenic bacteria by micro dilution method and culture method, respectively. Results: MIC values of Pg, Pi and Aa were 0.2 μg/ml whereas Fn showed sensitive at concentration of 3.125 μg/ml. MBC values mirrors the values same as that of MIC. Conclusion: Antimicrobial activity of pure green coffee bean extract against Pg, Pi, Fn and Aa suggests that it could be recommended as an adjunct to mechanical therapy in the management of periodontal disease. PMID:26097349

  6. Evaluation of antimicrobial action of Carie Care™ and Papacarie Duo™ on Aggregatibacter actinomycetemcomitans a major periodontal pathogen using polymerase chain reaction

    PubMed Central

    Kush, Anil; Thakur, Rachna; Patil, Sandya Devi S.; Paul, Santhosh T.; Kakanur, Madhu

    2015-01-01

    Background: In the present scenario, we are made available with chemomechanical caries removal system containing a natural proteolytic enzyme for the ease in the excavation of infected dentine. The additive action for these agents is providing antimicrobial and anti-inflammatory properties. Aim: This study was undertaken for assessing the action of Carie Care™ and Papacarie Duo™ on Aggregatibacter actinomycetemcomitans. Materials and Methods: The samples were collected for cultivation of the periodontal pathogen from the clinical periodontal pockets using sterile paper points. The samples cultured under suitable conditions were analyzed with quantitative polymerase chain reaction targeting 16s r-DNA. The samples were divided into three groups namely, Group A: Control, Group B: With Papacarie Duo, Group C: With Carie Care. The pathogen inoculums plugs were inserted in the petri dishes containing chemically defined medium and the experimental gels at different concentrations and were incubated under optimal conditions. The inhibition of growth of the pathogen was studied visually. Results: There was visual inhibition of growth for Group B and C and also exhibited a dose-dependent effect also. Conclusion: Based on the results of the present study, Carie Care™ gel demonstrated better antimicrobial action against A. actinomycetemcomitans which is a major periodontal disease causing pathogen. PMID:26681861

  7. Inverse Association of Plasma IgG Antibody to Aggregatibacter actinomycetemcomitans and High C-Reactive Protein Levels in Patients with Metabolic Syndrome and Periodontitis

    PubMed Central

    Thanakun, Supanee; Pornprasertsuk-Damrongsri, Suchaya; Gokyu, Misa; Kobayashi, Hiroaki; Izumi, Yuichi

    2016-01-01

    The association between clinically diagnosed periodontitis, a common chronic oral infection, and metabolic syndrome has been previously reported. The aim of this study was to investigate the association of plasma IgG levels against Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia, C-reactive protein, and periodontal status with metabolic syndrome. Plasma IgG levels and C-reactive protein were measured by enzyme-linked immunosorbent assay, and salivary levels of A. actinomycetemcomitans and P. gingivalis were determined by quantitative real-time polymerase chain reaction. Among 127 individuals aged 35–76 years, 57 participants had metabolic syndrome and severe periodontitis, 25 had metabolic syndrome and an absence of severe periodontitis, 17 healthy individuals had severe periodontitis, and 28 healthy individuals were without severe periodontitis. Patients with metabolic syndrome had reduced humoral immune response to A. actinomycetemcomitans (p = 0.008), regardless of their salivary levels or periodontitis status compared with healthy participants. The IgG antibody response to P. gingivalis, regardless of their salivary levels or participants’ health condition, was significantly higher in severe periodontitis patients (p<0.001). Plasma IgG titers for P. intermedia were inconsistent among metabolic syndrome or periodontal participants. Our results indicate that the presence of lower levels of IgG antibodies to A. actinomycetemcomitans (OR = 0.1; 95%CI 0.0–0.7), but not P. gingivalis, a severe periodontitis status (OR = 7.8; 95%CI 1.1–57.0), high C-reactive protein levels (OR = 9.4; 95%CI 1.0–88.2) and body mass index (OR = 3.0; 95%CI 1.7–5.2), are associated with the presence of metabolic syndrome. The role of the decreased IgG antibody response to A. actinomycetemcomitans, increased C-reactive protein levels on the association between periodontal disease and metabolic syndrome in a group of Thai patients is

  8. Lipopolysaccharide of Aggregatibacter actinomycetemcomitans induces the expression of chemokines MCP-1, MIP-1α, and IP-10 via similar but distinct signaling pathways in murine macrophages.

    PubMed

    Park, Ok-Jin; Cho, Min-Kyung; Yun, Cheol-Heui; Han, Seung Hyun

    2015-09-01

    Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium frequently isolated from lesions of patients with localized aggressive periodontitis. Lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria, stimulates innate immune cells via Toll-like receptor 4 (TLR4) to initiate inflammatory responses. In this study, we purified LPS from A. actinomycetemcomitans (AaLPS) and investigated its ability to induce the expression of chemokines, which play an important role in recruitment of leukocytes to the infection site. AaLPS induced the expression of chemokines, MCP-1, MIP-1α, and IP-10 in murine macrophages, leading to the infiltration of peripheral blood mononuclear cells in a transwell system. Although TLR4 was essential for the induction of all these chemokines by AaLPS, MCP-1 and MIP-1α expressions were MyD88-dependent, but IP-10 expression was MyD88-independent, as determined using macrophages from mice deficient in TLR4 or MyD88. Furthermore, the activation of ERK and JNK were necessary for the expression of MCP-1 and MIP-1α, whereas p38 MAP kinase and JNK activations were required for IP-10 expression. In addition, IFN-β/STAT1 signaling was exclusively involved in IP-10 expression but not in MCP-1 or MIP-1α expression. AaLPS also activated the transcription factors, NF-κB, AP-1, NF-IL6, and ISRE, all of which are involved in chemokine gene expression. These results suggest that AaLPS induces the expression of chemokines MCP-1, MIP-1α, and IP-10 through TLR4 in murine macrophages. Further, the induction of MCP-1 and MIP-1α requires MyD88, ERK, and JNK, whereas the induction of IP-10 requires JNK, p38 MAP kinase, and IFN-β/STAT1.

  9. Alteration in abundance of specific membrane proteins of Aggregatibacter actinomycetemcomitans is attributed to deletion of the inner membrane protein MorC

    PubMed Central

    Smith, Kenneth P.; Fields, Julia G.; Voogt, Richard D.; Deng, Bin; Lam, Ying-Wai; Mintz, Keith P.

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an important pathogen in the etiology of human periodontal and systemic diseases. Inactivation of the gene coding for the inner membrane protein, morphogenesis protein C (MorC), results is pleotropic effects pertaining to the membrane structure and function of this bacterium. The role of this protein in membrane biogenesis is unknown. To begin to understand the role of this conserved protein, stable isotope dimethyl labeling in conjunction with mass spectrometry was used to quantitatively analyze differences in the membrane proteomes of the isogenic mutant and wild-type strain. A total of 613 proteins were quantified and 601 of these proteins were found to be equal in abundance between the two strains. The remaining 12 proteins were found in lesser (10) or greater (2) abundance in the membrane preparation of the mutant strain compared with the wild-type strain. The 12 proteins were ascribed functions associated with protein quality control systems, oxidative stress responses, and protein secretion. The potential relationship between these proteins and the phenotypes of the morC mutant strain is discussed. PMID:25684173

  10. Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Treponema denticola / Prevotella intermedia Co-Infection Are Associated with Severe Periodontitis in a Thai Population.

    PubMed

    Torrungruang, Kitti; Jitpakdeebordin, Supawadee; Charatkulangkun, Orawan; Gleebbua, Yingampa

    2015-01-01

    Periodontitis is a polymicrobial infection of tooth-supporting tissues. This cross-sectional study aimed to examine the associations between five target species and severe periodontitis in a Thai population. Using the CDC/AAP case definition, individuals diagnosed with no/mild and severe periodontitis were included. Quantitative analyses of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), and Prevotella intermedia (Pi) in subgingival plaque were performed using real-time polymerase chain reaction. The association between target species and severe periodontitis was examined using logistic regression analysis. The study subjects comprised 479 individuals with no/mild periodontitis and 883 with severe periodontitis. Bacterial prevalence and quantity were higher in subjects with severe periodontitis than in those with no/mild disease. In the fully adjusted model, all species except Tf showed a dose-dependent relationship with periodontitis. The mere presence of Pg, even in low amount, was significantly associated with severe periodontitis, while the amount of Aa, Td, and Pi had to reach the critical thresholds to be significantly associated with disease. Compared to individuals with low levels of both Td and Pi, high colonization by either Td or Pi alone significantly increased the odds of having severe periodontitis by 2.5 (95%CI 1.7-3.5) folds. The odds ratio was further increased to 14.8 (95%CI 9.2-23.8) in individuals who were highly colonized by both species. Moreover, the presence of Pg and high colonization by Aa were independently associated with severe periodontitis with odds ratios of 5.6 (95%CI 3.4-9.1) and 2.2 (95%CI 1.5-3.3), respectively. Our findings suggest that the presence of Pg and high colonization by Aa, Td, and Pi play an important role in severe periodontitis in this study population. We also demonstrate for the first time that individuals co-infected with Td and Pi

  11. Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Treponema denticola / Prevotella intermedia Co-Infection Are Associated with Severe Periodontitis in a Thai Population

    PubMed Central

    Torrungruang, Kitti; Jitpakdeebordin, Supawadee; Charatkulangkun, Orawan; Gleebbua, Yingampa

    2015-01-01

    Periodontitis is a polymicrobial infection of tooth-supporting tissues. This cross-sectional study aimed to examine the associations between five target species and severe periodontitis in a Thai population. Using the CDC/AAP case definition, individuals diagnosed with no/mild and severe periodontitis were included. Quantitative analyses of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), and Prevotella intermedia (Pi) in subgingival plaque were performed using real-time polymerase chain reaction. The association between target species and severe periodontitis was examined using logistic regression analysis. The study subjects comprised 479 individuals with no/mild periodontitis and 883 with severe periodontitis. Bacterial prevalence and quantity were higher in subjects with severe periodontitis than in those with no/mild disease. In the fully adjusted model, all species except Tf showed a dose-dependent relationship with periodontitis. The mere presence of Pg, even in low amount, was significantly associated with severe periodontitis, while the amount of Aa, Td, and Pi had to reach the critical thresholds to be significantly associated with disease. Compared to individuals with low levels of both Td and Pi, high colonization by either Td or Pi alone significantly increased the odds of having severe periodontitis by 2.5 (95%CI 1.7–3.5) folds. The odds ratio was further increased to 14.8 (95%CI 9.2–23.8) in individuals who were highly colonized by both species. Moreover, the presence of Pg and high colonization by Aa were independently associated with severe periodontitis with odds ratios of 5.6 (95%CI 3.4–9.1) and 2.2 (95%CI 1.5–3.3), respectively. Our findings suggest that the presence of Pg and high colonization by Aa, Td, and Pi play an important role in severe periodontitis in this study population. We also demonstrate for the first time that individuals co-infected with Td

  12. Detection of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans after Systemic Administration of Amoxicillin Plus Metronidazole as an Adjunct to Non-surgical Periodontal Therapy: A Systematic Review and Meta-Analysis

    PubMed Central

    Dakic, Aleksandar; Boillot, Adrien; Colliot, Cyrille; Carra, Maria-Clotilde; Czernichow, Sébastien; Bouchard, Philippe

    2016-01-01

    Objective: To evaluate the variations in the detection of Porphyromonas gingivalis and/or Aggregatibacter actinomycetemcomitans before and after systemic administration of amoxicillin plus metronidazole in association with non-surgical periodontal therapy (NSPT). Background: The adjunctive use of antibiotics has been advocated to improve the clinical outcomes of NSPT. However, no systematic review has investigated the microbiological benefit of this combination. Materials and Methods: An electronic search was conducted up to December 2015. Randomized clinical trials comparing the number of patients testing positive for P. gingivalis and/or A. actinomycetemcomitans before and after NSPT with (test group) or without (control group) amoxicillin plus metronidazole were included. The difference between groups in the variation of positive patients was calculated using the inverse variance method with a random effects model. Results: The frequency of patients positive for A. actinomycetemcomitans was decreased by 30% (p = 0.002) and by 25% (p = 0.01) in the test group compared to the control group at 3- and 6-month follow-up, respectively. Similar findings were observed when considering the frequency of patients positive for Porphyromonas gingivalis, with a reduction by 28% (p < 0.0001), 32% (p < 0.0001), and 34% (p = 0.03) in the test group compared to the control group at 3-, 6-, and 12-month follow-up, respectively. Conclusion: The systemic administration of amoxicillin plus metronidazole as an adjunct to NSPT significantly decreased the number of patients positive for P. gingivalis and A. actinomycetemcomitans compared with periodontal therapy alone or with a placebo. PMID:27594851

  13. Detection of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans after Systemic Administration of Amoxicillin Plus Metronidazole as an Adjunct to Non-surgical Periodontal Therapy: A Systematic Review and Meta-Analysis

    PubMed Central

    Dakic, Aleksandar; Boillot, Adrien; Colliot, Cyrille; Carra, Maria-Clotilde; Czernichow, Sébastien; Bouchard, Philippe

    2016-01-01

    Objective: To evaluate the variations in the detection of Porphyromonas gingivalis and/or Aggregatibacter actinomycetemcomitans before and after systemic administration of amoxicillin plus metronidazole in association with non-surgical periodontal therapy (NSPT). Background: The adjunctive use of antibiotics has been advocated to improve the clinical outcomes of NSPT. However, no systematic review has investigated the microbiological benefit of this combination. Materials and Methods: An electronic search was conducted up to December 2015. Randomized clinical trials comparing the number of patients testing positive for P. gingivalis and/or A. actinomycetemcomitans before and after NSPT with (test group) or without (control group) amoxicillin plus metronidazole were included. The difference between groups in the variation of positive patients was calculated using the inverse variance method with a random effects model. Results: The frequency of patients positive for A. actinomycetemcomitans was decreased by 30% (p = 0.002) and by 25% (p = 0.01) in the test group compared to the control group at 3- and 6-month follow-up, respectively. Similar findings were observed when considering the frequency of patients positive for Porphyromonas gingivalis, with a reduction by 28% (p < 0.0001), 32% (p < 0.0001), and 34% (p = 0.03) in the test group compared to the control group at 3-, 6-, and 12-month follow-up, respectively. Conclusion: The systemic administration of amoxicillin plus metronidazole as an adjunct to NSPT significantly decreased the number of patients positive for P. gingivalis and A. actinomycetemcomitans compared with periodontal therapy alone or with a placebo.

  14. Inhibition of fibroblast proliferation by Actinobacillus actinomycetemcomitans.

    PubMed Central

    Shenker, B J; Kushner, M E; Tsai, C C

    1982-01-01

    We have examined soluble sonic extracts of Actinobacillus actinomycetemcomitans for their ability to alter human and murine fibroblast proliferation. We found that extracts of all A. actinomycetemcomitans strains examined (both leukotoxic and nonleukotoxic) caused a dose-dependent inhibition of both murine and human fibroblast proliferation as assessed by DNA synthesis ([3H]thymidine incorporation). Addition of sonic extract simultaneously with [3H]thymidine had no effect on incorporation, indicating that suppression was not due to the presence of excessive amounts of cold thymidine. Inhibition of DNA synthesis was also paralleled by decreased RNA synthesis ([3H]uridine incorporation) and by a decrease in cell growth as assessed by direct cell counts; there was no effect on cell viability. The suppressive factor(s) is heat labile; preliminary purification and characterization studies indicate that it is a distinct and separate moiety from other A. actinomycetemcomitans mediators previously reported, including leukotoxin, immune suppressive factor, and endotoxin. Although it is not clear how A. actinomycetemcomitans acts to cause disease, we propose that one aspect of the pathogenicity of this organism rests in its ability to inhibit fibroblast growth, which in turn could contribute to the collagen loss associated with certain forms of periodontal disease, in particular juvenile periodontitis. PMID:7152684

  15. LFA-1-targeting Leukotoxin (LtxA; Leukothera®) causes lymphoma tumor regression in a humanized mouse model and requires caspase-8 and Fas to kill malignant lymphocytes*

    PubMed Central

    DiFranco, Kristina M.; Johnson-Farley, Nadine; Bertino, Joseph R.; Elson, David; Vega, Brian A.; Belinka, Benjamin A.; Kachlany, Scott C.

    2015-01-01

    Leukotoxin (LtxA) is a protein secreted from the oral bacterium Aggregatibacter actinomycetemcomitans. LtxA binds to the β2 integrin lymphocyte-associated function antigen-1 (LFA-1) on human white blood cells (WBCs), resulting in cell death. LtxA is currently under investigation as a novel therapy (Leukothera®) for treating hematologic malignancies and autoimmune diseases. We show here that LtxA has potent in vivo anti-lymphoma activity in mice. LtxA caused complete regression of B-cell tumors and promoted long-term survival of mice. The mechanism of LtxA-mediated killing of malignant lymphocytes was further examined. We found that LtxA kills malignant lymphocytes by a novel mechanism requiring the death receptor Fas and caspase-8, but not Fas ligand (FasL) or caspase-9. We also determined that LFA-1 and Fas are closely associated on the cell surface and this proximity of LFA-1 and Fas could explain how signaling through an integrin can lead to cell death. In addition to LFA-1, this work reveals a second surface protein, Fas, that is critical for LtxA-mediated cell death. Knowledge of the mechanism of cell death induced by LtxA will facilitate the development and understanding of this potent experimental therapeutic agent. PMID:25850729

  16. Classification, Identification, and Clinical Significance of Haemophilus and Aggregatibacter Species with Host Specificity for Humans

    PubMed Central

    2014-01-01

    SUMMARY The aim of this review is to provide a comprehensive update on the current classification and identification of Haemophilus and Aggregatibacter species with exclusive or predominant host specificity for humans. Haemophilus influenzae and some of the other Haemophilus species are commonly encountered in the clinical microbiology laboratory and demonstrate a wide range of pathogenicity, from life-threatening invasive disease to respiratory infections to a nonpathogenic, commensal lifestyle. New species of Haemophilus have been described (Haemophilus pittmaniae and Haemophilus sputorum), and the new genus Aggregatibacter was created to accommodate some former Haemophilus and Actinobacillus species (Aggregatibacter aphrophilus, Aggregatibacter segnis, and Aggregatibacter actinomycetemcomitans). Aggregatibacter species are now a dominant etiology of infective endocarditis caused by fastidious organisms (HACEK endocarditis), and A. aphrophilus has emerged as an important cause of brain abscesses. Correct identification of Haemophilus and Aggregatibacter species based on phenotypic characterization can be challenging. It has become clear that 15 to 20% of presumptive H. influenzae isolates from the respiratory tracts of healthy individuals do not belong to this species but represent nonhemolytic variants of Haemophilus haemolyticus. Due to the limited pathogenicity of H. haemolyticus, the proportion of misidentified strains may be lower in clinical samples, but even among invasive strains, a misidentification rate of 0.5 to 2% can be found. Several methods have been investigated for differentiation of H. influenzae from its less pathogenic relatives, but a simple method for reliable discrimination is not available. With the implementation of identification by matrix-assisted laser desorption ionization–time of flight mass spectrometry, the more rarely encountered species of Haemophilus and Aggregatibacter will increasingly be identified in clinical microbiology

  17. Isolation of Pasteurella haemolytica leukotoxin mutants.

    PubMed Central

    Chidambaram, M; Sharma, B; Petras, S F; Reese, C P; Froshauer, S; Weinstock, G M

    1995-01-01

    Two mutants of Pasteurella haemolytica A1 that do not produce leukotoxin were isolated. Following mutagenesis, colonies were screened with antiserum by a filter assay for absence of the secreted leukotoxin. The two mutants both appeared to produce normal amounts of other antigens, as judged by reactivity with polyclonal serum from an animal with pasteurellosis, and were not altered in beta-hemolytic activity as seen on blood agar plates. There was no evidence of either cell-associated or secreted leukotoxin protein when Western blots (immunoblots) were carried out with the polyclonal serum or with a monoclonal antibody directed against the leukotoxin. Southern blots revealed that both mutants show the wild-type restriction pattern at the leukotoxin locus, although the strain with the lktA2 mutation showed differences in other regions of the chromosome on analysis by pulsed-field gel electrophoresis. The strain with the lktA2 mutation grew more slowly than did the wild-type strain, while the strain with the lktA1 mutation was indistinguishable from the wild-type strain in its growth properties. The strain with the lktA1 mutation should be valuable in determining the role of the leukotoxin in virulence as well as in identifying other virulence factors of P. haemolytica. PMID:7868223

  18. Binding of Pasteurella haemolytica leukotoxin to bovine leukocytes.

    PubMed Central

    Brown, J F; Leite, F; Czuprynski, C J

    1997-01-01

    Pasteurella haemolytica is the principal bacterial pathogen in the bovine respiratory disease complex. This organism produces an exotoxin (referred to as leukotoxin) during logarithmic-phase growth that is a potent leukocyte-modulating agent. At low concentrations, it activates neutrophils and mononuclear phagocytes to release inflammatory mediators, while at the same time making these cells destined to undergo apoptotic cell death. At higher concentrations, the toxin causes rapid swelling and loss of cell viability. In this study, we demonstrated that toxin binding can be directly evaluated by flow cytometry with biologically active biotinylated leukotoxin. Leukotoxin binding was blocked by the addition of a neutralizing anti-leukotoxin monoclonal antibody and was not detected when bovine leukocytes were incubated with culture filtrates from a mutant strain of P. haemolytica that does not produce biologically active leukotoxin. In addition, treatment of bovine leukocytes with protease K eliminated subsequent binding of leukotoxin, suggesting that there is a protein on the leukocyte surface that is either a leukotoxin binding site or is required for stabilization of leukotoxin binding. We did not detect binding of biotinylated leukotoxin to porcine or human leukocytes, which have been reported previously to be resistant to the lytic effects of the leukotoxin. These findings suggest that there may be a specific binding site for P. haemolytica leukotoxin on bovine but not on porcine or human leukocytes and that it might be involved in the activation and lytic activities of the leukotoxin. PMID:9284143

  19. Monoclonal antibodies to Actinobacillus actinomycetemcomitans.

    PubMed Central

    Place, D A; Scidmore, N C; McArthur, W P

    1988-01-01

    Murine hybridoma cell lines were developed which synthesized monoclonal antibodies against Actinobacillus actinomycetemcomitans-associated antigens. Monoclonal antibodies specific for an antigen(s) common to all A. actinomycetemcomitans isolates tested but not detected on other gram-negative oral plaque microorganisms or other Actinobacillus species were identified. Monoclonal antibodies specific for each serotype group of A. actinomycetemcomitans which did not bind to other Actinobacillus species or oral plaque microorganisms were also identified. PMID:3356470

  20. Localization of Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Subunits during Intoxication of Live Cells

    PubMed Central

    Damek-Poprawa, Monika; Jang, Jae Yeon; Volgina, Alla; Korostoff, Jonathan

    2012-01-01

    The cytolethal distending toxin (Cdt), produced by some clinically important Gram-negative bacterial species, is related to the family of AB-type toxins. Three heterologous proteins (CdtA, CdtB, and CdtC) and a genotoxin mode of action distinguish the Cdt from others in this toxin class. Crystal structures of several species-specific Cdts have provided a basis for predicting subunit interactions and functions. In addition, empirical studies have yielded significant insights into the in vivo interactions of the Cdt subunits. However, there are still critical gaps in information about the intoxication process. In this study, a novel protein tagging technology was used to localize the subunits in Chinese hamster ovary cells (CHO-K1). A tetracysteine motif was engineered in each subunit, and in subunits with mutations in predicted functional domains, to permit detection with the fluorescein arsenical hairpin binding (FlAsH) dye Lumio green. Live-cell imaging, in conjunction with confocal microscopy, was used to capture the locations of the individual subunits in cells intoxicated, under various conditions, with hybrid heterotrimers. Using this approach, we observed the following. (i) The CdtA subunit remains on the cell surface of CHO cells in association with cholesterol-containing and cholesterol-depleted membrane. (ii) The CdtB subunit is exclusively in the cytosol and, after longer exposure times, localizes to the nucleus. (iii) The CdtC subunit is present on the cell surface and, to a greater extent, in the cytosol. These observations suggest that CdtC, but not CdtA, functions as a chaperone for CdtB entry into cells. PMID:22645284

  1. Antigenic and virulence properties of Pasteurella haemolytica leukotoxin mutants.

    PubMed Central

    Petras, S F; Chidambaram, M; Illyes, E F; Froshauer, S; Weinstock, G M; Reese, C P

    1995-01-01

    Antigenic properties of two mutants of Pasteurella haemolytica, strains 59B0071 and 59B0072, that do not produce detectable leukotoxin were investigated. Western blot (immunoblot) analysis with a number of polyclonal sera from animals recovering from pasteurellosis revealed that both mutants secreted a variety of antigens that were also present in cultures of several wild-type strains. These antigens ranged from about 100 to 15 kDa. Mutant strain 59B0071 was found to be totally deficient in leukotoxin, as judged not only by Western blotting but also by cytotoxicity assays with bovine lymphoma (BL-3) cells or bovine polymorphonuclear cells as targets. The mutant strain 59B0071 had normal levels of a secreted sialylglycoprotease, however. When strains were tested for virulence in goat and cattle challenge experiments, a reduction in mortality and lung lesions was observed with the mutant 59B0071 in comparison with results obtained with wild-type strains. These results are consistent with an important role for leukotoxin in P. haemolytica virulence and suggest that leukotoxin-negative mutants may be useful tools in the investigation of other virulence properties involved in P. haemolytica infections. PMID:7868224

  2. CCR5 is a receptor for Staphylococcus aureus leukotoxin ED

    PubMed Central

    III, Francis Alonzo; Kozhaya, Lina; Rawlings, Stephen A.; Reyes-Robles, Tamara; DuMont, Ashley L.; Myszka, David G.; Landau, Nathaniel; Unutmaz, Derya; Torres, Victor J.

    2012-01-01

    Pore-forming toxins are critical virulence factors for many bacterial pathogens and are central to Staphylococcus aureus-mediated killing of host cells. S. aureus encodes pore-forming bi-component leukotoxins that are toxic toward neutrophils, but also specifically target other immune cells. Despite decades since the first description of Staphylococcal leukocidal activity, the host factors responsible for the selectivity of leukotoxins toward different immune cells remain unknown. Here we identified the HIV co-receptor, CCR5, as a cellular determinant required for cytotoxic targeting of subsets of myeloid cells and T lymphocytes by the S. aureus leukotoxin ED (LukED). We further demonstrate that LukED-dependent cell killing is blocked by CCR5 receptor antagonists, including the HIV drug maraviroc. Remarkably, CCR5-deficient mice are largely resistant to lethal S. aureus infection, highlighting the importance of CCR5 targeting in S. aureus pathogenesis. Thus, depletion of CCR5+ leukocytes by LukED suggests a novel S. aureus immune evasion mechanism that can be therapeutically targeted. PMID:23235831

  3. Killing of Actinobacillus actinomycetemcomitans by human lactoferrin.

    PubMed Central

    Kalmar, J R; Arnold, R R

    1988-01-01

    Actinobacillus actinomycetemcomitans is a fastidious, facultative gram-negative rod associated with endocarditis, certain forms of periodontal disease, and other focal infections. Human neutrophils have demonstrated bactericidal activity against A. actinomycetemcomitans, and much of the oxygen-dependent killing has been attributed to the myeloperoxidase-H2O2-halide system. However, the contribution of other neutrophil components to killing activity is obscure. Lactoferrin, an iron-binding glycoprotein, is a major constituent of neutrophil-specific granules and is also found in mucosal secretions. In this report, we show that human lactoferrin is bactericidal for A. actinomycetemcomitans. Killing activity required an unsaturated (iron- and anion-free) molecule that produced a 2-log decrease in viability within 120 min at 37 degrees C at a concentration of 1.9 microM. Besides exhibiting concentration dependence, killing kinetics were affected by minor variations in temperature and pH. Magnesium, a divalent cation thought to stabilize lipopolysaccharide interactions on the surface of gram-negative organisms, enhanced lactoferrin killing of A. actinomycetemcomitans, while other cations, such as potassium and calcium, had no effect. Our data suggest that lactoferrin contributes to killing of A. actinomycetemcomitans by human neutrophils and that it may also play a significant role in innate secretory defense against this potential periodontopathogen. PMID:3417349

  4. Regulation of expression of the Pasteurella haemolytica leukotoxin determinant.

    PubMed Central

    Strathdee, C A; Lo, R Y

    1989-01-01

    The Pasteurella haemolytica leukotoxin determinant is composed of four contiguous genes encoded on the same DNA strand and denoted lktCABD, in the order of their genetic organization. To gain a better understanding of the expression and regulation of the leukotoxin, the transcripts and promoters of the lkt determinant were mapped. Northern (RNA) blot analysis revealed two sets of transcripts. One set was 3.7 and 3.4 kilobases long, encoded lktCA, and comprised approximately 90% of the transcripts, whereas the other set was 7.4 and 7.1 kilobases long and encoded lktCABD. Two promoters were present, and each had features similar to the Escherichia coli consensus promoter sequences. Both promoters were located upstream from lktC; they were separated by 258 base pairs, as mapped by primer extension analysis. These results suggest a mechanism of expression similar to that of the related E. coli hemolysin. Transcription initiated upstream from lktC at either promoter and continued through lktC and lktA to a rho-independent transcriptional termination signal in the lktA-lktB intercistronic region. This signal attenuated expression by terminating 90% of transcription to generate the 3.7- and 3.4-kilobase lktCA transcripts. The remaining readthrough transcription generated full-length 7.4- and 7.1-kilobase lktCABD transcripts. Expression of the leukotoxin was greatly reduced by growth at 30 degrees C, pH 6.5, and Fe2+ limitation. These conditions also modulated the expression of a number of other secreted proteins, which suggests that all of these secreted proteins are controlled by the same regulatory mechanism. Images PMID:2478522

  5. Extraction and partial characterization of a leukotoxin from a plaque-derived Gram-negative microorganism.

    PubMed Central

    Tsai, C C; McArthur, W P; Baehni, P C; Hammond, B F; Taichman, N S

    1979-01-01

    The plaque-derived gram-negative microorganism Y4 identified as a member of the genus Actinobacillus, was tested for a soluble cytotoxic factor(s). Sonication or incubation of viable Y4 microorganisms in distilled water or normal human serum resulted in liberation of a soluble material which was cytotoxic in vitro for human polymorphonuclear leukocytes (PMNs). The Y4 soluble sonic extract was also cytotoxic to human peripheral blood monocytes. However, human lymphocytes, platelets, and fibroblasts, as well as rabbit, rat, and mouse leukocytes and chicken embryo fibroblasts, were not killed by exposure to the Y4 sonic extract. No hemolytic activity was detected in the Y4 sonic extract. No hemolytic activity was detected in the Y4 sonic extract. Consequently, the factor(s) in the Y4 sonic extract was referred to as Y4 leukotoxin. The Y4 leukotoxin was inactive at 4 degrees C, heat sensitive (56 degrees C, 30 min), and inactivated by proteases. The cytotoxic effect of Y4 leukotoxin on PMNs was dose, time, and temperature dependent. The leukotoxin did not bind to viable PMNs at 4 degrees C but did bind to dead PMN membrane components at both 4 and 37 degrees C. The addition of bovine serum albumin (51 mg/ml) to PMN-Y4 leukotoxin cultures inhibited the release of lactate dehydrogenase from the PMNs, but did not prevent the death of the cells as indicated by electron microscopy. Lysosomal markers were released in parallel to the cytoplasmic enzyme lactate dehydrogenase from Y4 leukotoxin-treated PMNs. The addition of 0.02 M ethylenedinitrilotetraacetic acid to these cultures inhibited release of lysosomal markers but enhanced the release of lactate dehydrogenase. These results suggested that a soluble leukotoxin with specificity for only human PMNs and monocytes can be liberated from viable Y4. What role this leukotoxin plays in the pathogenicity of the Y4 microorganism is not yet known. However, this leukotoxin is one of the first materials from a plaque

  6. Identification of Fur-regulated genes in Actinobacillus actinomycetemcomitans.

    PubMed

    Haraszthy, Violet I; Jordan, Shawn F; Zambon, Joseph J

    2006-03-01

    Actinobacillus actinomycetemcomitans is an oral pathogen that causes aggressive periodontitis as well as sometimes life-threatening, extra-oral infections. Iron regulation is thought to be important in the pathogenesis of A. actinomycetemcomitans infections and, consistent with this hypothesis, the fur gene has recently been identified and characterized in A. actinomycetemcomitans. In this study, 14 putatively Fur-regulated genes were identified by Fur titration assay (Furta) in A. actinomycetemcomitans, including afuA, dgt, eno, hemA, tbpA, recO and yfe - some of which are known to be Fur regulated in other species. A fur mutant A. actinomycetemcomitans strain was created by selecting for manganese resistance in order to study the Fur regulon. Comparisons between the fur gene sequences revealed that nucleotide 66 changed from C in the wild-type to T in the mutant strain, changing leucine to isoleucine. The fur mutant strain expressed a nonfunctional Fur protein as determined by Escherichia coli-based ferric uptake assays and Western blotting. It was also more sensitive to acid stress and expressed higher levels of minC than the wild-type strain. minC, which inhibits cell division in other bacterial species and whose regulation by iron has not been previously described, was found to be Fur regulated in A. actinomycetemcomitans by Furta, by gel shift assays, and by RT-qPCR assays for gene expression. PMID:16514158

  7. Distinction between pore assembly by staphylococcal alpha-toxin versus leukotoxins.

    PubMed

    Joubert, Olivier; Voegelin, Joëlle; Guillet, Valérie; Tranier, Samuel; Werner, Sandra; Colin, Didier A; Dalla Serra, Mauro; Keller, Daniel; Monteil, Henri; Mourey, Lionel; Prévost, Gilles

    2007-01-01

    The staphylococcal bipartite leukotoxins and the homoheptameric alpha-toxin belong to the same family of beta-barrel pore-forming toxins despite slight differences. In the alpha-toxin pore, the N-terminal extremity of each protomer interacts as a deployed latch with two consecutive protomers in the vicinity of the pore lumen. N-terminal extremities of leukotoxins as seen in their three-dimensional structures are heterogeneous in length and take part in the beta-sandwich core of soluble monomers. Hence, the interaction of these N-terminal extremities within structures of adjacent monomers is questionable. We show here that modifications of their N-termini by two different processes, using fusion with glutathione S-transferase (GST) and bridging of the N-terminal extremity to the adjacent beta-sheet via disulphide bridges, are not deleterious for biological activity. Therefore, bipartite leukotoxins do not need a large extension of their N-terminal extremities to form functional pores, thus illustrating a microheterogeneity of the structural organizations between bipartite leukotoxins and alpha-toxin.

  8. Crystal structures of the components of the Staphylococcus aureus leukotoxin ED

    PubMed Central

    Nocadello, S.; Minasov, G.; Shuvalova, L.; Dubrovska, I.; Sabini, E.; Bagnoli, F.; Grandi, G.; Anderson, W. F.

    2016-01-01

    Staphylococcal leukotoxins are a family of β-barrel, bicomponent, pore-forming toxins with membrane-damaging functions. These bacterial exotoxins share sequence and structural homology and target several host-cell types. Leukotoxin ED (LukED) is one of these bicomponent pore-forming toxins that Staphylococcus aureus produces in order to suppress the ability of the host to contain the infection. The recent delineation of the important role that LukED plays in S. aureus pathogenesis and the identification of its protein receptors, combined with its presence in S. aureus methicillin-resistant epidemic strains, establish this leukocidin as a possible target for the development of novel therapeutics. Here, the crystal structures of the water-soluble LukE and LukD components of LukED have been determined. The two structures illustrate the tertiary-structural variability with respect to the other leukotoxins while retaining the conservation of the residues involved in the interaction of the protomers in the bipartite leukotoxin in the pore complex. PMID:26894539

  9. Cloning and expression of the leukotoxin gene of Pasteurella haemolytica A1 in Escherichia coli K-12.

    PubMed Central

    Lo, R Y; Shewen, P E; Strathdee, C A; Greer, C N

    1985-01-01

    A clone bank of Pasteurella haemolytica A1 was constructed by partial digestion of the genomic DNA with Sau3A and ligation of 5- to 10-kilobase-pair fragments into the BamHI site of the plasmid vector pBR322. After transformation into Escherichia coli K-12, a total of 4 X 10(3) recombinant clones was obtained. These were screened for the production of P. haemolytica soluble antigens by a colony enzyme-linked immunosorbent assay blot method with a rabbit antiserum raised against the soluble antigens. The clones producing P. haemolytica soluble antigens were then analyzed for the production of the leukotoxin by a cytotoxicity assay with cells from a bovine leukemia-derived B-lymphocyte cell line as the target cells. Positive clones were identified, and subsequent restriction analysis of the recombinant plasmids showed that the same 6.3 kilobase pairs of insert DNA was cloned in either of the two orientations into the plasmid vector pBR322. One of the clones was selected for further characterization of the leukotoxin as produced in E. coli. Tests for heat lability and target cell species specificity with canine, porcine, and human peripheral blood lymphocytes indicated that the activity of the cloned leukotoxin was identical to that of the P. haemolytica leukotoxin. Furthermore, the E. coli-produced leukotoxin was also neutralized by bovine or rabbit antiserum known to have antitoxic activity. When cellular proteins from the E. coli clones were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis, a 100,000-dalton protein was identified which corresponded to one of the soluble antigens found in the leukotoxic culture supernatant of P. haemolytica. These results demonstrated that the gene(s) for the P. haemolytica leukotoxin have been cloned and that the leukotoxin was expressed in E. coli. Images PMID:3905610

  10. Complete Closed Genome Sequences of a Mannheimia haemolytica Serotype A1 Leukotoxin Deletion Mutant and Its Wild-Type Parent Strain

    PubMed Central

    Harhay, Gregory P.; Smith, Timothy P. L.; Bono, James L.; Chitko-McKown, Carol G.

    2015-01-01

    Mannheimia haemolytica is a bacterial pathogen that secretes leukotoxin (LktA) which binds to leukocyte membranes via CD18, causing bacterial pneumonia in ruminants. We report the complete closed genome sequences of a leukotoxin mutant and its parent strain that are frequently used in respiratory disease studies. PMID:25953160

  11. Recurrent infective endocarditis due to Aggregatibacter aphrophilus and Staphylococcus lugdunensis.

    PubMed

    Hidalgo-García, L; Hurtado-Mingo, A; Olbrich, P; Moruno-Tirado, A; Neth, O; Obando, I

    2015-03-01

    Uncommon microorganisms are increasingly being recognized as causative agents of paediatric infectious endocarditis (IE). We report a 4-year old girl with congenital heart disease, who suffered from 2 IE episodes secondary to Aggregatibacter aphrophilus (formerly Haemophilus aphrophilus) and Staphylococcus lugdunensis, both rarely reported pathogens in this age group. The patient was initially successfully treated with prolonged intravenous antibiotic courses, however removal of the Contegra valved conduit during the second episode was required due to recurrence of fever and development of pulmonary embolism despite completion of antibiotic therapy. A. aphrohilus is a member of the fastidious gram negative microorganisms of the HACEK group (Haemophilus spp., Aggregatibacter spp, Cardiobaterium hominis, Eikenella corrodens and Kingella kingae), that colonize the oropharynx and are a recognised cause of IE. Prognosis of children with IE due to HACEK group members varies, half of them suffering from complications and mortality rates of 10-12.5%. Although S. lugdunensis belongs to coagulase negative staphylococci (CONS), it behaves more like S. aureus species rather than CONS. This microorganism is a well-described cause of endocarditis in adult patients, associated with high requirements of surgical procedures and mortality (42-78%). In conclusion, paediatric IE can be caused by uncommon microorganisms associated with severe complications and potential fatality. The isolation of S. lugdunensis or A. aphrophilus in febrile patients should be considered clinically relevant and cardiac involvement must be ruled out. Those patients with proved IE will require prolonged intravenous antibiotic courses and in complicated cases surgical intervention.

  12. Complete closed genome sequences of a Mannheimia haemolytica serotype A1 leukotoxin deletion mutant and its wild type parent strain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mannheimia haemolytica is a bacterial pathogen associated with bovine respiratory disease complex (BRDC). It secretes a leukotoxin that binds to CD18 on leukocyte membranes and causes acute inflammation and lung injury characteristic of BRDC. We report the complete closed genome sequences of a leu...

  13. Mannheimia haemolytica and Its Leukotoxin Cause Macrophage Extracellular Trap Formation by Bovine Macrophages

    PubMed Central

    Aulik, Nicole A.; Hellenbrand, Katrina M.

    2012-01-01

    Human and bovine neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of extracellular trapping and killing of pathogens. Recently, we reported that bovine neutrophils release NETs in response to the important respiratory pathogen Mannheimia haemolytica and its leukotoxin (LKT). Here, we demonstrate macrophage extracellular trap (MET) formation by bovine monocyte-derived macrophages exposed to M. haemolytica or its LKT. Both native fully active LKT and noncytolytic pro-LKT (produced by an lktC mutant of M. haemolytica) stimulated MET formation. Confocal and scanning electron microscopy revealed a network of DNA fibrils with colocalized histones in extracellular traps released from bovine macrophages. Formation of METs required NADPH oxidase activity, as previously demonstrated for NET formation. METs formed in response to LKT trapped and killed a portion of the M. haemolytica cells. Bovine alveolar macrophages, but not peripheral blood monocytes, also formed METs in response to M. haemolytica cells. MET formation was not restricted to bovine macrophages. We also observed MET formation by the mouse macrophage cell line RAW 264.7 and by human THP-1 cell-derived macrophages, in response to Escherichia coli hemolysin. The latter is a member of the repeats-in-toxin (RTX) toxin family related to the M. haemolytica leukotoxin. This study demonstrates that macrophages, like neutrophils, can form extracellular traps in response to bacterial pathogens and their exotoxins. PMID:22354029

  14. Mannheimia haemolytica and its leukotoxin cause macrophage extracellular trap formation by bovine macrophages.

    PubMed

    Aulik, Nicole A; Hellenbrand, Katrina M; Czuprynski, Charles J

    2012-05-01

    Human and bovine neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of extracellular trapping and killing of pathogens. Recently, we reported that bovine neutrophils release NETs in response to the important respiratory pathogen Mannheimia haemolytica and its leukotoxin (LKT). Here, we demonstrate macrophage extracellular trap (MET) formation by bovine monocyte-derived macrophages exposed to M. haemolytica or its LKT. Both native fully active LKT and noncytolytic pro-LKT (produced by an lktC mutant of M. haemolytica) stimulated MET formation. Confocal and scanning electron microscopy revealed a network of DNA fibrils with colocalized histones in extracellular traps released from bovine macrophages. Formation of METs required NADPH oxidase activity, as previously demonstrated for NET formation. METs formed in response to LKT trapped and killed a portion of the M. haemolytica cells. Bovine alveolar macrophages, but not peripheral blood monocytes, also formed METs in response to M. haemolytica cells. MET formation was not restricted to bovine macrophages. We also observed MET formation by the mouse macrophage cell line RAW 264.7 and by human THP-1 cell-derived macrophages, in response to Escherichia coli hemolysin. The latter is a member of the repeats-in-toxin (RTX) toxin family related to the M. haemolytica leukotoxin. This study demonstrates that macrophages, like neutrophils, can form extracellular traps in response to bacterial pathogens and their exotoxins. PMID:22354029

  15. Lytic sensitivity of Actinobacillus actinomycetemcomitans Y4 to lysozyme.

    PubMed Central

    Iacono, V J; Boldt, P R; MacKay, B J; Cho, M I; Pollock, J J

    1983-01-01

    The ability of both human and hen egg white lysozymes to lyse Actinobacillus actinomycetemcomitans Y4 was investigated. Lysis was followed optically at 540 nm by measuring the percent reduction in turbidity of freshly harvested log-phase cells suspended in Tris-maleate buffers within a wide range of pH (5.2 to 8.5) and molarity (0.01 to 0.2 M) and containing various amounts of enzyme and EDTA. In several instances, treated microorganisms were subsequently examined in thin sections by electron microscopy. Reductions in turbidity and clearing of suspensions occurred with small amounts of lysozyme (less than 1 microgram) under relatively alkaline conditions and at low ionic strength and in the presence of small amounts of EDTA (greater than 0.01 mM). Under the most alkaline conditions, EDTA alone effected turbidity reductions similar to those observed in the presence of lysozyme, which suggested that EDTA not only increased outer membrane permeability but also caused cell lysis. Ultrastructural analysis did not always correspond to turbidimetric observations. Cell lysis was virtually complete in suspensions containing both lysozyme and EDTA. However, in contrast to turbidimetric findings, a significant percentage of cells (greater than 25%) was lysed in the presence of lysozyme alone. Furthermore, significant damage occurred in the presence of EDTA alone. Spheroplast-like cell ghosts were present which surrounded condensed cytoplasm or relatively clear spaces. These findings further support the concept of the requirement for electron microscopy to assess lytic damage in addition to turbidimetric and biochemical methods. Our results are the first to demonstrate the remarkable sensitivity of A. actinomycetemcomitans Y4 to lysozyme and to show that EDTA not only affects outer membrane permeability but effects cell lysis, possibly through activation of autolytic enzymes at the cytoplasmic membrane. The exquisite sensitivity of A. actinomycetemcomitans Y4 to lysis could be

  16. Efficacy of recombinant leukotoxin in protection against pneumonic challenge with live Pasteurella haemolytica A1.

    PubMed Central

    Conlon, J A; Shewen, P E; Lo, R Y

    1991-01-01

    The recombinant leukotoxin (rLKT) of the bacterium Pasteurella haemolytica A1 was examined for its ability to protect cattle from experimental challenge with logarithmic-phase P. haemolytica. Six different vaccines were utilized in the experiment: P. haemolytica culture supernatant, P. haemolytica culture supernatant enriched with rLKT, rLKT alone, P. haemolytica culture supernatant enriched with Escherichia coli supernatant not containing LKT, E. coli supernatant alone, and phosphate-buffered saline. rLKT alone showed no protective capacity against development of clinical signs of respiratory disease or against development of postmortem lung lesions after experimental challenge. It was, however, shown to enhance the efficacy of the culture supernatant vaccine and decrease clinical signs and pneumonic lesions. The complexity of protective immunity in this disease is emphasized in this study, and, although LKT is an important virulence factor of the organism, an immune response to LKT alone does not protect animals against disease. PMID:1987075

  17. In vitro activity of azithromycin compared with that of erythromycin against Actinobacillus actinomycetemcomitans.

    PubMed Central

    Pajukanta, R; Asikainen, S; Saarela, M; Alaluusua, S; Jousimies-Somer, H

    1992-01-01

    The in vitro susceptibility of Actinobacillus actinomycetemcomitans to azithromycin, a new macrolide antibiotic of a new class known as azalides, was compared with that of erythromycin by the agar dilution method on Mueller-Hinton Haemophilus test medium. Eighty-two A. actinomycetemcomitans strains, 79 recent clinical isolates obtained from 40 periodontally healthy or diseased subjects, and 3 type strains were included in the study. Erythromycin showed poor in vitro activity against A. actinomycetemcomitans. Azithromycin, however, was highly effective against A. actinomycetemcomitans: all strains were inhibited at 2.0 micrograms/ml. Azithromycin exhibited the best in vitro activity against the serotype a subpopulation of A. actinomycetemcomitans: 100% of the strains were inhibited at 1.0 micrograms/ml. The lowest MICs were, however, recorded by serotype b strains. Since azithromycin has favorable pharmacokinetic properties, including excellent distribution into tissues, it could be expected to pass into gingival crevicular fluid at levels sufficient to inhibit A. actinomycetemcomitans in vivo. Therefore, it is a good candidate for future clinical trials in A. actinomycetemcomitans-associated periodontitis. PMID:1329617

  18. Immunosuppressive factor from Actinobacillus actinomycetemcomitans down regulates cytokine production.

    PubMed Central

    Kurita-Ochiai, T; Ochiai, K

    1996-01-01

    A cytoplasmic soluble fraction of Actinobacillus actinomycetemcomitans Y4 was isolated and characterized as suppressing mitogen-stimulated proliferation of and cytokine production by C3H/HeN mouse splenic T cells. This factor, designated suppressive factor 1 (SF1), was isolated from the supernatant of sonicated whole bacteria and purified by Q-Sepharose Fast Flow column chromatography, DEAE-Sepharose Fast Flow column chromatography, hydroxyapatite high-pressure liquid chromatography (HPLC), and Protein Pack 300 & 125 gel filtration HPLC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the purified SF1 migrated as a single band corresponding to a molecular mass of 14 kDa. This molecule was protease labile, heat resistant, and noncytotoxic. N'-terminal sequence analysis revealed no homology with any known peptides of periodontopathic bacteria or with any host-derived growth factors. Purified SF1 suppressed the proliferation of mouse splenic T cells which had been stimulated with concanavalin A, as well as suppressing the production of interleukin-2 (IL-2), gamma interferon, IL-4, and IL-5 from CD4+ T cells as 0.1 microgram/ml or more. These data suggest that SF1 produced by the periodontal pathogen A. actinomycetemcomitans functions as a virulence factor by down regulating T-cell proliferation and cytokine production at local defense sites. PMID:8557373

  19. A longitudinal microbiological investigation of Actinobacillus actinomycetemcomitans and Eikenella corrodens in juvenile periodontitis.

    PubMed Central

    Mandell, R L

    1984-01-01

    Longitudinal clinical and microbiological monitoring of subjects with localized juvenile periodontitis indicated that Actinobacillus actinomycetemcomitans and Eikenella corrodens were significantly associated (P less than 0.05) with active tissue destruction. PMID:6381313

  20. Role of high-avidity binding of human neutrophil myeloperoxidase in the killing of Actinobacillus actinomycetemcomitans.

    PubMed Central

    Miyasaki, K T; Zambon, J J; Jones, C A; Wilson, M E

    1987-01-01

    The binding of the neutrophil enzyme myeloperoxidase (MPO) to microbial surfaces is believed to be the first step in its microbicidal activity. The MPO-H2O2-Cl- system is responsible for most oxidative killing of Actinobacillus actinomycetemcomitans by human neutrophils. There appear to be three forms of MPO (MPO I, II, and III), all of which can kill this organism in the presence of H2O2 and chloride. In this study, we characterized the binding of native human neutrophil MPO to A. actinomycetemcomitans by an elution procedure dependent on the cationic detergent cetyltrimethylammonium bromide. Binding of native MPO was rapid and reached apparent equilibrium within 1 min. A proportion of binding under equilibrium conditions was saturable and highly avid, with a capacity of 4,500 sites per cell and a dissociation constant of 7.9 X 10(-10) M. At equal protein concentrations, more MPO III bound than MPO II, and more MPO II bound than MPO I. The high-avidity interaction was inhibitable with yeast mannan and with the serotype-defining mannan of A. actinomycetemcomitans. Binding was also partially reversible with yeast mannan. MPO bound to the high-avidity sites did not oxidize guaiacol but oxidized chloride, as detected by the chlorination of taurine. MPO bound to the high-avidity sites was incapable of killing A. actinomycetemcomitans alone in the presence of H2O2 and Cl-, but potentiated killing when sufficient additional MPO was provided. The killing of A. actinomycetemcomitans by the MPO-H2O2-Cl- system was inhibited by yeast mannan and a serotype-defining mannan of A. actinomycetemcomitans. We conclude that high-avidity binding of MPO to the surface of A. actinomycetemcomitans is a mannan-specific interaction and that MPO bound to the high-avidity sites is essential but not alone sufficient to kill A. actinomycetemcomitans. PMID:3032796

  1. Conjugal transfer of broad-host-range incompatibility group P and Q plasmids from Escherichia coli to Actinobacillus actinomycetemcomitans.

    PubMed Central

    Goncharoff, P; Yip, J K; Wang, H; Schreiner, H C; Pai, J A; Furgang, D; Stevens, R H; Figurski, D H; Fine, D H

    1993-01-01

    The first example of conjugal transfer of DNA from Escherichia coli to the periodontal pathogen Actinobacillus actinomycetemcomitans is presented. Derivatives of the incompatibility group P (IncP) plasmid RK2 successfully transferred from an E. coli donor to an A. actinomycetemcomitans recipient. The resulting A. actinomycetemcomitans transconjugants transferred the plasmids back to E. coli recipients. The IncP transfer functions were also used in trans to mobilize the IncQ plasmid pBK1 from E. coli to A. actinomycetemcomitans. The IncP and IncQ plasmids both transferred into A. actinomycetemcomitans at high frequencies (0.3 to 0.5 transconjugants per donor) and showed no gross deletions, insertions, or rearrangements. Determinations of MICs of various antibiotics for the A. actinomycetemcomitans transconjugant strains demonstrated the expression of ampicillin, chloramphenicol, and kanamycin resistance determinants. Images PMID:8335386

  2. Cellular fatty acid composition of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

    PubMed

    Braunthal, S D; Holt, S C; Tanner, A C; Socransky, S S

    1980-06-01

    Strains of Actinobacillus actinomycetemcomitans isolated from deep pockets of patients with juvenile periodontitis were analyzed for their content of cellular fatty acids. Oral Haemophilus strains, morphologically and biochemically similar to Haemophilus aphrophilus, were also examined for their content of cellular fatty acids. The extractable lipids of the actinobacilli represented approximately 10% of the cell dry weight, with the bound lipids representing 2 to 5%. The major fatty acids consisted of myristic (C14:0) and palmitic (C16:0) acids and a C16:1 acid, possibly palmitoleic acid, accounting for 21, 35, and 31% of the total extractable fatty acids, respectively. Haemophilus strains had a similar cellular fatty acid content. PMID:7430333

  3. Cellular fatty acid composition of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

    PubMed Central

    Braunthal, S D; Holt, S C; Tanner, A C; Socransky, S S

    1980-01-01

    Strains of Actinobacillus actinomycetemcomitans isolated from deep pockets of patients with juvenile periodontitis were analyzed for their content of cellular fatty acids. Oral Haemophilus strains, morphologically and biochemically similar to Haemophilus aphrophilus, were also examined for their content of cellular fatty acids. The extractable lipids of the actinobacilli represented approximately 10% of the cell dry weight, with the bound lipids representing 2 to 5%. The major fatty acids consisted of myristic (C14:0) and palmitic (C16:0) acids and a C16:1 acid, possibly palmitoleic acid, accounting for 21, 35, and 31% of the total extractable fatty acids, respectively. Haemophilus strains had a similar cellular fatty acid content. PMID:7430333

  4. Actinobacillus actinomycetemcomitans contamination of toothbrushes from patients harbouring the organism.

    PubMed

    Müller, H P; Lange, D E; Müller, R F

    1989-07-01

    The main ecological niche of Actinobacillus actinomycetemcomitans (A.a.) seems to be the periodontal pocket, but it can also be isolated from supragingival plaque, buccal and tongue mucosa, or saliva. We examined toothbrushes from 21 patients, all identified as harbouring moderate to large numbers of A.a. in subgingival plaque, for contamination with this organism. 29% of the toothbrushes presented by our patients yielded detectable numbers of A.a. Immediately after toothbrushing this figure rose to 62%, but dropped to 50% after 1 h. Numbers of isolated A.a. on toothbrushes were weakly correlated with the degree of periodontal destruction, and significantly more numbers of A.a. on toothbrushes could be detected if the organism was found on mucous membranes or in saliva. There was no association with gingival inflammation, supragingival plaque nor mean numbers of isolated subgingival A.a. PMID:2760252

  5. Introns in the Cytolethal Distending Toxin Gene of Actinobacillus actinomycetemcomitans

    PubMed Central

    Tan, Kai Soo; Ong, Grace; Song, Keang Peng

    2005-01-01

    In eukaryotic cells, genes are interrupted by intervening sequences called introns. Introns are transcribed as part of a precursor RNA that is subsequently removed by splicing, giving rise to mature mRNA. However, introns are rarely found in bacteria. Actinobacillus actinomycetemcomitans is a periodontal pathogen implicated in aggressive forms of periodontal disease. This organism has been shown to produce cytolethal distending toxin (CDT), which causes sensitive eukaryotic cells to become irreversibly blocked at the G2/M phase of the cell cycle. In this study, we report the presence of introns within the cdt gene of A. actinomycetemcomitans. By use of reverse transcription-PCR, cdt transcripts of 2.123, 1.572, and 0.882 kb (RTA1, RTA2, and RTA3, respectively) were detected. In contrast, a single 2.123-kb amplicon was obtained by PCR with the genomic DNA. Similar results were obtained when a plasmid carrying cdt was cloned into Escherichia coli. Sequence analysis of RTA1, RTA2, and RTA3 revealed that RTA1 had undergone splicing, giving rise to RTA2 and RTA3. Two exon-intron boundaries, or splice sites, were identified at positions 863 to 868 and 1553 to 1558 of RTA1. Site-directed and deletion mutation studies of the splice site sequence indicated that sequence conservation was important in order for accurate splicing to occur. The catalytic region of the cdt RNA was located within the cdtC gene. This 0.56-kb RNA behaved independently as a catalytically active RNA molecule (a ribozyme) in vitro, capable of splicing heterologous RNA in both cis and trans configurations. PMID:15629928

  6. microRNAs responsive to A. actinomycetemcomitans and P. gingivalis LPS modulate expression of genes regulating innate immunity in human macrophages

    PubMed Central

    Naqvi, Afsar R.; Fordham, Jezrom B.; Khan, Asma; Nares, Salvador

    2013-01-01

    microRNAs (miRNA) are a class of small noncoding RNAs that regulate post-transcriptional expression of their respective target genes and are responsive to various stimuli, including lipopolysaccharide (LPS). Here we examined the early (4h) miRNA responses of THP1-differentiated macrophages challenged with LPS derived from the periodontal pathogens, Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) or environmentally modified LPS obtained from Pg grown in cigarette smoke extract. Predicted miRNA-gene target interactions for LPS-responsive miR-29b and let-7f were confirmed using dual-luciferase assays and by transfection experiments using miRNA mimics and inhibitors. Convergent and divergent miRNA profiles were observed in treated samples where differences in miRNA levels related to the type, concentration and incubation times of LPS challenge. Dual-luciferase experiments revealed miR-29b targeting of IL-6Rα and IFN-γ inducible protein 30 (IFI30) and let-7f targeting of suppressor of cytokine signaling 4 (SOCS4) and Thrombospondin-1 (TSP-1). Transfection experiments confirmed miR-29b and let-7f modulation of IL-6R and SOCS4 protein expression levels, respectively. Thus, we demonstrate convergent/divergent miRNA responses to wild type and its environmentally-modified LPS and demonstrate miRNA targeting of key genes linked to inflammation and immunity. Our data indicate that these LPS-responsive miRNAs may play a key role in fine-tuning the host response to periodontal pathogens. PMID:24062196

  7. Periodontitis‐associated pathogens P. gingivalis and A. actinomycetemcomitans activate human CD14+ monocytes leading to enhanced Th17/IL‐17 responses

    PubMed Central

    Cheng, Wan‐Chien; van Asten, Saskia D.; Burns, Lachrissa A.; Evans, Hayley G.; Walter, Gina J.; Hashim, Ahmed; Hughes, Francis J.

    2016-01-01

    The Th17/IL‐17 pathway is implicated in the pathogenesis of periodontitis (PD), however the mechanisms are not fully understood. We investigated the mechanism by which the periodontal pathogens Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) promote a Th17/IL‐17 response in vitro, and studied IL‐17+ CD4+ T‐cell frequencies in gingival tissue and peripheral blood from patients with PD versus periodontally healthy controls. Addition of Pg or Aa to monocyte/CD4+ T‐cell co‐cultures promoted a Th17/IL‐17 response in vitro in a dose‐ and time‐dependent manner. Pg or Aa stimulation of monocytes resulted in increased CD40, CD54 and HLA‐DR expression, and enhanced TNF‐α, IL‐1β, IL‐6 and IL‐23 production. Mechanistically, IL‐17 production in Pg‐stimulated co‐cultures was partially dependent on IL‐1β, IL‐23 and TLR2/TLR4 signalling. Increased frequencies of IL‐17+ cells were observed in gingival tissue from patients with PD compared to healthy subjects. No differences were observed in IL‐17+ CD4+ T‐cell frequencies in peripheral blood. In vitro, Pg induced significantly higher IL‐17 production in anti‐CD3 mAb‐stimulated monocyte/CD4+ T‐cell co‐cultures from patients with PD compared to healthy controls. Our data suggest that periodontal pathogens can activate monocytes, resulting in increased IL‐17 production by human CD4+ T cells, a process that appears enhanced in patients with PD. PMID:27334899

  8. Mannheimia haemolytica and its leukotoxin cause neutrophil extracellular trap formation by bovine neutrophils.

    PubMed

    Aulik, Nicole A; Hellenbrand, Katrina M; Klos, Heather; Czuprynski, Charles J

    2010-11-01

    Mannheimia haemolytica is an important member of the bovine respiratory disease complex, which is characterized by abundant neutrophil infiltration into the alveoli and fibrin deposition. Recently several authors have reported that human neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of trapping and killing pathogens. Here, we demonstrate that the leukotoxin (LKT) of M. haemolytica causes NET formation by bovine neutrophils in a CD18-dependent manner. Using an unacylated, noncytotoxic pro-LKT produced by an ΔlktC mutant of M. haemolytica, we show that binding of unacylated pro-LKT stimulates NET formation despite a lack of cytotoxicity. Inhibition of LKT binding to the CD18 chain of lymphocyte function-associated antigen 1 (LFA-1) on bovine neutrophils reduced NET formation in response to LKT or M. haemolytica cells. Further investigation revealed that NETs formed in response to M. haemolytica are capable of trapping and killing a portion of the bacterial cells. NET formation was confirmed by confocal microscopy and by scanning and transmission electron microscopy. Prior exposure of bovine neutrophils to LKT enhanced subsequent trapping and killing of M. haemolytica cells in bovine NETs. Understanding NET formation in response to M. haemolytica and its LKT provides a new perspective on how neutrophils contribute to the pathogenesis of bovine respiratory disease. PMID:20823211

  9. Sequence diversity, cytotoxicity and antigenic similarities of the leukotoxin of isolates of Mannheimia species from mastitis in domestic sheep.

    PubMed

    Omaleki, Lida; Browning, Glenn F; Barber, Stuart R; Allen, Joanne L; Srikumaran, Subramaniam; Markham, Philip F

    2014-11-01

    Species within the genus Mannheimia are among the most important causes of ovine mastitis. Isolates of these species can express leukotoxin A (LktA), a primary virulence factor of these bacteria. To examine the significance of variation in the LktA, the sequences of the lktA genes in a panel of isolates from cases of ovine mastitis were compared. The cross-neutralising capacities of rat antisera raised against LktA of one Mannheimia glucosida, one haemolytic Mannheimia ruminalis, and two Mannheimia haemolytica isolates were also examined to assess the effect that variation in the lktA gene can have on protective immunity against leukotoxins with differing sequences. The lktA nucleotide distance between the M. haemolytica isolates was greater than between the M. glucosida isolates, with the M. haemolytica isolates divisible into two groups based on their lktA sequences. Comparison of the topology of phylogenetic trees of 16S rDNA and lktA sequences revealed differences in the relationships between some isolates, suggesting horizontal gene transfer. Cross neutralisation data obtained with monospecific anti-LktA rat sera were used to derive antigenic similarity coefficients for LktA from the four Mannheimia species isolates. Similarity coefficients indicated that LktA of the two M. haemolytica isolates were least similar, while LktA from M. glucosida was most similar to those for one of the M. haemolytica isolates and the haemolytic M. ruminalis isolate. The results suggested that vaccination with the M. glucosida leukotoxin would generate the greatest cross-protection against ovine mastitis caused by Mannheimia species with these alleles.

  10. Expression cloning of a periodontitis-associated apoptotic effector, cagE homologue, in Actinobacillus actinomycetemcomitans.

    PubMed

    Teng, Yen-Tung A; Hu, Wenqi

    2003-04-18

    To study anti-bacterial immunity and to identify critical bacterial antigens associated with specific periodontal infection, we screened the genomic library of Actinobacillus actinomycetemcomitans, a major Gram(-) anaerobe causing human periodontitis, by expression cloning using disease-associated periodontal CD4(+)T cells derived from HuPBL-engrafted NOD/SCID mice. Here, we report one of the novel genes identified and designated, cagE homologue (in short: cagE) of A. actinomycetemcomitans, which encodes a putative bacterial type IV secretion system with significant homology to Helicobacter pylori CagE and Agrobacterium tumefaciens VirB4. All serum samples from A. actinomycetemcomitans-infected periodontitis patients, but not from the healthy controls, readily recognized CagE by ELISA and Western blot, suggesting its biological and clinical significance. The CagE protein, upon secretion, elicited significant apoptosis on primary human epithelia, endothelia, osteoblasts, and T cells by 4-12h in vitro. Importantly, both cagE(-) mutant strain and N-terminus truncated CagE protein drastically reduced (p<0.001) the induction of apoptosis on human epithelia in vitro. These data strongly suggest that a novel effector protein, CagE in A. actinomycetemcomitans, induces apoptosis of human cells and destructive immunity, thereby it may play an important role in the pathogenesis of A. actinomycetemcomitans-mediated infections. PMID:12684047

  11. The gingival immune response to Actinobacillus actinomycetemcomitans in juvenile periodontitis.

    PubMed

    Hall, E R; Falkler, W A; Martin, S A; Suzuki, J B

    1991-12-01

    The established and advanced lesions of juvenile periodontitis-localized form (JP) are predominated by B-lymphocytes and plasma cells. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. Actinobacillus actinomycetemcomitans (A.a.) is implicated as a primary etiologic agent in JP. An in vitro gingival explant culture system was utilized to study the specificity of immunoglobulins produced by diseased JP tissues. A dot-immunobinding assay demonstrated that 46% of the supernatant fluids (SF) from explant cultures of diseased tissues (n = 39) were positive for the presence of antibody to A.a. Y4, while 61% of autologous JP sera (n = 39) tested positive. For rapidly progressive (RP) and adult periodontitis (AP) SF, 50% and 40% were positive for A.a. Y4, respectively. Seventeen percent of SF from healthy tissue were positive for A.a. Y4. There was no significant difference between JP SF reactivities to A.a. Y4 when compared to reactivities of SF from AP and RP patients. Only 10% of JP SF were positive for Porphyromonas asaccharolytica, a non-oral control microorganism. The de novo biosynthesis of antibody in JP tissue, reactive with A.a. Y4, was demonstrated with Staph Protein A isolated 14C-labeled IgG (SPAG) and the use of a dot-immunobinding assay and autoradiography. The in vitro gingival tissue explant culture system described provides a useful model for the study of the synthesis and specificity of localized immunoglobulins produced by diseased tissues of JP patients. PMID:1765941

  12. Lymphocyte function-associated antigen 1 is a receptor for Pasteurella haemolytica leukotoxin in bovine leukocytes.

    PubMed

    Jeyaseelan, S; Hsuan, S L; Kannan, M S; Walcheck, B; Wang, J F; Kehrli, M E; Lally, E T; Sieck, G C; Maheswaran, S K

    2000-01-01

    Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) causes cell type- and species-specific effects in ruminant leukocytes. Recent studies indicate that P. haemolytica Lkt binds to bovine CD18, the common subunit of all beta2 integrins. We designed experiments with the following objectives: to identify which member of the beta2 integrins is a receptor for Lkt; to determine whether Lkt binding to the receptor is target cell (bovine leukocytes) specific; to define the relationships between Lkt binding to the receptor, calcium elevation, and cytolysis; and to determine whether a correlation exists between Lkt receptor expression and the magnitude of target cell cytolysis. We compared Lkt-induced cytolysis in neutrophils from control calves and from calves with bovine leukocyte adhesion deficiency (BLAD), because neutrophils from BLAD-homozygous calves exhibit reduced beta2 integrin expression. The results demonstrate for the first time that Lkt binds to bovine CD11a and CD18 (lymphocyte function-associated antigen 1 [LFA-1]). The binding was abolished by anti-CD11a or anti-CD18 monoclonal antibody (MAb). Lkt-induced calcium elevation in bovine alveolar macrophages (BAMs) was inhibited by anti-CD11a or anti-CD18 MAb (65 to 94% and 37 to 98%, respectively, at 5 and 50 Lkt units per ml; P < 0.05). Lkt-induced cytolysis in neutrophils and BAMs was also inhibited by anti-CD11a or anti-CD18 MAb in a concentration-dependent manner. Lkt bound to porcine LFA-1 but did not induce calcium elevation or cytolysis. In neutrophils from BLAD calves, Lkt-induced cytolysis was decreased by 44% compared to that of neutrophils from control calves (P < 0.05). These results indicate that LFA-1 is a Lkt receptor, Lkt binding to LFA-1 is not target cell specific, Lkt binding to bovine LFA-1 correlates with calcium elevation and cytolysis, and bovine LFA-1 expression correlates with the magnitude of Lkt-induced target cell cytolysis. PMID:10603370

  13. Phylogenetic diversity of Pasteurellaceae and horizontal gene transfer of leukotoxin in wild and domestic sheep.

    PubMed

    Kelley, Scott T; Cassirer, E Frances; Weiser, Glen C; Safaee, Shirin

    2007-01-01

    Wild and domestic animal populations are known to be sources and reservoirs of emerging diseases. There is also a growing recognition that horizontal genetic transfer (HGT) plays an important role in bacterial pathogenesis. We used molecular phylogenetic methods to assess diversity and cross-transmission rates of Pasteurellaceae bacteria in populations of bighorn sheep, Dall's sheep, domestic sheep and domestic goats. Members of the Pasteurellaceae cause an array of deadly illnesses including bacterial pneumonia known as "pasteurellosis", a particularly devastating disease for bighorn sheep. A phylogenetic analysis of a combined dataset of two RNA genes (16S ribosomal RNA and RNAse P RNA) revealed remarkable evolutionary diversity among Pasteurella trehalosi and Mannheimia (Pasteurella) haemolytica bacteria isolated from sheep and goats. Several phylotypes appeared to associate with particular host species, though we found numerous instances of apparent cross-transmission among species and populations. Statistical analyses revealed that host species, geographic locale and biovariant classification, but not virulence, correlated strongly with Pasteurellaceae phylogeny. Sheep host species correlated with P. trehalosi isolates phylogeny (PTP test; P=0.002), but not with the phylogeny of M. haemolytica isolates, suggesting that P. trehalosi bacteria may be more host specific. With regards to populations within species, we also discovered a strong correlation between geographic locale and isolate phylogeny in the Rocky Mountain bighorn sheep (PTP test; P=0.001). We also investigated the potential for HGT of the leukotoxin A (lktA) gene, which produces a toxin that plays an integral role in causing disease. Comparative analysis of the combined RNA gene phylogeny and the lktA phylogenies revealed considerable incongruence between the phylogenies, suggestive of HGT. Furthermore, we found identical lktA alleles in unrelated bacterial species, some of which had been isolated

  14. Actinobacillus actinomycetemcomitans Keratitis After Glaucoma Infiltration Surgery: A Clinical Report and Literature Review.

    PubMed

    Hong, Jiaxu; Xu, Jianjiang; Cao, Wenjun; Ji, Jian; Sun, Xinghuai

    2016-01-01

    Actinobacillus actinomycetemcomitans infection is a rare and easily misdiagnosed ocular disease. In this article, the authors report a chronic, purulent, and difficult-to-treat case of A actinomycetemcomitans keratitis following a glaucoma infiltration surgery.A 56-year-old man with a long-standing history of open-angle glaucoma in both eyes presented with a 12-week history of ocular pain, redness, and blurred vision in his right eye. He underwent a glaucoma infiltration surgery in his right eye 6 months ago. Three months postoperatively, he developed peripheral corneal stromal opacities associated with a white, thin, cystic bleb, and conjunctival injection. These opacities grew despite topical treatment with topical tobramycin, levofloxacin, natamycin, amikacin, and metronidazole eye drops.Multiple corneal scrapings revealed no organisms, and no organisms grew on aerobic, anaerobic, fungal, or mycobacterial cultures. The patient's right eye developed a severe purulent corneal ulcer with a dense hypopyon and required a corneal transplantation. Histopathologic analysis and 16S ribosomalribonucleic acid polymerase chain reaction sequencing revealed A actinomycetemcomitans as the causative organism. Postoperatively, treatment was initiated with topical levofloxacin and cyclosporine, as well as oral levofloxacin and cyclosporine. Graft and host corneal transparency were maintained at the checkup 1 month after surgery.Although it is a rare cause of corneal disease, A actinomycetemcomitans should be suspected in patients with keratitis refractory to topical antibiotic therapy. Delay in diagnosis and appropriate treatment can result in vision loss. PMID:26817919

  15. Actinobacillus actinomycetemcomitans Keratitis After Glaucoma Infiltration Surgery: A Clinical Report and Literature Review.

    PubMed

    Hong, Jiaxu; Xu, Jianjiang; Cao, Wenjun; Ji, Jian; Sun, Xinghuai

    2016-01-01

    Actinobacillus actinomycetemcomitans infection is a rare and easily misdiagnosed ocular disease. In this article, the authors report a chronic, purulent, and difficult-to-treat case of A actinomycetemcomitans keratitis following a glaucoma infiltration surgery.A 56-year-old man with a long-standing history of open-angle glaucoma in both eyes presented with a 12-week history of ocular pain, redness, and blurred vision in his right eye. He underwent a glaucoma infiltration surgery in his right eye 6 months ago. Three months postoperatively, he developed peripheral corneal stromal opacities associated with a white, thin, cystic bleb, and conjunctival injection. These opacities grew despite topical treatment with topical tobramycin, levofloxacin, natamycin, amikacin, and metronidazole eye drops.Multiple corneal scrapings revealed no organisms, and no organisms grew on aerobic, anaerobic, fungal, or mycobacterial cultures. The patient's right eye developed a severe purulent corneal ulcer with a dense hypopyon and required a corneal transplantation. Histopathologic analysis and 16S ribosomalribonucleic acid polymerase chain reaction sequencing revealed A actinomycetemcomitans as the causative organism. Postoperatively, treatment was initiated with topical levofloxacin and cyclosporine, as well as oral levofloxacin and cyclosporine. Graft and host corneal transparency were maintained at the checkup 1 month after surgery.Although it is a rare cause of corneal disease, A actinomycetemcomitans should be suspected in patients with keratitis refractory to topical antibiotic therapy. Delay in diagnosis and appropriate treatment can result in vision loss.

  16. Identification of genomic clonal types of Actinobacillus actinomycetemcomitans by restriction endonuclease analysis.

    PubMed Central

    Han, N; Hoover, C I; Winkler, J R; Ng, C Y; Armitage, G C

    1991-01-01

    To evaluate its utility in discriminating different strains, restriction endonuclease analysis was applied to 12 strains of Actinobacillus actinomycetemcomitans (3 serotype a, 5 serotype b, and 4 serotype c strains). DNA isolated from each strain was digested by 12 different restriction endonucleases, and the electrophoretic banding patterns of the resulting DNA fragments were compared. The DNA fragment patterns produced by SalI, XhoI, and XbaI for the 12 A. actinomycetemcomitans strains were simple (less than 30 bands) and allowed us to recognize easily 10 distinct genomic clonal types. The three serotype a strains exhibited distinctly different clonal types from one another, the five serotype b strains exhibited an additional four distinct clonal types, and the four serotype c strains showed another three different clonal types. The other endonucleases tested were less useful in typing A. actinomycetemcomitans. We conclude that restriction endonuclease analysis is a powerful tool for typing and discerning genetic heterogeneity and homogeneity among A. actinomycetemcomitans strains. It should, therefore, be very useful for epidemiologic studies. Images PMID:1761677

  17. Actinobacillus actinomycetemcomitans toxin induces both cell cycle arrest in the G2/M phase and apoptosis.

    PubMed

    Ohguchi, M; Ishisaki, A; Okahashi, N; Koide, M; Koseki, T; Yamato, K; Noguchi, T; Nishihara, T

    1998-12-01

    We found that the culture supernatant of the periodontopathic bacterium Actinobacillus actinomycetemcomitans had a cytotoxic effect on several cell lines. In this study, we purified the toxin from the culture supernatant of A. actinomycetemcomitans Y4 by a four-step procedure: ammonium sulfate precipitation, POROS HQ/M column chromatography, polymyxin B matrix column chromatography, and Mono-Q column chromatography. The purified toxin gave two major bands of protein with molecular masses of 80 and 85 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mechanism of cell death of the B-cell hybridoma cell line HS-72 was examined by observing changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of degraded chromosomal DNA, indicating the induction of apoptosis. Overexpression of human Bcl-2 suppressed apoptosis in HS-72 cells, indicating that the toxin from A. actinomycetemcomitans induces apoptosis by a Bcl-2-inhibitable mechanism. Flow cytometric analysis revealed that the toxin caused cell cycle arrest in the G2/M phase and apoptosis in HS-72 cells. In addition, aurintricarboxylic acid, a DNA endonuclease inhibitor, markedly decreased the percentage of apoptotic cells but had no effect on cell cycle arrest in the G2/M phase. Taken together, these findings suggest that the toxin from A. actinomycetemcomitans could mediate the development of periodontal diseases through cell cycle arrest in the G2/M phase and apoptosis in B lymphocytes of periodontal tissue. PMID:9826381

  18. Nonspecific Adherence by Actinobacillus actinomycetemcomitans Requires Genes Widespread in Bacteria and Archaea

    PubMed Central

    Kachlany, Scott C.; Planet, Paul J.; Bhattacharjee, Mrinal K.; Kollia, Evyenia; DeSalle, Rob; Fine, Daniel H.; Figurski, David H.

    2000-01-01

    The gram-negative coccobacillus, Actinobacillus actinomycetemcomitans, is the putative agent for localized juvenile periodontitis, a particularly destructive form of periodontal disease in adolescents. This bacterium has also been isolated from a variety of other infections, notably endocarditis. Fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms, a property likely to be critical for colonization of teeth and other surfaces. Here we report the identification of a locus of seven genes required for nonspecific adherence of A. actinomycetemcomitans to surfaces. The recently developed transposon IS903φkan was used to isolate mutants of the rough clinical isolate CU1000 that are defective in tight adherence to surfaces (Tad−). Unlike wild-type cells, Tad− mutant cells adhere poorly to surfaces, fail to form large autoaggregates, and lack long, bundled fibrils. Nucleotide sequencing and genetic complementation analysis revealed a 6.7-kb region of the genome with seven adjacent genes (tadABCDEFG) required for tight adherence. The predicted TadA polypeptide is similar to VirB11, an ATPase involved in macromolecular transport. The predicted amino acid sequences of the other Tad polypeptides indicate membrane localization but no obvious functions. We suggest that the tad genes are involved in secretion of factors required for tight adherence of A. actinomycetemcomitans. Remarkably, complete and highly conserved tad gene clusters are present in the genomes of the bubonic plague bacillus Yersinia pestis and the human and animal pathogen Pasteurella multocida. Partial tad loci also occur in strikingly diverse Bacteria and Archaea. Our results show that the tad genes are required for tight adherence of A. actinomycetemcomitans to surfaces and are therefore likely to be essential for colonization and pathogenesis. The occurrence of similar genes in a wide array of microorganisms indicates that they have important functions. We propose that tad

  19. Iron-Chelating Activity of Tetracyclines and Its Impact on the Susceptibility of Actinobacillus actinomycetemcomitans to These Antibiotics

    PubMed Central

    Grenier, Daniel; Huot, Marie-Pierre; Mayrand, Denis

    2000-01-01

    Three tetracyclines (tetracycline, doxycycline, and minocycline) were found to possess iron-chelating activity in a colorimetric siderophore assay. Determination of MICs indicated that the activity of doxycycline against the periodontopathogen Actinobacillus actinomycetemcomitans was only slightly influenced by the presence of an excess of iron that likely saturates the antibiotic. On the other hand, the MICs of doxycycline and minocycline were significantly lower for A. actinomycetemcomitans cultivated under iron-poor conditions than under iron-rich conditions. PMID:10681353

  20. Cellular fatty acid and soluble protein composition of Actinobacillus actinomycetemcomitans and related organisms.

    PubMed Central

    Calhoon, D A; Mayberry, W R; Slots, J

    1981-01-01

    The cellular fatty acid and protein content of twenty-five representative strains of Actinobacillus actinomycetecomitans isolated from juvenile and adult periodontitis patients was compared to that of 15 reference strains of oral and nonoral Actinobacillus species and Haemophilus aphrophilus. Trimethylsilyl derivatives of the fatty acid methyl esters were analyzed by gas-liquid chromatography. The predominant fatty acids of all 40 strains examined were 14:0, 3-OH 14:0, 16 delta, and 16:0. Actinobacillus seminis (ATCC 15768) was unlike the other strains examined because of a greater amount of 14:0 detected. The soluble protein analysis using polyacrylamide gel electrophoresis revealed that A. actinomycetemcomitans, H. aphrophilus, and nonoral Actinobacillus species possessed distinct protein profiles attesting to the validity of separating these organisms into different species. Established biotypes of A. actinomycetemcomitans could not be differentiated on the basis of fatty acid or protein profiles. PMID:7287893

  1. Microbial ecology of Actinobacillus actinomycetemcomitans, Eikenella corrodens and Capnocytophaga spp. in adult periodontitis.

    PubMed

    Müller, H P; Heinecke, A; Borneff, M; Knopf, A; Kiencke, C; Pohl, S

    1997-08-01

    Information on intraoral distribution of putative periodontal pathogens might be essential for controlling different forms of periodontal disease. Colonization may be either promoted or impeded by other bacteria competing in the subgingival ecosystem. In recent investigations microbial associations between dental organisms have been determined in a multitude of subgingival plaque samples within multiple patients and described by odds ratios, in most circumstances without taking into account the correlated structure of the observations within a single individual. The present investigation had 3 major objectives: (i) to describe the intraoral distribution of some facultatively anaerobic, Gram-negative rods, i.e. Actinobacillus actinomycetemcomitans, Eikenella corrodens-like organisms and Capnocytophaga spp., in a multitude of subgingival and extracrevicular samples of 10 adult subjects with A. actinomycetemcomitans-associated periodontitis; (ii) to analyse possible inconsistencies of microbial associations between these periodontal organisms; and (iii) to determine factors increasing the likelihood of isolating these bacteria in a given subgingival site by employing Generalized Estimation Equation (GEE) methods. Clinical examinations were carried out at 6 sites of every tooth present. In each subject, 13 extracrevicular (2 cheek mucosa, 3 tongue, 4 gingival, 2 tonsillar samples, 1 palatinal, 1 saliva sample) and between 22 and 44 subgingival samples from deepest sites of every tooth present (n = 296) were selectively cultivated for A. actinomycetemcomitans, E. corrodens and Capnocytophaga spp. In extracrevicular material, A. actinomycetemcomitans, Capnocytophaga spp. and E. corrodens were isolated in 9, 10 and 6 patients, and from 65, 82 and 15% samples, respectively. The organisms were recovered from 51, 62 and 27% subgingival plaque samples, respectively. Heterogeneity tests did not reveal significant inconsistencies of microbial associations between bacteria in

  2. In vitro activity of antibiotics alone and in combination against Actinobacillus actinomycetemcomitans.

    PubMed

    Yogev, R; Shulman, D; Shulman, S T; Glogowski, W G

    1986-01-01

    The MICs for 90% of the organisms tested (MIC90S) of 11 antibiotics against 24 clinical isolates of Actinobacillus actinomycetemcomitans were determined by the MIC 2000 system. The lowest MIC90S (16 micrograms/ml) were observed with ceftriaxone and rifampin. The next lowest MIC90S were found with cephapirin, tetracycline, and chloramphenicol (3.12 micrograms/ml). The MIC90S of penicillin, ampicillin, ticarcillin, piperacillin, and amikacin were each greater than or equal to 12.5 micrograms/ml. Antibiotic synergy was studied by the killing curve method and was defined as a greater than or equal to 2 log10 reduction in CFU when two antibiotics were used in combination at one-fourth the MBC for each compared with the effect of each antibiotic alone at one-half the MBC. Synergism between rifampin and penicillin, cephapirin, or ceftriaxone was tested for with 12 A. actinomycetemcomitans strains. In 7 of 37 instances, synergism was demonstrated for the combinations rifampin plus ceftriaxone (n = 3) or rifampin plus penicillin (n = 4); in 9 instances, an additive effect was noted, and impaired killing with drug combinations compared with the effect of a single antibiotic was suggested in 4 strains. The majority of strains were indifferent to the combinations. Similarly, variable results were observed when the combination of trimethoprim and cephapirin was tested against eight A. actinomycetemcomitans strains. Our data suggest that rifampin and cephapirin are the most active of the 11 antibiotics studied against A. actinomycetemcomitans. In addition, in vitro synergism between rifampin and other antibiotics or between trimethoprim and cephapirin was not consistently demonstrable.

  3. Molecular Characterization of an Outer Membrane Protein of Actinobacillus actinomycetemcomitans Belonging to the OmpA Family

    PubMed Central

    White, Peter A.; Nair, Sean P.; Kim, Mi-Jurng; Wilson, Michael; Henderson, Brian

    1998-01-01

    The major outer membrane protein (OMP) of Actinobacillus actinomycetemcomitans is an OmpA homolog that demonstrates electrophoretic heat modifiability. The gene encoding this protein was isolated from a genomic library of A. actinomycetemcomitans NCTC 9710 by immunoscreening with serum from a patient with localized juvenile periodontitis. Expression of the cloned gene in Escherichia coli and subsequent Western blot analysis revealed a protein with an approximate molecular mass of 34 kDa. The amino acid sequence predicted from the cloned gene demonstrated that the mature protein had a molecular mass of 34,911 Da and significant identity to members of the OmpA family of proteins. We have named the major OMP of A. actinomycetemcomitans Omp34, and its corresponding gene has been named omp34. PMID:9423883

  4. Acylation Enhances, but Is Not Required for, the Cytotoxic Activity of Mannheimia haemolytica Leukotoxin in Bighorn Sheep

    PubMed Central

    Batra, Sai A.; Shanthalingam, Sudarvili; Munske, Gerhard R.; Raghavan, Bindu; Kugadas, Abirami; Bavanthasivam, Jegarubee; Highlander, Sarah K.

    2015-01-01

    Mannheimia haemolytica causes pneumonia in domestic and wild ruminants. Leukotoxin (Lkt) is the most important virulence factor of the bacterium. It is encoded within the four-gene lktCABD operon: lktA encodes the structural protoxin, and lktC encodes a trans-acylase that adds fatty acid chains to internal lysine residues in the protoxin, which is then secreted from the cell by a type 1 secretion system apparatus encoded by lktB and lktD. It has been reported that LktC-mediated acylation is necessary for the biological effects of the toxin. However, an LktC mutant that we developed previously was only partially attenuated in its virulence for cattle. The objective of this study was to elucidate the role of LktC-mediated acylation in Lkt-induced cytotoxicity. We performed this study in bighorn sheep (Ovis canadensis) (BHS), since they are highly susceptible to M. haemolytica infection. The LktC mutant caused fatal pneumonia in 40% of inoculated BHS. On necropsy, a large number of necrotic polymorphonuclear leukocytes (PMNs) were observed in the lungs. Lkt from the mutant was cytotoxic to BHS PMNs in an in vitro cytotoxicity assay. Flow cytometric analysis of mutant Lkt-treated PMNs revealed the induction of necrosis. Scanning electron microscopic analysis revealed the presence of pores and blebs on mutant-Lkt-treated PMNs. Mass spectrometric analysis confirmed that the mutant secreted an unacylated Lkt. Taken together, these results suggest that acylation is not necessary for the cytotoxic activity of M. haemolytica Lkt but that it enhances the potency of the toxin. PMID:26216418

  5. Unconventional N-Linked Glycosylation Promotes Trimeric Autotransporter Function in Kingella kingae and Aggregatibacter aphrophilus

    PubMed Central

    Rempe, Katherine A.; Spruce, Lynn A.; Porsch, Eric A.; Seeholzer, Steven H.; Nørskov-Lauritsen, Niels

    2015-01-01

    ABSTRACT Glycosylation is a widespread mechanism employed by both eukaryotes and bacteria to increase the functional diversity of their proteomes. The nontypeable Haemophilus influenzae glycosyltransferase HMW1C mediates unconventional N-linked glycosylation of the adhesive protein HMW1, which is encoded in a two-partner secretion system gene cluster that also encodes HMW1C. In this system, HMW1 is modified in the cytoplasm by sequential transfer of hexose residues. In the present study, we examined Kingella kingae and Aggregatibacter aphrophilus homologues of HMW1C that are not encoded near a gene encoding an obvious acceptor protein. We found both homologues to be functional glycosyltransferases and identified their substrates as the K. kingae Knh and the A. aphrophilus EmaA trimeric autotransporter proteins. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed multiple sites of N-linked glycosylation on Knh and EmaA. Without glycosylation, Knh and EmaA failed to facilitate wild-type levels of bacterial autoaggregation or adherence to human epithelial cells, establishing that glycosylation is essential for proper protein function. PMID:26307167

  6. Purification and characterization of the serotype c antigen from Actinobacillus actinomycetemcomitans.

    PubMed Central

    Zambon, J J; Slots, J; Miyasaki, K; Linzer, R; Cohen, R; Levine, M; Genco, R J

    1984-01-01

    The serotype c antigen from Actinobacillus actinomycetemcomitans was purified with fractional ethanol precipitation of cell-free culture supernatant, sequential ion-exchange chromatography, and gel filtration chromatography. The preparation obtained demonstrated a single precipitin line in immunodiffusion, immunoelectrophoresis, and crossed immunoelectrophoresis when rabbit antisera to serotype c whole bacterial cells were used. No immunological reaction was detected with antisera to serotype c lipopolysaccharide, indicating that lipopolysaccharide was not present in the preparation. The serotype c antigen was composed of 95% carbohydrate, 2% protein, and 3.1% phosphate. Gas chromatographic analysis of the antigen obtained from growth in either complex or chemically defined media revealed that the carbohydrate constituent was composed of 84 to 90.1% mannose, 4.8 to 16% glucose, 1.9% N-acetylglucosamine, 1.4% fucose, and 0.2% galactose. The present data suggest that A. actinomycetemcomitans serotype c antigen is predominantly a mannose-containing carbohydrate suggestive of a mannan. Images PMID:6423542

  7. Crystallization and preliminary X-ray crystallographic analysis of MacA from Actinobacillus actinomycetemcomitans

    SciTech Connect

    Piao, Shunfu; Xu, Yongbin; Ha, Nam-Chul

    2008-05-01

    A periplasmic membrane-fusion protein MacA from Actinobacillus actinomycetemcomitans, an essential component of the multidrug efflux pump in Gram-negative bacteria, was crystallized. Periplasmic membrane-fusion proteins (MFPs) are an essential component of the multidrug efflux pump in Gram-negative bacteria. They play a crucial role in bridging the outer membrane porin TolC and two distinct types of inner membrane transporters. The MFP MacA bridges the inner membrane ABC-type multidrug transporter MacB and the outer membrane porin TolC. MacA from the pathogenic bacterium Actinobacillus actinomycetemcomitans was expressed in Escherichia coli B834 (DE3) and the recombinant protein was purified using Ni–NTA affinity, Q anion-exchange and gel-filtration chromatography. The purified MacA protein was crystallized using the vapour-diffusion method. A MAD diffraction data set was collected to a resolution of 3.0 Å at 100 K. The crystal belongs to space group P622, with unit-cell parameters a = b = 109.2, c = 255.4 Å, α = β = 90, γ = 120°, and contains one molecule in the asymmetric unit.

  8. Antibodies to S. aureus LukS-PV Attenuated Subunit Vaccine Neutralize a Broad Spectrum of Canonical and Non-Canonical Bicomponent Leukotoxin Pairs

    PubMed Central

    Adhikari, Rajan P.; Kort, Thomas; Shulenin, Sergey; Kanipakala, Tulasikumari; Ganjbaksh, Nader; Roghmann, Mary-Claire; Holtsberg, Frederick W.; Aman, M. Javad

    2015-01-01

    S. aureus vaccine development has proven particularly difficult. The conventional approach to achieve sterile immunity through opsonophagocytic killing has been largely unsuccessful. S. aureus is highly toxigenic and a great body of evidence suggests that a successful future vaccine for this organism should target extracellular toxins which are responsible for host tissue destruction and immunosuppression. Major staphylococcal toxins are alpha toxin (a single subunit hemolysin) along with a group of bicomponent pore-forming toxins (BCPFT), namely Panton-Valentine leukocidin (PVL), gamma hemolysins (HlgCB and AB), LukAB and LukED. In our previous report, an attenuated mutant of LukS-PV (PVL- S subunit) named as “LukS-mut9” elicited high immunogenic response as well as provided a significant protection in a mouse sepsis model. Recent discovery of PVL receptors shows that mice lack receptors for this toxin, thus the reported protection of mice with the PVL vaccine may relate to cross protective responses against other homologous toxins. This manuscript addresses this issue by demonstrating that polyclonal antibody generated by LukS-mut9 can neutralize other canonical and non-canonical leukotoxin pairs. In this report, we also demonstrated that several potent toxins can be created by non-canonical pairing of subunits. Out of 5 pairs of canonical and 8 pairs of non-canonical toxins tested, anti-LukS-mut9 polyclonal antibodies neutralized all except for LukAB. We also studied the potential hemolytic activities of canonical and noncanonical pairs among biocomponent toxins and discovered that a novel non-canonical pair consisting of HlgA and LukD is a highly toxic combination. This pair can lyse RBC from different species including human blood far better than alpha hemolysin. Moreover, to follow-up our last report, we explored the correlation between the levels of pre-existing antibodies to new sets of leukotoxins subunits and clinical outcomes in adult patients with S

  9. Actinobacillus actinomycetemcomitans in Brazilian insulin-dependent individuals with diabetes mellitus.

    PubMed

    Feitosa, A C; de Uzeda, M; Novaes, A B

    1992-01-01

    The prevalence of Actinobacillus actinomycetemcomitans (Aa) in subgingival plaque specimens from 26 insulin-dependent diabetes mellitus patients, 11-25 years of age, was determined between January 1987 and December 1989. One hundred and thirty subgingival plaque samples were collected with sterile periodontal curettes. The specimens were weighted, diluted, inoculated on trypticase-soy-serum-bacitracin-vancomycin agar medium (TSBV) and incubated under microacrophilic conditions. Aa was isolated from 2.3% of healthy periodontal areas in these patients, while the microorganism was found in 12.5% of the sites with gingivitis and in 2.6% of the periodontal pockets examined. Although biochemical tests used for the characterization of Aa strains showed homogeneous results, different biotypes were isolated from one or more periodontal sites in the same patient.

  10. Morphology and ultrastructure of oral strains of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

    PubMed Central

    Holt, S C; Tanner, A C; Socransky, S S

    1980-01-01

    Selected human oral and nonoral strains of the genera Actinobacillus and Haemophilus were examined by transmission and scanning electron microscopy. The strains examined were morphologically identical to recognized Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus. By transmission electron microscopy, the cells were typically gram negative in morphology, with several strains possessing some extracellular ruthenium red-staining polymeric material. Numerous vesicular structures, morphologically identical to lipopolysaccharide vesicles, were seen to originate from and be continuous with the surface of the outer membrane. Large numbers of these vesicles were also found in the external environment. Scanning electron microscopic observations revealed that both actinobacilli and haemophili possessed surface projections and an amorphous surface material which connected and covered adjacent cells. Images Fig. 6 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 7 PMID:7439996

  11. Characterization of the lipopolysaccharide from Actinobacillus actinomycetemcomitans Y4 and N27.

    PubMed Central

    Kiley, P; Holt, S C

    1980-01-01

    The lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans strains Y4 and N27 was isolated by the phenol-water procedure. Morphologically, the molecule consisted of ribbon and branched filaments which comprised 3% of the cellular dry weight. Chemical analysis of the isolated and purified LPSs of both strains showed them to consist of carbohydrate, lipid, 2-keto-3-deoxyoctonate, heptose, hexosamine, and phosphate. The major fatty acids of the lipid A moiety were saturated C14 and beta-OH C14 compounds. Rhamnose, fucose, galactose, glucose, heptose, glucosamine, and galactosamine comprised the monosaccharide portion of the LPS. Biological activity studies revealed both LPS molecules to be active in the Schwartzman reaction and in in vitro 45Ca bone resorption, as well as in macrophage activation and lethality and in platelet aggregation. Images Fig. 1 Fig. 2 Fig. 4 PMID:7228391

  12. Actinobacillus actinomycetemcomitans Y4 capsular-polysaccharide-like polysaccharide promotes osteoclast-like cell formation by interleukin-1 alpha production in mouse marrow cultures.

    PubMed Central

    Nishihara, T; Ueda, N; Amano, K; Ishihara, Y; Hayakawa, H; Kuroyanagi, T; Ohsaki, Y; Nagata, K; Noguchi, T

    1995-01-01

    The mechanism of osteoclast-like cell formation induced by periodontopathic bacterium Actinobacillus actinomycetemcomitans Y4 (serotype b) capsular-polysaccharide-like polysaccharide (capsular-like polysaccharide) was examined in a mouse bone marrow culture system. When mouse bone marrow cells were cultured with A. actinomycetemcomitans Y4 capsular-like polysaccharide for 9 days, many multinucleated cells were formed. The multinucleated cells showed several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP) and the ability to resorb the calcified dentine. In this study, we examined the effects of antisera to interleukins on the formation of osteoclast-like cells induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. Monospecific anti-mouse recombinant interleukin-1 alpha (rIL-1 alpha) serum completely inhibited the formation of osteoclast-like cells in the presence of A. actinomycetemcomitans Y4 capsular-like polysaccharide. However, anti-mouse rIL-1 beta and anti-mouse rIL-6 sera showed no effect on osteoclast-like cell formation. IL-1 receptor antagonist significantly inhibited the osteoclast-like cell formation mediated by A. actinomycetemcomitans Y4 capsular-like polysaccharide in mouse marrow cultures. The bioactive IL-1 was detected in the culture media of mouse bone marrow cells stimulated with A. actinomycetemcomitans Y4 capsular-like polysaccharide. These results indicate that IL-1 alpha is involved in the mechanism of the formation of osteoclast-like cells induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. We sought to determine whether osteoclast-like cell formation induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide could be modulated by the protein kinase inhibitors H8 and HA1004. The formation of osteoclast-like cells was suppressed by H8 and HA1004. These findings suggest that the signals by protein kinases may regulate osteoclast-like cell formation induced by A

  13. Genetic and Functional Analyses of the Actinobacillus actinomycetemcomitans AfeABCD Siderophore-Independent Iron Acquisition System

    PubMed Central

    Rhodes, Eric R.; Tomaras, Andrew P.; McGillivary, Glen; Connerly, Pamela L.; Actis, Luis A.

    2005-01-01

    The Actinobacillus actinomycetemcomitans afeABCD iron transport system, the expression of which is controlled by iron and Fur, was identified in three different isolates. The protein products of this locus are related to bacterial ABC transporters involved in metal transport. Transformation of the Escherichia coli 1017 iron acquisition mutant with a plasmid harboring afeABCD promoted cell growth under iron-chelated conditions. However, insertion disruption of each of the afeABCD coding regions abolished this growth-relieving effect. The replacement of the parental afeA allele with the derivative afeA::EZ::TN drastically reduced the ability of A. actinomycetemcomitans cells to grow under iron-chelated conditions. PMID:15908408

  14. Bacteriocin production by Actinobacillus actinomycetemcomitans isolated from the oral cavity of humans with periodontal disease, periodontally healthy subjects and marmosets.

    PubMed

    Lúcia, Lima Francisca; Farias, Flávio F; Eustáquio, Costa José; Auxiliadora, Maria; Carvalho, R; Alviano, Celuta S; Farias, Luiz M

    2002-01-01

    The ability of Actinobacillus actinomycetemcomitans to produce bacteriocin has rarely been reported. Antagonistic substance production may confer an important ecological advantage for the producer microorganisms, especially in a competitive ecosystem such as the oral cavity. In the present study, 75 A. actinomycetemcomitans strains isolated from the oral cavity of human patients with periodontal disease, periodontally healthy subjects and marmosets, as well as two reference strains (A. actinomycetemcomitans ATCC 29523 and FDC Y4) were evaluated for auto-, iso-, and heteroantagonistic activity. Fifty-one (68.00%) strains exhibited antagonistic activity; heteroantagonism was observed more often than isoantagonism. Isolated strains antagonized 17 different species of gram-positive and gram-negative bacteria from the oral and nonoral microbiota. Sensitivity to heat and to proteolytic enzymes constituted strong evidence that the antagonistic substance has a proteic nature. Taken together, our data enabled us to confirm that the antagonistic substance detected was a bacteriocin. The wide spectrum of activity indicates the possibility that more than one antagonistic substance is produced and that these substances play an important role in the ecological balance of the oral ecosystem.

  15. 16S rDNA PCR-denaturing gradient gel electrophoresis in determining proportions of coexisting Actinobacillus actinomycetemcomitans strains.

    PubMed

    Ihalin, Riikka; Asikainen, Sirkka

    2006-06-01

    Certain serotypes of Actinobacillus actinomycetemcomitans seem to prefer coexistence in vivo. The 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) was tested for its capability to distinguish coexisting A. actinomycetemcomitans strains of different serotypes or genetic lineages and to determine their proportions in vitro. The migration pattern of the PCR amplicon from serotype c differed from those of the other serotypes. Contrary to the strains of serotypes c, d, and e, strains of serotypes a, b, and f consistently demonstrated intra-serotype migration patterns similar to each other. Since the migration patterns differed between serotype c and b strains a strain of each was used to determine their proportional representation in a strain mixture. The strains were distinguishable from each other above the 5% PCR-DGGE detection level (12.5 ng DNA/1.5 x 10(6) cells). DGGE provides a promising tool for in vitro studies on the coexistence of different genetic lineages of A. actinomycetemcomitans.

  16. Evidence that the serotype b antigenic determinant of Actinobacillus actinomycetemcomitans Y4 resides in the polysaccharide moiety of lipopolysaccharide.

    PubMed

    Wilson, M E; Schifferle, R E

    1991-04-01

    A high-molecular-weight polysaccharide-containing antigen was isolated from a phenol-water extract of Actinobacillus actinomycetemcomitans ATCC 43718 (formerly Y4) by gel permeation chromatography in lipopolysaccharide (LPS)-disaggregating buffer. The polysaccharide antigen formed a precipitin band with rabbit serotype b-specific antiserum but not with rabbit antisera to serotype a or c. Electroblotted serotype b antigen was probed with serum from a patient with localized juvenile periodontitis (LJP), resulting in a diffuse "smear" in the upper region of the lane. By utilizing an enzyme-linked immunosorbent assay, it was demonstrated that the geometric mean immunoglobulin G antibody titer to the serotype b polysaccharide was significantly higher in sera from LJP patients than in sera from periodontally healthy individuals. Moreover, LJP antibody titers to the serotype b polysaccharide exhibited age-dependent variation. Double immunodiffusion analysis revealed that the serotype b antigen formed a line of identity with low-molecular-weight LPS following reaction with serotype b-specific antiserum. Incubation of LJP serum in the presence of a lipid-free polysaccharide moiety obtained by mild acid hydrolysis of LPS from A. actinomycetemcomitans Y4 markedly reduced immunoglobulin G titer to the serotype b antigen. In contrast, solubilized lipid A was only weakly inhibitory. The results of this study indicate that the serotype b-specific determinant of A. actinomycetemcomitans resides in the polysaccharide moiety of LPS and represents a major target for immunoglobulin G antibody in serum of LJP subjects colonized by this organism.

  17. Immunodominant antigen of Actinobacillus actinomycetemcomitans Y4 in high-responder patients.

    PubMed Central

    Califano, J V; Schenkein, H A; Tew, J G

    1989-01-01

    This study was undertaken to look for characteristics of the immunodominant antigen(s) of Actinobacillus actinomycetemcomitans Y4 that might help explain the high antibody titers in periodontitis patients. Radioimmunoassays (RIA) were performed on sera from 481 patients; sera from the 32 patients with the highest anti-Y4 titers (above 128,000 RIA U/ml) were further analyzed. Y4 antigen was boiled for 45 min or treated with papain, and antibody responses were analyzed by RIA and Western blotting (immunoblotting). In addition, carbohydrate was purified from Y4 and examined by Western blotting. The results indicated that the immunodominant antigen of Y4 in high responders was stable after papain treatment or boiling for 45 min. Papain or boiling eliminated protein bands but a large diffuse band persisted on Western blots. With increasing dilutions of sera, bands on Western blots corresponding to protein antigens disappeared, while the large diffuse band resembling that of carbohydrate persisted. Partially purified Y4 carbohydrate contained the large diffuse band. Double-immunodiffusion analysis indicated that rabbit serotype b-specific antiserum and patient sera recognized the same antigen. When the carbohydrate extract was passed over a lipid A-binding column to remove lipopolysaccharide, the smear corresponding to the immunodominant antigen was still present on Western blots. The immunodominant antigen of Y4 in high-responder individuals appears to be a carbohydrate and is possibly the capsular polysaccharide. Images PMID:2496034

  18. Augmentation of Actinobacillus actinomycetemcomitans Invasion of Human Oral Epithelial Cells and Up-Regulation of Interleukin-8 Production by Saliva CD14

    PubMed Central

    Takayama, Atsuko; Satoh, Aya; Ngai, Tomoko; Nishimura, Takashi; Ikawa, Keiji; Matsuyama, Takami; Shimauchi, Hidetoshi; Takada, Haruhiko; Sugawara, Shunji

    2003-01-01

    It has recently been shown that human salivary glands constitutively express CD14, an important molecule in innate immunity, and that a soluble form of CD14 is secreted in saliva. The concentration of CD14 in parotid (a serous gland) saliva was comparable to that in normal serum and 10-fold the amount in whole saliva, although the physiological function of saliva CD14 remained unclear. Actinobacillus actinomycetemcomitans is a periodontopathic bacterium and is able to invade oral epithelial cells. The present study showed that upon exposure to live A. actinomycetemcomitans Y4 for 2 h, human oral epithelial HSC-2 cells produced interleukin-8 (IL-8) for a further 24 h and whole saliva augmented the production induced by A. actinomycetemcomitans Y4. Parotid saliva showed a more pronounced effect on the production of IL-8 than whole saliva. Neither saliva preparation itself had IL-8-inducing activity. Parotid saliva exhibited antibacterial activity against a low concentration of A. actinomycetemcomitans Y4, but recombinant CD14 did not show the activity. The internalization of A. actinomycetemcomitans Y4 into HSC-2 cells was inhibited by cytochalasin B, indicating that the process was actin dependent, and depletion of CD14 from parotid saliva inhibited the invasion and, as a consequence, inhibited production of IL-8. Furthermore, human recombinant CD14 augmented invasion and IL-8 production. These results suggest that saliva CD14 promoted the invasion of oral epithelial cells by A. actinomycetemcomitans and consequently augmented the production of IL-8, playing an important role in innate immunity in the oral cavity. PMID:14500479

  19. Taming C-terminal peptides of Staphylococcus aureus leukotoxin M for B-cell response: Implication in improved subclinical bovine mastitis diagnosis and protective efficacy in vitro.

    PubMed

    Padmaja, Radhakrishnan Jayasree; Halami, Prakash Motiram

    2016-09-01

    Leukotoxin M/F'-PV (LukM/F'-PV) produced by bovine mastitis causing Staphylococcus aureus structurally comprises three domains, the β-sandwich, rim and stem domain. The rim and stem domains interacting with target cell membrane lipid rafts contributes to the virulent trait of the toxin. In the present study, two facts were hypothesized that neutralization of these domains will ebb LukM/F'-PV leukotoxicity. Secondly, the neutralizing antibodies can improve the leukotoxin detection sensitivity in bovine mastitis milk samples. The in silico mapping of S. aureus LukM C-termini comprising these domains predicted seven linear B-cell antigenic epitopes. The immune response of C-terminal truncated recombinant peptides rCtM19 (19 kDa; near carboxy-terminal) having four epitopes and rCtM15 (15 kDa; C-terminal) with three epitopes were evaluated for their diagnostic and neutralization potential. Anti-rCtM19 and anti-rCtM15 antibodies with enhanced immunogenicity had the most striking outcome in IgG-ELISA for detecting native determinants of leukotoxin. For the obtained ELISA values, ROC curve inferred a cut-off score of >0.102 OD405. The assay sensitivity in the range of 90-96% along with 100% specificity and AUC of 0.93-0.98 categorized subclinical and clinical from healthy bovine milk samples. As observed through in vitro neutralization and LDH assays, C-terminus specific antibodies (1:42 titer) deactivating leukotoxicity abolished LukM from interacting with lipid bilayer and LukF for forming pores on bovine neutrophil membrane. As a proof of concept, it was proved that peptide antibodies can be a more specific serodiagnostic and passive therapeutic molecules.

  20. The survival rate of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Bacteroides forsythus following 4 randomized treatment modalities.

    PubMed

    Shiloah, J; Patters, M R; Dean, J W; Bland, P; Toledo, G

    1997-08-01

    The overall goal of this clinical study was to determine the short-term anti-infective effects of four randomized treatment modalities on Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Bacteroides forsythus (Bf) and determine the effects of bacterial survival on treatment outcomes in patients with adult periodontitis. Twelve adult patients requiring therapy for moderate periodontitis were selected for this study. All patients had at least one tooth in each quadrant that had an inflamed pocket of probing depth > or =5 mm with probing attachment loss that harbored at least one of the following three periodontal pathogens: Aa, Pg, or Bf. The number of target organisms per site was determined pre-operatively, at 1 week, and 1 month and 3 months postoperatively utilizing DNA probes. One quadrant in each patient was randomly assigned to each one of the following four treatment groups: 1) scaling and root planing (SRP group); 2) pocket reduction through osseous surgery and apically-positioned flap (OS group); 3) modified Widman flap (MWF group); and 4) modified Widman flap and topical application of saturated citric acid at pH 1 for 3 minutes (CA group). The 4 treatment modalities were performed in one appointment. No postoperative antibiotics were used. Patients were instructed to supplement their daily oral hygiene with chlorohexidine oral rinse during the study. The results of this investigation indicated that: 1) none of the treatment modalities was effective in eliminating the target species; 2) the incidence of infected sites for all groups was 100% preoperatively; 62.5%, 33.3%, and 31.3% at 1 week, and 1 and 3 months postoperatively, respectively; 3) these infected sites lost 1.1 +/- 0.4 mm of probing attachment compared to gain of 0.0 +/- 0.3 mm for uninfected sites; 4) the infected sites had higher plaque and bleeding on probing 0.9 +/- 0.3, 73 +/- 12%, respectively, compared to 0.3 +/- 0.1 and 30 +/- 8% for the uninfected sites

  1. Site-specific subgingival colonization by Actinobacillus actinomycetemcomitans in orthodontic patients.

    PubMed

    Paolantonio, M; Festa, F; di Placido, G; D'Attilio, M; Catamo, G; Piccolomini, R

    1999-04-01

    A high prevalence of Actinobacillus actinomycetemcomitans (Aa) in subgingival plaque in patients for orthodontia already has been observed. The present study had the following aims: 1) to ascertain a direct relationship between the orthodontic appliance placement and the subgingival colonization by Aa, and 2) to determine whether the Aa growth specifically occurred on teeth with braces attached or whether the presence of orthodontic appliances could also cause the isolation of Aa in teeth free from therapeutic appliances. Twenty-four young systemically and periodontally healthy subjects with malaligned and crowded teeth in the anterior sextants of both dental arches participated in this study. After 1 session of ultrasonic scaling with oral hygiene instructions during the first experimental session, the mesiobuccal sites of the first molars and the distobuccal sites of the lateral incisors in both dental arches in each participant were subjected to clinical and microbiologic examination for the recovery of Aa. Clinical examination consisted of recording the presence of plaque and the examination of gingival bleeding on probing and probing depth. Microbiologic sampling was obtained with the insertion of 3 sterile paper points at the deepest part of each gingival sulcus. Altogether, 192 periodontal sites were examined. After the examinations, the patients received fixed orthodontic appliances in only 1 dental arch (test sites) and the other one was left free from appliances (control sites). Clinical examination and microbiologic sampling were repeated in the same experimental test and control sites after 4, 8, and 12 weeks. At the 12-week session, the orthodontic appliance was removed from the test arch, and, 4 weeks later, a further clinical and microbiologic examination was performed. The results showed that, during the period with orthodontic appliances, the presence of plaque scores and the gingival bleeding on probing scores were increased significantly and that

  2. Leukotoxin family genes in Staphylococcus aureus isolated from domestic animals and prevalence of lukM-lukF-PV genes by bacteriophages in bovine isolates.

    PubMed

    Yamada, Tomoko; Tochimaru, Naoko; Nakasuji, Sachiko; Hata, Eiji; Kobayashi, Hideki; Eguchi, Masashi; Kaneko, Jun; Kamio, Yoshiyuki; Kaidoh, Toshio; Takeuchi, Shotaro

    2005-09-30

    Leukotoxin family genes in Staphylococcus aureus isolated from domestic animals were examined by polymerase chain reaction. LukS and lukF genes were detected in all 48 avian and 72 porcine isolates of S. aureus. LukE and lukD genes, located in a putative staphylococcal pathogenicity island (Sapln3/Saplm3), were recognized in 44 (91.7%) of 48 avian isolates, but these genes were not detected in porcine isolates. In 297 bovine isolates collected from mastitic cow's milk and bulk milk from dairy farms in two regions, lukM and lukF-PV(P83) genes in addition to lukS-lukF and lukE-lukD genes were detected in 100 (62.5%) of the 160 isolates from Ishikawa and in118 (86.1%) of the 137 isolates from Hokkaido. When the lysogeny of S. aureus bovine isolates was examined by treatment with mitomycin C, clearing of the culture due to cell lysis was observed in 34 (91.9%) of 37 lukM-lukF-PV(P83) genes--positive isolates. In addition, we isolated a novel lukM-lukF-PV(P83)-carrying (designated phiLukM), and revealed that the lukM-lukF-PV(P83) genes were located very close to an amidase gene on the temperate phage genomes. These results suggest horizontal transmission of lukM-lukF-PV(P83) genes by temperate bacteriophages in S. aureus of bovine origin.

  3. Immunoglobulin allotypes and immunoglobulin G subclass responses to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in early-onset periodontitis.

    PubMed Central

    Choi, J I; Ha, M H; Kim, J H; Kim, S J

    1996-01-01

    The present study was performed to estimate the observed frequencies of the immunoglobulin heavy-chain (Gm) and light-chain (Km) allotypes among patients with early-onset periodontitis (EOP) and their effect on the IgG2 subclass responses against Actinobacillus actinomycetemcomitans Y4 and Porphyromonas gingivalis 381, respectively. Sixty-nine EOP patients, including 11 with localized juvenile periodontitis (LJP), 19 who had LJP, 15 with LJP-rapidly progressing periodontitis (RPP), and 24 with RPP, were examined for the Gm and Km allotypes by a hemagglutination inhibition test. Levels of immunoglobulin G2 (IgG2) antibodies against the two organisms were determined by enzyme-linked immunosorbent assay. Fifty race- and age-matched, periodontally healthy subjects were also included as a control group. The observed frequencies of the Gm haplotype afnb and Km(1) were significantly higher in the RPP and LJP groups, respectively. The G2m(n)+ group of those with RPP and the Km(1)+ group of those with LJP had significantly higher levels of IgG2 antibodies to A. actinomycetemcomitans and P. gingivalis, respectively. The results indicate that linkage disequilibrium of the G2m(n) locus in RPP patients or the Km(1) locus in LJP patients may be associated with high IgG2 antibody responses to the respective bacteria. It was reasoned that the IgG2 antibody responses are associated with the immunoglobulin allotypes. The function of IgG2 antibodies in their reaction to different bacterial antigens may be interpreted as either protective or nonprotective in the two different types of EOP (i.e., LJP and RPP). PMID:8926092

  4. Analytical performance of an immunologic-based periodontal bacterial test for simultaneous detection and differentiation of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia.

    PubMed

    Snyder, B; Ryerson, C C; Corona, H; Grogan, E A; Reynolds, H S; Contestable, P B; Boyer, B P; Mayer, J; Mangan, T; Norkus, N; Zambon, J J; Genco, R J

    1996-05-01

    The analytical performance of a membrane-based immunoassay for the simultaneous detection and differentiation of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia (including Prevotella nigrescens) was investigated. Positive reactions were observed for 71 of 71 reference strains and recent oral isolates of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. No cross-reactivity was observed with 39 other common oral and environmental species. The specificity of the test was unaffected by the presence of potential oral interferents including whole blood, white blood cells, mucin, saliva, toothpastes, and oral rinses. A proficiency test by dental professionals using a standardized set of unknown simulated samples yielded a sensitivity of 97% (116/120) and a 100% specificity (240/ 240). An additional group including dental professionals and high school students was shown to be 99% proficient (1385/1397) in distinguishing proper from improper test function when processing control samples with normal test devices and devices with simulated error conditions. Comparisons to a culture standard for 104 subgingival plaque samples collected from 26 adult periodontitis patients yielded > 98% specificity for each of the test bacteria. In addition, the detection threshold for the test was determined to be equivalent to 10(4) cultivable test bacteria when compared to the culture standard. The data indicate that this membrane immunoassay is a valid and easy-to-use method for the detection of A. actinomycetemcomitans, P. gingivalis, and P. intermedia in subgingival plaque, at levels above the detection threshold of the test.

  5. Actinobacillus actinomycetemcomitans serotype b-specific polysaccharide antigen stimulates production of chemotactic factors and inflammatory cytokines by human monocytes.

    PubMed Central

    Yamaguchi, N; Yamashita, Y; Ikeda, D; Koga, T

    1996-01-01

    Serotype b-specific polysaccharide antigen (SPA) was extracted from whole cells of Actinobacillus actinomycetemcomitans Y4 by autoclaving and purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300. SPA induced the release of monocyte and leukocyte chemotactic factors by human monocytes. Polymyxin B had almost no effect on the release of monocyte chemotactic factor, but a monoclonal antibody against SPA markedly inhibited it. Human monocytes stimulated with SPA exhibited the increased mRNA expression of monocyte chemoattractant protein 1 (MCP-1) and a neutrophil chemotactic factor, interleukin-8 (IL-8). On the other hand, SPA induced the release of IL-1, IL-6, and tumor necrosis factor (TNF) and enhanced the expression of IL-1alpha, IL-1beta, IL-6, and TNF alpha (TNF-alpha) mRNAs. Human monocytes expressed MCP-1 and IL-8 mRNAs when stimulated by human recombinant IL-1alpha, I1-1beta, IL-6, and TNF-alpha, suggesting that these inflammatory cytokines induced by SPA might participate in the production of chemotactic factors in human monocytes. PMID:8698480

  6. Characterization of an antiproliferative surface-associated protein from Actinobacillus actinomycetemcomitans which can be neutralized by sera from a proportion of patients with localized juvenile periodontitis.

    PubMed Central

    White, P A; Wilson, M; Nair, S P; Kirby, A C; Reddi, K; Henderson, B

    1995-01-01

    The gentle agitation of suspensions of Actinobacillus actinomycetemcomitans serotype a, b, or c in saline resulted in the release of a proteinaceous surface-associated material (SAM) which produced a dose-dependent inhibition of tritiated thymidine incorporation by the osteoblast-like cell line MG63 in culture. This cell line was sensitive to low concentrations of SAM (50% inhibitory concentration, 200 ng/ml for serotype c). Immunoglobulin G antibodies to constituents of the SAM were found in the blood of patients with localized juvenile periodontitis (LJP). Sera from 9 of 16 patients with LJP significantly neutralized the antiproliferative activity of the SAM, while sera from 15 controls, with no evidence of periodontal disease, were unable to neutralize this activity. Neutralization was not directly related to the patient's antibody titer to the whole SAM. Characterization of the antiproliferative activity in the SAM demonstrated that it was not cytotoxic and was heat and trypsin sensitive. The active component separated in a well-defined peak in anion-exchange high-performance liquid chromatography (HPLC) which, when further analyzed by size exclusion HPLC, revealed a single active peak, which had an apparent molecular mass of approximately 8 kDa. The lipopolysaccharide from A. actinomycetemcomitans was only weakly active. SAM from Porphyromonas gingivalis W50 and Eikenella corrodens NCTC 10596 did not exhibit any antiproliferative activity with this cell line, even at concentrations as high as 10 micrograms/ml. This study has shown that SAM from A. actinomycetemcomitans contains a potent antiproliferative protein whose activity can be neutralized by antibodies in the sera from some patients with LJP. PMID:7790076

  7. Cell cycle-specific growth inhibitory effect on human gingival fibroblasts of a toxin isolated from the culture medium of Actinobacillus actinomycetemcomitans.

    PubMed

    Helgeland, K; Nordby, O

    1993-05-01

    A toxin isolated from the growth medium of Actinobacillus actinomycetemcomitans by ammonium sulfate precipitation was shown to inhibit irreversibly the multiplication of human gingival fibroblasts. DNA histograms from flow cytometric measurements showed that the cells accumulated preferentially in the G2 phase of the cell cycle. Such cells exhibited sheetlike protrusions, and an increased frequency of micronuclei was evident in cells treated with low concentrations of the toxin. Toxin-treated cells were viable for several weeks, as shown by staining with trypan blue and fluorescein diacetate, and the general cell metabolism as measured by oxygen consumption was unimpaired. PMID:8496779

  8. Buccal microbiology analyzed by infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    de Abreu, Geraldo Magno Alves; da Silva, Gislene Rodrigues; Khouri, Sônia; Favero, Priscila Pereira; Raniero, Leandro; Martin, Airton Abrahão

    2012-01-01

    Rapid microbiological identification and characterization are very important in dentistry and medicine. In addition to dental diseases, pathogens are directly linked to cases of endocarditis, premature delivery, low birth weight, and loss of organ transplants. Fourier Transform Infrared Spectroscopy (FTIR) was used to analyze oral pathogens Aggregatibacter actinomycetemcomitans ATCC 29523, Aggregatibacter actinomycetemcomitans-JP2, and Aggregatibacter actinomycetemcomitans which was clinically isolated from the human blood-CI. Significant spectra differences were found among each organism allowing the identification and characterization of each bacterial species. Vibrational modes in the regions of 3500-2800 cm-1, the 1484-1420 cm-1, and 1000-750 cm-1 were used in this differentiation. The identification and classification of each strain were performed by cluster analysis achieving 100% separation of strains. This study demonstrated that FTIR can be used to decrease the identification time, compared to the traditional methods, of fastidious buccal microorganisms associated with the etiology of the manifestation of periodontitis.

  9. Bacterial RTX toxins allow acute ATP release from human erythrocytes directly through the toxin pore.

    PubMed

    Skals, Marianne; Bjaelde, Randi G; Reinholdt, Jesper; Poulsen, Knud; Vad, Brian S; Otzen, Daniel E; Leipziger, Jens; Praetorius, Helle A

    2014-07-01

    ATP is as an extracellular signaling molecule able to amplify the cell lysis inflicted by certain bacterial toxins including the two RTX toxins α-hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans. Inhibition of P2X receptors completely blocks the RTX toxin-induced hemolysis over a larger concentration range. It is, however, at present not known how the ATP that provides the amplification is released from the attacked cells. Here we show that both HlyA and LtxA trigger acute release of ATP from human erythrocytes that preceded and were not caused by cell lysis. This early ATP release did not occur via previously described ATP-release pathways in the erythrocyte. Both HlyA and LtxA were capable of triggering ATP release in the presence of the pannexin 1 blockers carbenoxolone and probenecid, and the HlyA-induced ATP release was found to be similar in erythrocytes from pannexin 1 wild type and knock-out mice. Moreover, the voltage-dependent anion channel antagonist TRO19622 had no effect on ATP release by either of the toxins. Finally, we showed that both HlyA and LtxA were able to release ATP from ATP-loaded lipid (1-palmitoyl-2-oleoyl-phosphatidylcholine) vesicles devoid of any erythrocyte channels or transporters. Again we were able to show that this happened in a non-lytic fashion, using calcein-containing vesicles as controls. These data show that both toxins incorporate into lipid vesicles and allow ATP to be released. We suggest that both toxins cause acute ATP release by letting ATP pass the toxin pores in both human erythrocytes and artificial membranes.

  10. Antibacterial activity of the lactoperoxidase system combined with edible Laminaria hot-water extract as a source of halide ions.

    PubMed

    Shin, Kouichirou; Nakano, Manabu; Yamauchi, Koji; Toida, Tomohiro; Iwatsuki, Keiji

    2012-01-01

    Hot-water extracts prepared from nine out of 12 samples of dried edible Laminaria reduced the viable numbers of Aggregatibacter actinomycetemcomitans, Staphylococcus aureus, and Esherichia coli below the detection limit after incubation for 5 min when combined with lactoperoxidase, glucose oxidase, and glucose. Some extracts showed higher bactericidal activity and a higher OI(-) concentration in the assay mixture after ultrafiltration.

  11. Simultaneous measurement of the viability, aggregation, and live and dead adherence of Streptococcus crista, Streptococcus mutans and Actinobacillus actinomycetemcomitans in human saliva in relation to indices of caries, dental plaque and periodontal disease.

    PubMed

    Rudney, J D; Staikov, R K

    2002-05-01

    Salivary proteins have multiple functions and many share similar functions, which may be why it has been difficult to relate variations in their concentrations to oral health and ecology. An alternative is to focus on variations in the major functions of saliva. An hydroxyapatite-coated microplate model has been developed that simultaneously measures saliva-promoted bacterial viability, bacterial aggregation, and live and dead bacterial adherence, while simulating oral temperature and shearing forces from swallowing. That model was applied to resting whole and stimulated parotid saliva from 149 individuals, using representative strains of Streptococcus crista, S. mutans, and Actinobacillus actinomycetemcomitans. Two major factors were defined by multivariate analysis (this was successful only for whole-saliva). One factor was correlated with aggregation, live adherence and dead adherence for all three strains; the other was correlated with total viability of all three strains. Participants were grouped <25th percentile and >75th percentile for each factor. Those groups were compared for clinical indices of oral health. Caries scores were significantly lower in those with high scores for aggregation-adherence, regardless of whether total viability scores were low or high. Live bacteria always predominated on surfaces when live and dead adherence scores were expressed as ratios. However, participants with high scores for aggregation-adherence showed significantly more dead adherent bacteria than those with low scores (these ratios were uncorrelated with total viability). This finding may indicate that extreme differences in the ability to kill bacteria on surfaces can influence caries risk.

  12. OCCURRENCE OF ACTINOBACILLUS ACTINOMYCETEMCOMITANS IN PATIENTS WITH CHRONIC PERIODONTITIS, AGGRESSIVE PERIODONTITIS, HEALTHY SUBJECTS AND CHILDREN WITH GINGIVITIS IN TWO CITIES OF THE STATE OF SÃO PAULO, BRAZIL

    PubMed Central

    Jardim, Elerson Gaetti; Bosco, Joseane Maria Dias; Lopes, Angélica Marquezim; Landucci, Luís Fernando; Jardim, Ellen Cristina Gaetti; Carneiro, Sílvia Rosana Soares

    2006-01-01

    The aim of this study was to determine the frequency of isolation of Actinobacillus actinomycetemcomitans (Aa) in 100 patients with chronic periodontitis, 14 patients with aggressive periodontitis, 142 pre-school children with gingivitis and 134 periodontally healthy subjects. Samples of subgingival plaque were taken using sterilized paper points introduced into periodontal pockets or gingival crevice for 60 seconds and inoculated on TSBV agar, which was incubated under anaerobiosis at 37°C, for 4 days. Microbial identification was performed through biochemical methods and morphocellular and morphocolonial analysis. Aa was detected in 40.3% of healthy subjects, 68% of patients with chronic periodontitis, 92.86% of patients with aggressive periodontitis and 40.14% of children with gingivitis. The rate of recovery of Aa in the tested human groups proved to be higher than previously reported and in agreement with participation of this facultative anaerobe as a member of native microbiota of the periodontium and its relation with aggressive and chronic periodontitis in Brazil. PMID:19089064

  13. Quercetin inhibits inflammatory bone resorption in a mouse periodontitis model.

    PubMed

    Napimoga, Marcelo H; Clemente-Napimoga, Juliana T; Macedo, Cristina G; Freitas, Fabiana F; Stipp, Rafael N; Pinho-Ribeiro, Felipe A; Casagrande, Rubia; Verri, Waldiceu A

    2013-12-27

    Periodontitis is a disease that leads to bone destruction and represents the main cause of tooth loss in adults. The development of aggressive periodontitis has been associated with increased inflammatory response that is induced by the presence of a subgingival biofilm containing Aggregatibacter actinomycetemcomitans. The flavonoid quercetin (1) is widespread in vegetables and fruits and exhibits many biological properties for possible medical and clinical applications such as its anti-inflamatory and antioxidant effects. Thus, in the present study, the properties of 1 have been evaluated in bone loss and inflammation using a mouse periodontitis model induced by A. actinomycetemcomitans infection. Subcutaneous treatment with 1 reduced A. actinomycetemcomitans-induced bone loss and IL-1β, TNF-α, IL-17, RANKL, and ICAM-1 production in the gingival tissue without affecting bacterial counts. These results demonstrated that quercetin exhibits protective effects in A. actinomycetemcomitans-induced periodontitis in mice by modulating cytokine and ICAM-1 production.

  14. Enhancing the stability and antibiofilm activity of DspB by immobilization on carboxymethyl chitosan nanoparticles.

    PubMed

    Tan, Yulong; Ma, Su; Liu, Chenguang; Yu, Wengong; Han, Feng

    2015-09-01

    A β-N-acetyl-glucosaminidase (DspB) from Aggregatibacter actinomycetemcomitans CU1000 has been proved to inhibit and detach the biofilms formed by Staphylococcus epidermidis, Staphylococcus aureus and A. actinomycetemcomitans. However, the application of this enzyme is limited by its poor stability. In the present study, a β-N-acetyl-glucosaminidase encoding gene, dspB, was cloned from A. actinomycetemcomitans HK1651 and expressed in Escherichia coli. The recombinant DspB was loaded on hydrogel nanoparticles, which was prepared by using linoleic acid (LA) modified carboxymethyl chitosan (CMCS) after sonication. The nanoparticles were almost saturated by DspB at 0.3 mg/ml, which gave a loading capacity of 76.7%. The immobilization enhanced thermal stability, storage stability and reusability of DspB significantly. Moreover, it also increased antibiofilm activity due to the dual mechanism, including the improvement of the enzyme stability and the antibiofilm activity of CMCS nanoparticles. PMID:26302845

  15. [USE OF THE REAL-TIME PCR FOR STUDY OF THE PERIODONTAL MICROBIOME IN PATIENTS WITH COMBINED PATHOLOGY OF GASTRODUODENAL ZONE AND CHRONIC PERIODONTITIS].

    PubMed

    Shibaeva, A V; Ayvazova, R A; Rebrikov, D V; Trubnikova, E V; Kudykina, Yu K; Belyakova, A V; Zaripova, R S; Shevelev, A B

    2016-01-01

    The total of 54 patients with chronic periodontitis of different severity was tested using real-time PCR (Dentoflor kit). The group included 38 patients with chronic gastritis. For the first time, a higher prevalence of Treponema denticola in periodontium of males in comparison with females was demonstrated. The patients with chronic gastritis had more human genome DNA at their periodontium than healthy individuals. Non-parametric statistical analysis demonstrated high association of periodontium colonization with. T. forsythensis and T. denticola (but not Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia) with the severity of the chronic periodontitis. PMID:27183718

  16. Impact of CDT Toxin on Human Diseases

    PubMed Central

    Faïs, Tiphanie; Delmas, Julien; Serres, Arnaud; Bonnet, Richard; Dalmasso, Guillaume

    2016-01-01

    Cytolethal distending toxin (CDT) is found in Gram-negative bacteria, especially in certain Proteobacteria such as the Pasteurellaceae family, including Haemophilus ducreyi and Aggregatibacter (Actinobacillus) actinomycetemcomitans, in the Enterobacteriaceae family and the Campylobacterales order, including the Campylobacter and Helicobacter species. In vitro and in vivo studies have clearly shown that this toxin has a strong effect on cellular physiology (inflammation, immune response modulation, tissue damage). Some works even suggest a potential involvement of CDT in cancers. In this review, we will discuss these different aspects. PMID:27429000

  17. Inhibitory activity of Aloe vera gel on some clinically isolated cariogenic and periodontopathic bacteria.

    PubMed

    Fani, Mohammadmehdi; Kohanteb, Jamshid

    2012-03-01

    Aloe vera is a medicinal plant with anti-inflammatory, antimicrobial, antidiabetic and immune-boosting properties. In the present study we investigated the inhibitory activities of Aloe vera gel on some cariogenic (Streptococcus mutans), periodontopathic (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis) and an opportunistic periodontopathogen (Bacteroides fragilis) isolated from patients with dental caries and periodontal diseases. Twenty isolates of each of these bacteria were investigated for their sensitivity to Aloe vera gel using the disk diffusion and microdilution methods. S. mutans was the species most sensitive to Aloe vera gel with a MIC of 12.5 µg/ml, while A. actinomycetemcomitans, P. gingivalis, and B. fragilis were less sensitive, with a MIC of 25-50 µg/ml (P < 0.01). Based on our present findings it is concluded that Aloe vera gel at optimum concentration could be used as an antiseptic for prevention of dental caries and periodontal diseases.

  18. Occurrence of periodontal pathogens among patients with chronic periodontitis

    PubMed Central

    Farias, B.C.; Souza, P.R.E.; Ferreira, B.; Melo, R.S.A.; Machado, F.B.; Gusmão, E.S.; Cimões, R.

    2012-01-01

    The aim of the present study was to evaluate the presence of the periodontal pathogens that form the red complex (Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola) and Aggregatibacter actinomycetemcomitans in patients with chronic periodontitis. The sample consisted of 29 patients with a clinical and radiographic diagnosis of chronic periodontitis based on the criteria of the American Academy of Periodontology (3). Samples for microbiological analysis were collected from the four sites of greatest probing depth in each patient, totaling 116 samples. These samples were processed using conventional polymerase chain reaction, which achieved the following positive results: 46.6% for P. gingivalis, 41.4% for T. forsythia, 33.6% for T. denticola and 27.6% for A. actinomycetemcomitans. P. gingivalis and T. forsythia were more prevalent (p < 0.05) in periodontal pockets ≥ 8 mm. The combinations T. forsythia + P. gingivalis (23.2%) and T. forsythia + P. gingivalis + T. denticola (20.0%) were more frequent in sites with a probing depth ≥ 8 mm. Associations with the simultaneous presence of A. actinomycetemcomitans + P. gingivalis, A. actinomycetemcomitans + T. forsythia, P. gingivalis + T. forsythia and T. forsythia + T. denticola were statistically significant (p < 0.05). It was concluded that the red complex pathogens are related to chronic periodontitis, presenting a higher occurrence in deep periodontal pockets. Moreover, the simultaneous presence of these bacteria in deep sites suggests a symbiotic relationship between these virulent species, favoring, in this way, a further progression of periodontal disease. PMID:24031906

  19. Essential Oils from Ugandan Aromatic Medicinal Plants: Chemical Composition and Growth Inhibitory Effects on Oral Pathogens.

    PubMed

    Ocheng, Francis; Bwanga, Freddie; Joloba, Moses; Softrata, Abier; Azeem, Muhammad; Pütsep, Katrin; Borg-Karlson, Anna-Karin; Obua, Celestino; Gustafsson, Anders

    2015-01-01

    The study assessed the growth inhibitory effects of essential oils extracted from ten Ugandan medicinal plants (Bidens pilosa, Helichrysum odoratissimum, Vernonia amygdalina, Hoslundia opposita, Ocimum gratissimum, Cymbopogon citratus, Cymbopogon nardus, Teclea nobilis, Zanthoxylum chalybeum, and Lantana trifolia) used traditionally in the management of oral diseases against oral pathogens. Chemical compositions of the oils were explored by GC-MS. Inhibitory effects of the oils were assessed on periodontopathic Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans and cariogenic Streptococcus mutans and Lactobacillus acidophilus using broth dilution methods at concentrations of 1%, 0.1%, and 0.01%. The most sensitive organism was A. actinomycetemcomitans. Its growth was markedly inhibited by six of the oils at all the concentrations tested. Essential oil from C. nardus exhibited the highest activity with complete growth inhibition of A. actinomycetemcomitans and P. gingivalis at all the three concentrations tested, the major constituents in the oil being mainly oxygenated sesquiterpenes. Most of the oils exhibited limited effects on L. acidophilus. We conclude that essential oils from the studied plants show marked growth inhibitory effects on periodontopathic A. actinomycetemcomitans and P. gingivalis, moderate effects on cariogenic S. mutans, and the least effect on L. acidophilus. The present study constitutes a basis for further investigations and development of certain oils into alternative antiplaque agents. PMID:26170872

  20. A Commensal Bacterium Promotes Virulence of an Opportunistic Pathogen via Cross-Respiration

    PubMed Central

    Stacy, Apollo; Fleming, Derek; Lamont, Richard J.; Rumbaugh, Kendra P.

    2016-01-01

    ABSTRACT Bacteria rarely inhabit infection sites alone, instead residing in diverse, multispecies communities. Despite this fact, bacterial pathogenesis studies primarily focus on monoculture infections, overlooking how community interactions influence the course of disease. In this study, we used global mutant fitness profiling (transposon sequencing [Tn-seq]) to determine the genetic requirements for the pathogenic bacterium Aggregatibacter actinomycetemcomitans to cause disease when coinfecting with the commensal bacterium Streptococcus gordonii. Our results show that S. gordonii extensively alters A. actinomycetemcomitans requirements for virulence factors and biosynthetic pathways during infection. In addition, we discovered that the presence of S. gordonii enhances the bioavailability of oxygen during infection, allowing A. actinomycetemcomitans to shift from a primarily fermentative to a respiratory metabolism that enhances its growth yields and persistence. Mechanistically, respiratory metabolism enhances the fitness of A. actinomycetemcomitans in vivo by increasing ATP yields via central metabolism and creating a proton motive force. Our results reveal that, similar to cross-feeding, where one species provides another species with a nutrient, commensal bacteria can also provide electron acceptors that promote the respiratory growth and fitness of pathogens in vivo, an interaction that we term cross-respiration. PMID:27353758

  1. An in-vitro evaluation of the efficacy of garlic extract as an antimicrobial agent on periodontal pathogens: A microbiological study

    PubMed Central

    Shetty, Sunaina; Thomas, Biju; Shetty, Veena; Bhandary, Rahul; Shetty, Raghavendra M.

    2013-01-01

    With the rise in bacterial resistance to antibiotics, there is considerable interest in the development of other classes of antimicrobials for the control of infection. Garlic (Allium sativum Linn.) has been used as medicine since ancient times and has long been known to have antibacterial, antifungal, and antiviral properties. This study was undertaken to assess the inhibitory effect of garlic on Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, to assess the time-kill curve of P. gingivalis and A. actinomycetemcomitans, and to determine the antiproteolytic activity of garlic on P. gingivalis. Ethanolic garlic extract (EGE) and aqueous garlic extract (AGE) were prepared and the inhibitory effects of these extracts for two periodontal pathogens (P. gingivalis and A. actinomycetemcomitans) were tested. Antiproteolytic activity on protease of P. gingivalis was determined. 25 microliter (μl), 50 μl, and 75 μl of AGE showed 16 mm, 20 mm, and 25 mm zone of inhibition, respectively, on P. gingivalis. The AGE showed greater bacteriostatic activity against the P. gingivalis with minimum inhibitory concentration determined at 16.6 μl/ml. The time-kill assay of AGE and EGE were compared for P. gingivalis and A. actinomycetemcomitans. AGE showed better antiproteolytic activity on total protease of P. gingivalis compared to the EGE. Thus, the study concludes the antimicrobial activity of garlic extract against periodontal pathogens, P. gingivalis, A. actinomycetemcomitans. Its action against P. gingivalis includes inhibition of total protease activity, and this raises the possibility that garlic may have therapeutic use for periodontitis and possibly other oral infections. PMID:24695825

  2. [Study of mutual dependence of periodontal and colonic microbiome in health and pathology using NSG-sequencing].

    PubMed

    Petrukhina, N B; Zorina, O A; Shikh, E V; Shibaeva, A V; Shevelev, A B

    2016-01-01

    By using NGS-sequencing libraries of DNA from periodontal swabs with primers specific to V6 region of 16S rDNA prevalence of bacterial genera and species in periodontal and colonic microbiota of patients with periodontitis of different severity and healthy donors was analyzed. Hyper-colonization of the colon with Akkermansia muciniphila was found to be the most important maker of negative predisposition to periodontitis (t=133,7 at р=10(-6)). This result is in a good agreement with communications about positive impact of hyper-colonization of the colon with this species on type 2 diabetes, obesity, atopic dermatitis, and antibiotic-induced diarrhea associated with Clostridium dificile. Analysis of the periodontal protectors at the periodontium elucidated a number of close taxonomic relatives of the periodontal pathogens by Socransky, e.g. Aggregatibacter segnis and Aggregatibacter aphrophilus are closely related to Aggregatibacter actinomycetemcomitans; Treponema vencentii is a relative of Treponema denticola; Prevotella baroniae, Prevotella salivae and Prevotella spp. are relatives of Prevotella intermedia; Campylobacter concisus is a relative of Campylobacter jejuni, causative agent of enterocolitis. PMID:27239990

  3. Bacterial Heat Shock Protein GroEL (Hsp64) Exerts Immunoregulatory Effects on T Cells by Utilizing Apoptosis

    PubMed Central

    Nalbant, Ayten; Kant, Melis

    2016-01-01

    Aggregatibacter actinomycetemcomitans (Aa) expresses a 64-kDa GroEL protein belonging to the heat shock family of proteins. This protein has been shown to influence human host cells, but the apoptotic capacity of the GroEL protein regarding T cells is not yet known. The purpose of this study was to investigate the ability of A. actinomycetemcomitans GroEL (AaGroEL) protein to induce human peripheral blood T-cell apoptosis. Endogenous, purified AaGroEL protein was used as an antigen. In AaGroEL-treated T cells, the data indicated that phosphatidylserine exposure, an early apoptotic event, was dose- and time-dependent. The AaGroEL-treated T cells were also positive for active caspase-3 in a dose-dependent manner. The rate of AaGroEL-induced apoptosis was suppressed by the addition of the general caspase inhibitor Z-VAD-FMK. Furthermore, cleaved caspase-8 bands (40/36 kDa and 23 kDa) were identified in cells responding to AaGroEL. DNA fragmentation was also detected in the AaGroEL-treated T cells. Overall, we demonstrated that the endogenous GroEL from A. actinomycetemcomitans has the capacity to induce T-cell apoptosis. PMID:27736933

  4. Effects of ozone nano-bubble water on periodontopathic bacteria and oral cells - in vitro studies

    NASA Astrophysics Data System (ADS)

    Hayakumo, Sae; Arakawa, Shinichi; Takahashi, Masayoshi; Kondo, Keiko; Mano, Yoshihiro; Izumi, Yuichi

    2014-10-01

    The aims of the present study were to evaluate the bactericidal activity of a new antiseptic agent, ozone nano-bubble water (NBW3), against periodontopathogenic bacteria and to assess the cytotoxicity of NBW3 against human oral cells. The bactericidal activities of NBW3 against representative periodontopathogenic bacteria, Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) were evaluated using in vitro time-kill assays. The cytotoxicity of NBW3 was evaluated using three-dimensional human buccal and gingival tissue models. The numbers of colony forming units (CFUs)/mL of P. gingivalis and A. actinomycetemcomitans exposed to NBW3 dropped to below the lower limit of detection (<10 CFUs mL-1) after only 0.5 min of exposure. There were only minor decreases in the viability of oral tissue cells after 24 h of exposure to NBW3. These results suggest that NBW3 possesses potent bactericidal activity against representative periodontopathogenic bacteria and is not cytotoxic to cells of human oral tissues. The use of NBW3 as an adjunct to periodontal therapy would be promising.

  5. Oxygen as a Virulence Determinant in Polymicrobial Infections.

    PubMed

    Selleck, Elizabeth M; Gilmore, Michael S

    2016-01-01

    Infections caused by multiple organisms, or polymicrobial infections, are likely more common than is broadly appreciated. Interaction among microbial communities (and with their host) can change the infection landscape by subverting immunity, providing nutrients and inhibiting competing microbes. Stacy et al. (A. Stacy, D. Fleming, R. J. Lamont, K. P. Rumbaugh, and M. Whiteley, mBio 7:e00782-16, 2016, http://dx.doi.org/10.1128/mBio.00782-16) described a novel mechanism that results in synergistic growth of oral microbes Aggregatibacter actinomycetemcomitans and Streptococcus gordonii The authors used whole-genome fitness profiling by transposon sequencing (Tn-seq) to identify genes differentially required for growth in vitro versus in a mono- or coinfection in a thigh abscess model. They found that coinfection with S. gordonii allowed A. actinomycetemcomitans to shift from an anaerobic to an aerobic mode of growth. This shift involved the production of a terminal electron acceptor H2O2 by S. gordonii and increased A. actinomycetemcomitans persistence-an interaction termed "cross-respiration." PMID:27531913

  6. Oxygen as a Virulence Determinant in Polymicrobial Infections

    PubMed Central

    2016-01-01

    ABSTRACT Infections caused by multiple organisms, or polymicrobial infections, are likely more common than is broadly appreciated. Interaction among microbial communities (and with their host) can change the infection landscape by subverting immunity, providing nutrients and inhibiting competing microbes. Stacy et al. (A. Stacy, D. Fleming, R. J. Lamont, K. P. Rumbaugh, and M. Whiteley, mBio 7:e00782-16, 2016, http://dx.doi.org/10.1128/mBio.00782-16) described a novel mechanism that results in synergistic growth of oral microbes Aggregatibacter actinomycetemcomitans and Streptococcus gordonii. The authors used whole-genome fitness profiling by transposon sequencing (Tn-seq) to identify genes differentially required for growth in vitro versus in a mono- or coinfection in a thigh abscess model. They found that coinfection with S. gordonii allowed A. actinomycetemcomitans to shift from an anaerobic to an aerobic mode of growth. This shift involved the production of a terminal electron acceptor H2O2 by S. gordonii and increased A. actinomycetemcomitans persistence—an interaction termed “cross-respiration.” PMID:27531913

  7. Antimicrobial Activity of Protamine against Oral Microorganisms.

    PubMed

    Kim, Yeon-Hee; Kim, Sang Moo; Lee, Si Young

    2015-01-01

    Protamine is an arginine-rich polycationic protein extracted from sperm cells of vertebrates including fishes such as salmon. The purpose of this study was to investigate the suppressive effects of protamine on the growth of oral pathogens for possible usage in dental materials. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by the microdilution method. Twelve strains of oral viridans streptococci, Actinomyces naeslundii, Actinomyces odontolyticus, Enterococcus faecalis, Lactobacillus acidophilus, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Candida albicans were suppressed by protamine. MIC and MBC values were between 0.009 ~ 20 mg/mL and 0.019 ~ 80 mg/mL, respectively. The bactericidal activities of protamine against susceptible bacterial species were dependent on the concentration of protamine and incubation time. Based on the results of this study, protamine would be a useful compound for the development of antimicrobial agents against oral pathogens in dental materials.

  8. Synthesis of new carolacton derivatives and their activity against biofilms of oral bacteria.

    PubMed

    Stumpp, N; Premnath, P; Schmidt, T; Ammermann, J; Dräger, G; Reck, M; Jansen, R; Stiesch, M; Wagner-Döbler, I; Kirschning, A

    2015-05-28

    Carolacton, a secondary metabolite isolated from the extracts of Sorangium cellulosum, causes membrane damage and cell death in biofilms of the caries- and endocarditis-associated bacterium Streptococcus mutans. Here, we report the total synthesis of several derivatives of carolacton. All new structural modifications introduced abolished its biological activity, including subtle ones, such as inversion of configuration at C9. However, a bicyclic bislactone derivative as well as the methyl ester of carolacton resulted in compounds with prodrug properties. Their inhibitory activity on S. mutans was proven to be based on enzymatic hydrolysis by S. mutans which provided native carolacton resulting in biofilm damage in vivo. Moreover, we demonstrate that carolacton acts also on S. gordonii, S. oralis and the periodontitis pathogen Aggregatibacter actinomycetemcomitans, causing elongated cells and growth inhibition.

  9. Nonsyndromic localized aggressive periodontitis of primary dentition: A rare case report.

    PubMed

    Muppa, Radhika; Nallanchakrava, Srinivas; Chinta, Mahesh; Manthena, Ravi Teja

    2016-01-01

    Periodontitis is an inflammatory disease of the gingiva and tissues of the periodontium. It is characterized by pocket formation and destruction of supporting alveolar bone. Periodontal diseases of aggressive nature are not very common in children. They are usually associated with systemic conditions. The present case report is of a 5-year-old male child who reported with rapid attachment loss and bony defects of the gingiva and supporting structures. His family and medical history gave no contribution for the diagnosis. Blood investigations did not reveal any abnormality. The microbial examination of culture revealed the presence of periodontal pathogen Aggregatibacter actinomycetemcomitans. The treatment objective in the present case was to prevent the further progress of the condition, restore esthetic and function in the child which would psychologically benefit the child. PMID:27307682

  10. Synthesis of new carolacton derivatives and their activity against biofilms of oral bacteria.

    PubMed

    Stumpp, N; Premnath, P; Schmidt, T; Ammermann, J; Dräger, G; Reck, M; Jansen, R; Stiesch, M; Wagner-Döbler, I; Kirschning, A

    2015-05-28

    Carolacton, a secondary metabolite isolated from the extracts of Sorangium cellulosum, causes membrane damage and cell death in biofilms of the caries- and endocarditis-associated bacterium Streptococcus mutans. Here, we report the total synthesis of several derivatives of carolacton. All new structural modifications introduced abolished its biological activity, including subtle ones, such as inversion of configuration at C9. However, a bicyclic bislactone derivative as well as the methyl ester of carolacton resulted in compounds with prodrug properties. Their inhibitory activity on S. mutans was proven to be based on enzymatic hydrolysis by S. mutans which provided native carolacton resulting in biofilm damage in vivo. Moreover, we demonstrate that carolacton acts also on S. gordonii, S. oralis and the periodontitis pathogen Aggregatibacter actinomycetemcomitans, causing elongated cells and growth inhibition. PMID:25902328

  11. Anti-inflammatory, Antioxidant and Antimicrobial Effects of Artemisinin Extracts from Artemisia annua L.

    PubMed Central

    Kim, Wan-Su; Choi, Woo Jin; Lee, Sunwoo; Kim, Woo Joong; Lee, Dong Chae; Sohn, Uy Dong; Shin, Hyoung-Shik

    2015-01-01

    The anti-inflammatory, antioxidant, and antimicrobial properties of artemisinin derived from water, methanol, ethanol, or acetone extracts of Artemisia annua L. were evaluated. All 4 artemisinin-containing extracts had anti-inflammatory effects. Of these, the acetone extract had the greatest inhibitory effect on lipopolysaccharide-induced nitric oxide (NO), prostaglandin E2 (PGE2), and proinflammatory cytokine (IL-1β , IL-6, and IL-10) production. Antioxidant activity evaluations revealed that the ethanol extract had the highest free radical scavenging activity, (91.0±3.2%), similar to α-tocopherol (99.9%). The extracts had antimicrobial activity against the periodontopathic microorganisms Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum subsp. animalis, Fusobacterium nucleatum subsp. polymorphum, and Prevotella intermedia. This study shows that Artemisia annua L. extracts contain anti-inflammatory, antioxidant, and antimicrobial substances and should be considered for use in pharmaceutical products for the treatment of dental diseases. PMID:25605993

  12. Antibacterial Efficacy of Exogenous Nitric Oxide on Periodontal Pathogens

    PubMed Central

    Backlund, C.J.; Sergesketter, A.R.; Offenbacher, S.; Schoenfisch, M.H.

    2014-01-01

    Current treatments for periodontitis (e.g., scaling/root planing and chlorhexidine) have limited efficacy since they fail to suppress microbial biofilms satisfactorily over time, and the use of adjunctive antimicrobials can promote the emergence of antibiotic-resistant organisms. Herein, we report the novel application of nitric oxide (NO)-releasing scaffolds (i.e., dendrimers and silica particles) as anti-periodontopathogenic agents. The effectiveness of macromolecular NO release was demonstrated by a 3-log reduction in periodontopathogenic Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis viability. In contrast, Streptococcus mutans and Streptococcus sanguinis, caries-associated organisms, were substantially less sensitive to NO treatment. Both dendrimer- and silica-based NO release exhibited substantially less toxicity to human gingival fibroblasts at concentrations necessary to eradicate periodontopathogens than did clinical concentrations of chlorhexidine. These results suggest the potential utility of macromolecular NO-release scaffolds as a novel platform for the development of periodontal disease therapeutics. PMID:25139363

  13. Bactericidal Activity and Mechanism of Photoirradiated Polyphenols against Gram-Positive and -Negative Bacteria.

    PubMed

    Nakamura, Keisuke; Ishiyama, Kirika; Sheng, Hong; Ikai, Hiroyo; Kanno, Taro; Niwano, Yoshimi

    2015-09-01

    The bactericidal effect of various types of photoirradiated polyphenols against Gram-positive and -negative bacteria was evaluated in relation to the mode of action. Gram-positive bacteria (Enterococcus faecalis, Staphylococcus aureus, and Streptococcus mutans) and Gram-negative bacteria (Aggregatibacter actinomycetemcomitans, Escherichia coli, and Pseudomonas aeruginosa) suspended in a 1 mg/mL polyphenol aqueous solution (caffeic acid, gallic acid, chlorogenic acid, epigallocatechin, epigallocatechin gallate, and proanthocyanidin) were exposed to LED light (wavelength, 400 nm; irradiance, 260 mW/cm(2)) for 5 or 10 min. Caffeic acid and chlorogenic acid exerted the highest bactericidal activity followed by gallic acid and proanthocyanidin against both Gram-positive and -negative bacteria. It was also demonstrated that the disinfection treatment induced oxidative damage of bacterial DNA, which suggests that polyphenols are incorporated into bacterial cells. The present study suggests that blue light irradiation of polyphenols could be a novel disinfection treatment.

  14. Nonsyndromic localized aggressive periodontitis of primary dentition: A rare case report

    PubMed Central

    Muppa, Radhika; Nallanchakrava, Srinivas; Chinta, Mahesh; Manthena, Ravi Teja

    2016-01-01

    Periodontitis is an inflammatory disease of the gingiva and tissues of the periodontium. It is characterized by pocket formation and destruction of supporting alveolar bone. Periodontal diseases of aggressive nature are not very common in children. They are usually associated with systemic conditions. The present case report is of a 5-year-old male child who reported with rapid attachment loss and bony defects of the gingiva and supporting structures. His family and medical history gave no contribution for the diagnosis. Blood investigations did not reveal any abnormality. The microbial examination of culture revealed the presence of periodontal pathogen Aggregatibacter actinomycetemcomitans. The treatment objective in the present case was to prevent the further progress of the condition, restore esthetic and function in the child which would psychologically benefit the child. PMID:27307682

  15. Effectiveness of ozone against periodontal pathogenic microorganisms.

    PubMed

    Huth, Karin C; Quirling, Martina; Lenzke, Stefanie; Paschos, Ekaterini; Kamereck, Klaus; Brand, Korbinian; Hickel, Reinhard; Ilie, Nicoleta

    2011-06-01

    Ozone has been proposed as an adjunct antiseptic in periodontitis therapy. The aim of this study was to investigate the antimicrobial effectiveness of gaseous/aqueous ozone, in comparison with that of the established antiseptic chlorhexidine digluconate (CHX), against periodontal microorganisms. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, and Parvimonas micra in planktonic or biofilm cultures were exposed, for 1 min, to gaseous ozone, aqueous ozone, CHX, or phosphate-buffered saline (control). None of the agents was able to substantially reduce the A. actinomycetemcomitans count in biofilm cultures. In contrast, P. gingivalis, T. forsythia, and P. micra could be eliminated by 2% CHX or by ozone gas at 53 gm(-3) . Significantly greater antimicrobial effects were observed against planktonic cultures than against biofilm-associated bacteria. The rate of killing was influenced by the species of bacteria, and by the type and concentration of agent. There were no significant differences in the effectiveness of aqueous ozone (20 μg ml(-1) ) or gaseous ozone (≥ 4 gm(-3) ) compared with 2% CHX but they were more effective than 0.2% CHX. Therefore, high-concentrated gaseous and aqueous ozone merit further investigation as antiseptics in periodontitis therapy. A safe system for applying gaseous ozone into the periodontal pocket that avoids inhalation still needs to be developed.

  16. Determination of periodontopathogens in patients with Cri du chat syndrome

    PubMed Central

    Ballesta-Mudarra, Sofía; Torres-Lagares, Daniel; Rodríguez-Caballero, Ángela; Yáñez-Vico, Rosa M.; Solano-Reina, Enrique; Perea-Pérez, Evelio

    2013-01-01

    Objectives: Cri du chat syndrome is a genetic alteration associated with some oral pathologies. However, it has not been described previously any clinical relationship between the periodontal disease and the syndrome. The purpose of this comparative study was to compare periodontopathogenic flora in a group with Cri du chat syndrome and another without the síndrome, to assess a potential microbiological predisposition to suffer a periodontitis. Study Design: The study compared nineteen subjects with Cri du chat Syndrome with a control group of nineteen patients without it. All patients were clinically evaluated by periodontal probing, valuing the pocket depth, the clinical attachmente level and bleeding on probing. There were no significant differences between both groups. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Treponema denticola were detected by multiplex-PCR using 16S rDNA (microIDENT). Results: When A. actinomycetemcomitans, P. gingivalis, P. intermedia and T. denticola were compared, no statistically significant differences were found between the two groups (p>0.05). The value of T. forsythia was significantly higher for Cri du chat syndrome (31.6%) than for the control group (5.3%). The odds ratio for T. forsythia was 8.3. Conclusions: In the present study T. forsythia is associated with Cri du chat syndrome subjects and not with healthy subjects. Key words:Cri du Chat syndrome, periodontal health, microbiology, special care dentistry. PMID:24121919

  17. Antibacterial activity of moxifloxacin on bacteria associated with periodontitis within a biofilm.

    PubMed

    Tsaousoglou, Phoebus; Nietzsche, Sandor; Cachovan, Georg; Sculean, Anton; Eick, Sigrun

    2014-02-01

    The activity of moxifloxacin was compared with ofloxacin and doxycycline against bacteria associated with periodontitis within a biofilm (single strain and mixed population) in vitro. MICs and minimal bactericidal concentrations (MBCs) of moxifloxacin, ofloxacin and doxycyline were determined against single strains and mixed populations in a planktonic state. Single-species biofilms of two Porphyromonas gingivalis and two Aggregatibacter actinomycetemcomitans strains and a multispecies biofilm consisting of 12 species were formed for 3 days. The minimal biofilm eradication concentrations (MBECs) were determined after exposing the biofilms to the antibacterials (0.002-512 µg ml(-1)) for 18 h, addition of nutrient broth for 3 days and subsequent subcultivation. Photographs were taken using confocal laser-scanning microscopy and scanning electron microscopy. The MICs and MBCs did not differ between ofloxacin and moxifloxacin against A. actinomycetemcomitans, whilst moxifloxacin was more active than the other tested antibacterials against anaerobes and the mixed population. The single-species biofilms were eradicated by moderate concentrations of the antibacterials, and the lowest MBECs were always found for moxifloxacin (2-8 µg ml(-1)). MBECs against the multispecies biofilms were 128, >512 and >512 µg ml(-1) for moxifloxacin, ofloxacin and doxycycline, respectively. In summary, moxifloxacin in a topical formulation may have potential as an adjunct to mechanical removal of the biofilms.

  18. The Comparative Evaluation of the Antimicrobial Effect of Propolis with Chlorhexidine against Oral Pathogens: An In Vitro Study

    PubMed Central

    Akca, Gülçin; Topçu, Fulya Toksoy; Macit, Enis; Pikdöken, Levent; Özgen, I. Şerif

    2016-01-01

    This study aimed to compare the antimicrobial effectiveness of ethanolic extract of propolis (EEP) to chlorhexidine gluconate (CHX) on planktonic Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus, Lactobacillus salivarius subsp. salivarius, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Staphylococcus aureus, Enterococcus faecalis, Actinomyces israelii, Candida albicans, and their single-species biofilms by agar dilution and broth microdilution test methods. Both agents inhibited the growth of all planktonic species. On the other hand, CHX exhibited lower minimum bactericidal concentrations than EEP against biofilms of A. actinomycetemcomitans, S. aureus, and E. faecalis whereas EEP yielded a better result against Lactobacilli and P. intermedia. The bactericidal and fungicidal concentrations of both agents were found to be equal against biofilms of Streptecocci, P. gingivalis, A. israelii, and C. albicans. The results of this study revealed that propolis was more effective in inhibiting Gram-positive bacteria than the Gram-negative bacteria in their planktonic state and it was suggested that EEP could be as effective as CHX on oral microorganisms in their biofilm state. PMID:26949701

  19. Determination of porphyrins in oral bacteria by liquid chromatography electrospray ionization tandem mass spectrometry.

    PubMed

    Fyrestam, Jonas; Bjurshammar, Nadja; Paulsson, Elin; Johannsen, Annsofi; Östman, Conny

    2015-09-01

    Biofilms in the oral cavity can be visualized by fluorescence and a common assumption is that the endogenously produced porphyrins in certain bacteria give rise to this fluorescence. Porphyrin content in oral bacteria has been sparingly investigated, and non-selective detection techniques such as utilizing the Soret fluorescence band of porphyrins are often used. In the present study, a quantitative and selective method for the determination of porphyrins in oral bacteria has been developed and validated using high performance liquid chromatography-tandem mass spectrometry. Lysis of bacteria using Tris-EDTA buffer together with ultrasonication showed high microbial killing efficiency ≥99.98%, and sample clean-up using C18-solid phase extraction resulted in low matrix effects ≤14% for all analytes. Using this method, the porphyrin content was determined in the two oral pathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, as well as for baker's yeast, Saccharomyces cerevisiae. Uroporphyrin, 7-carboxylporphyrin, 6-carboxylporphyrin, coproporphyrin, and protoporphyrin IX were identified in the investigated microorganisms, and it was shown that the porphyrin profile differs between the two bacteria, as well as for S. cerevisiae. To our knowledge, this is the first time the porphyrin profile has been determined for the bacterium A. actinomycetemcomitans. PMID:26168965

  20. The pathogenic persona of community-associated oral streptococci.

    PubMed

    Whitmore, Sarah E; Lamont, Richard J

    2011-07-01

    The mitis group streptococci (MGS) are widespread in the oral cavity and are traditionally associated with oral health. However, these organisms have many attributes that contribute to the development of pathogenic oral communities. MGS adhere rapidly to saliva-coated tooth surfaces, thereby providing an attachment substratum for more overtly pathogenic organisms such as Porphyromonas gingivalis, and the two species assemble into heterotypic communities. Close physical association facilitates physiologic support, and pathogens such as Aggregatibacter actinomycetemcomitans display resource partitioning to favour carbon sources generated by streptococcal metabolism. MGS exchange information with community members through a number of interspecies signalling systems including AI-2 and contact dependent mechanisms. Signal transduction systems induced in P. gingivalis are based on protein dephosphorylation mediated by the tyrosine phosphatase Ltp1, and converge on a LuxR-family transcriptional regulator, CdhR. Phenotypic responses in P. gingivalis include regulation of hemin uptake systems and gingipain activity, processes that are intimately linked to the virulence of the organism. Furthermore, communities of S. gordonii with P. gingivalis or with A. actinomycetemcomitans are more pathogenic in animal models than the constituent species alone. We propose that MGS should be considered accessory pathogens, organisms whose pathogenic potential only becomes evident in the context of a heterotypic microbial community.

  1. Determination of porphyrins in oral bacteria by liquid chromatography electrospray ionization tandem mass spectrometry.

    PubMed

    Fyrestam, Jonas; Bjurshammar, Nadja; Paulsson, Elin; Johannsen, Annsofi; Östman, Conny

    2015-09-01

    Biofilms in the oral cavity can be visualized by fluorescence and a common assumption is that the endogenously produced porphyrins in certain bacteria give rise to this fluorescence. Porphyrin content in oral bacteria has been sparingly investigated, and non-selective detection techniques such as utilizing the Soret fluorescence band of porphyrins are often used. In the present study, a quantitative and selective method for the determination of porphyrins in oral bacteria has been developed and validated using high performance liquid chromatography-tandem mass spectrometry. Lysis of bacteria using Tris-EDTA buffer together with ultrasonication showed high microbial killing efficiency ≥99.98%, and sample clean-up using C18-solid phase extraction resulted in low matrix effects ≤14% for all analytes. Using this method, the porphyrin content was determined in the two oral pathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, as well as for baker's yeast, Saccharomyces cerevisiae. Uroporphyrin, 7-carboxylporphyrin, 6-carboxylporphyrin, coproporphyrin, and protoporphyrin IX were identified in the investigated microorganisms, and it was shown that the porphyrin profile differs between the two bacteria, as well as for S. cerevisiae. To our knowledge, this is the first time the porphyrin profile has been determined for the bacterium A. actinomycetemcomitans.

  2. Prediagnostic plasma antibody levels to periodontopathic bacteria and risk of coronary heart disease.

    PubMed

    Ueno, Masayuki; Izumi, Yuichi; Kawaguchi, Yoko; Ikeda, Ai; Iso, Hiroyasu; Inoue, Manami; Tsugane, Shoichiro

    2012-01-01

    Many epidemiological studies have indicated that periodontitis is an important risk factor for coronary heart disease (CHD). We examined whether plasma antibody levels to 3 major periodontal pathogens, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia predicted the risk of CHD events. A nested case-control research design (case: n = 191, control: n = 382), by matching gender, age, study area, date of blood collection, and time since last meal at blood collection, was employed in a large cohort of Japanese community residents.Antibody levels of periodontopathic bacteria were associated with risk of CHD after adjusting for BMI, smoking status, alcohol intake, history of hypertension, history of diabetes mellitus, exercise during leisure time, and perceived mental stress. The association was different by age subgroup. For subjects aged 40-55 years, the medium (31.7-184.9 U/mL) or high tertile plasma antibody level (> 184.9 U/mL) of A. actinomycetemcomitans showed higher risk of CHD (medium: OR = 3.72; 95% CI = 1.20-11.56, high: OR = 4.64; 95% CI = 1.52-14.18) than the low tertile level (< 31.7 U/mL). The ORs of CHD incidence became higher with an increase in IgG level of A. actinomycetemcomitans (P for trend = 0.007). For subjects aged 56-69 years, the high tertile level (> 414.1 U/mL) of P. intermedia was associated with higher risk of CHD (OR = 2.65; 95% CI = 1.18-5.94) in a dose-response fashion (P for trend = 0.007). The possible role of periodontopathic bacteria as a risk factor for CHD incidence was suggested by the results of this study by the elevated antibody level to these bacteria with the increased risk of CHD. PMID:22878796

  3. Integration of non-oral bacteria into in vitro oral biofilms.

    PubMed

    Thurnheer, Thomas; Belibasakis, Georgios N

    2015-01-01

    Biofilms are polymicrobial communities that grow on surfaces in nature. Oral bacteria can spontaneously form biofilms on the surface of teeth, which may compromise the health of the teeth, or their surrounding (periodontal) tissues. While the oral bacteria exhibit high tropism for their specialized ecological niche, it is not clear if bacteria that are not part of the normal oral microbiota can efficiently colonize and grow within oral biofilms. By using an in vitro "supragingival" biofilm model of 6 oral species, this study aimed to investigate if 3 individual bacterial species that are not part of the normal oral microbiota (Eschericia coli, Staphylococcus aureus, Enterococcus faecails) and one not previously tested oral species (Aggregatibacter actinomycetemcomitans) can be incorporated into this established supragingival biofilm model. Staphylococcus aureus and A. actinomycetemcomitans were able to grow efficiently in the biofilm, without disrupting the growth of the remaining species. They localized in sparse small aggregates within the biofilm mass. Enterococcus faecalis and E. coli were both able to populate the biofilm at high numbers, and suppressed the growth of A. oris and S. mutants. Enterococcus faecalis was arranged in a chain-like conformation, whereas E. coli was densely and evenly spread throughout the biofilm mass. In conclusion, it is possible for selected species that are not part of the normal oral microbiota to be introduced into an oral biofilm, under the given experimental micro-environmental conditions. Moreover, the equilibrated incorporation of A. actinomycetemcomitans and S. aureus in this oral biofilm model could be a useful tool in the study of aggressive periodontitis and peri-implantitis, in which these organisms are involved, respectively.

  4. Innate Immune Response of Human Embryonic Stem Cell-Derived Fibroblasts and Mesenchymal Stem Cells to Periodontopathogens

    PubMed Central

    Seneviratne, Chaminda Jayampath

    2016-01-01

    Periodontitis involves complex interplay of bacteria and host immune response resulting in destruction of supporting tissues of the tooth. Toll-like receptors (TLRs) play a role in recognizing microbial pathogens and eliciting an innate immune response. Recently, the potential application of multipotent stem cells and pluripotent stem cells including human embryonic stem cells (hESCs) in periodontal regenerative therapy has been proposed. However, little is known about the impact of periodontopathogens on hESC-derived progenies. This study investigates the effects of heat-killed periodontopathogens, namely, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, on TLR and cytokine expression profile of hESC-derived progenies, namely, fibroblasts (hESC-Fib) and mesenchymal stem cells (hESC-MSCs). Additionally, the serotype-dependent effect of A. actinomycetemcomitans on hESC-derived progenies was explored. Both hESC-Fib and hESC-MSCs constitutively expressed TLR-2 and TLR-4. hESC-Fib upon exposure to periodontopathogens displayed upregulation of TLRs and release of cytokines (IL-1β, IL-6, and IL-8). In contrast, hESC-MSCs were largely nonresponsive to bacterial challenge, especially in terms of cytokine production. Further, exposure of hESC-Fib to A. actinomycetemcomitans serotype c was associated with higher IL-8 production than serotype b. In contrast, the hESC-MSCs displayed no serotype-dependent response. Differential response of the two hESC progenies implies a phenotype-dependent response to periodontopathogens and supports the concept of immunomodulatory properties of MSCs. PMID:27642305

  5. Antimicrobial efficacy of Tulsi leaf (Ocimum sanctum) extract on periodontal pathogens: An in vitro study

    PubMed Central

    Mallikarjun, Sajjanshetty; Rao, Ashwini; Rajesh, Gururaghavendran; Shenoy, Ramya; Pai, Mithun

    2016-01-01

    Background: Periodontitis is an infection of the periodontal complex with severe forms of disease associated with specific bacteria colonizing the subgingival area. Widespread use of drugs has resulted in the emergence of side effects, uncommon infections, and resistance. Plant medicine like Tulsi has been used in many clinical conditions, and it appears to be a suitable alternative to manage conditions affecting the oral cavity. Hence, the objective was to assess the in vitro antimicrobial activity of Tulsi leaves extract (Ocimum sanctum) on periodontal pathogens with doxycycline as standard, as doxycycline has been used as an adjunct to nonsurgical therapy in periodontitis patients. Materials and Methods: Ethanolic extract of Tulsi was prepared by cold extraction method. Extract was diluted with an inert solvent, dimethyl formamide, to obtain five different concentrations (0.5%, 1%, 2%, 5%, and 10%). Doxycycline was used as a positive control and dimethyl formamide, as a negative control. The extract and controls were subjected to the microbiological investigation against Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Porphyromonas gingivalis. Agar well diffusion method was employed to determine the concentration at which Tulsi gave an inhibition zone, similar to doxycycline. Data were analyzed using one-way analysis of variance and Tukey post-hoc test was used for inter- and intra-group comparisons. Results: At 5% and 10% concentrations, Tulsi extracts demonstrated antimicrobial activity against A. actinomycetemcomitans, similar to doxycycline with similar inhibition zones (P > 0.05). P. gingivalis and P. intermedia, however, exhibited resistance to Tulsi extract that showed significantly smaller inhibition zones (P < 0.05). Conclusions: Tulsi demonstrated effective antimicrobial property against A. actinomycetemcomitans, suggesting its possible use as an effective and affordable “adjunct” along with the standard care in the management of

  6. Innate Immune Response of Human Embryonic Stem Cell-Derived Fibroblasts and Mesenchymal Stem Cells to Periodontopathogens.

    PubMed

    Sriram, Gopu; Natu, Vaishali Prakash; Islam, Intekhab; Fu, Xin; Seneviratne, Chaminda Jayampath; Tan, Kai Soo; Cao, Tong

    2016-01-01

    Periodontitis involves complex interplay of bacteria and host immune response resulting in destruction of supporting tissues of the tooth. Toll-like receptors (TLRs) play a role in recognizing microbial pathogens and eliciting an innate immune response. Recently, the potential application of multipotent stem cells and pluripotent stem cells including human embryonic stem cells (hESCs) in periodontal regenerative therapy has been proposed. However, little is known about the impact of periodontopathogens on hESC-derived progenies. This study investigates the effects of heat-killed periodontopathogens, namely, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, on TLR and cytokine expression profile of hESC-derived progenies, namely, fibroblasts (hESC-Fib) and mesenchymal stem cells (hESC-MSCs). Additionally, the serotype-dependent effect of A. actinomycetemcomitans on hESC-derived progenies was explored. Both hESC-Fib and hESC-MSCs constitutively expressed TLR-2 and TLR-4. hESC-Fib upon exposure to periodontopathogens displayed upregulation of TLRs and release of cytokines (IL-1β, IL-6, and IL-8). In contrast, hESC-MSCs were largely nonresponsive to bacterial challenge, especially in terms of cytokine production. Further, exposure of hESC-Fib to A. actinomycetemcomitans serotype c was associated with higher IL-8 production than serotype b. In contrast, the hESC-MSCs displayed no serotype-dependent response. Differential response of the two hESC progenies implies a phenotype-dependent response to periodontopathogens and supports the concept of immunomodulatory properties of MSCs. PMID:27642305

  7. Innate Immune Response of Human Embryonic Stem Cell-Derived Fibroblasts and Mesenchymal Stem Cells to Periodontopathogens

    PubMed Central

    Seneviratne, Chaminda Jayampath

    2016-01-01

    Periodontitis involves complex interplay of bacteria and host immune response resulting in destruction of supporting tissues of the tooth. Toll-like receptors (TLRs) play a role in recognizing microbial pathogens and eliciting an innate immune response. Recently, the potential application of multipotent stem cells and pluripotent stem cells including human embryonic stem cells (hESCs) in periodontal regenerative therapy has been proposed. However, little is known about the impact of periodontopathogens on hESC-derived progenies. This study investigates the effects of heat-killed periodontopathogens, namely, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, on TLR and cytokine expression profile of hESC-derived progenies, namely, fibroblasts (hESC-Fib) and mesenchymal stem cells (hESC-MSCs). Additionally, the serotype-dependent effect of A. actinomycetemcomitans on hESC-derived progenies was explored. Both hESC-Fib and hESC-MSCs constitutively expressed TLR-2 and TLR-4. hESC-Fib upon exposure to periodontopathogens displayed upregulation of TLRs and release of cytokines (IL-1β, IL-6, and IL-8). In contrast, hESC-MSCs were largely nonresponsive to bacterial challenge, especially in terms of cytokine production. Further, exposure of hESC-Fib to A. actinomycetemcomitans serotype c was associated with higher IL-8 production than serotype b. In contrast, the hESC-MSCs displayed no serotype-dependent response. Differential response of the two hESC progenies implies a phenotype-dependent response to periodontopathogens and supports the concept of immunomodulatory properties of MSCs. PMID:27642305

  8. Sphingosine-1-Phosphate Receptor 2 Regulates Proinflammatory Cytokine Production and Osteoclastogenesis

    PubMed Central

    2016-01-01

    Sphingosine-1-phosphate receptor 2 (S1PR2) couples with the Gi, Gq, and G12/13 group of proteins, which modulate an array of cellular signaling pathways and affect immune responses to multiple stimuli. In this study, we demonstrated that knockdown of S1PR2 by a specific S1PR2 shRNA lentiviral vector significantly inhibited IL-1β, IL-6, and TNF-α protein levels induced by oral pathogen Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) in murine bone marrow-derived monocytes and macrophages (BMMs) compared with controls. In addition, knockdown of S1PR2 by the S1PR2 shRNA lentiviral vector suppressed p-PI3K, p-ERK, p-JNK, p-p38, and p-NF-κBp65 protein expressions induced by A. actinomycetemcomitans. Furthermore, bone marrow cells treated with the S1PR2 shRNA lentiviral vector inhibited osteoclastogenesis induced by RANKL compared with controls. The S1PR2 shRNA suppressed the mRNA levels of six osteoclastogenic factors including nuclear factor of activated T-cells cytoplasmic calcineurin-dependent 1 (NFATc1), cathepsin K (Ctsk), acid phosphatase 5 (Acp5), osteoclast-associated receptor (Oscar), dendritic cells specific transmembrane protein (Dcstamp), and osteoclast stimulatory transmembrane protein (Ocstamp) in bone marrow cells. We conclude that S1PR2 plays an essential role in modulating proinflammatory cytokine production and osteoclastogenesis. Blocking S1PR2 signaling might be a novel therapeutic strategy to treat inflammatory bone loss diseases. PMID:27224249

  9. Association between periodontal disease and plasma levels of cholesterol and triglycerides

    PubMed Central

    Lafaurie, Gloria Inés; Millán, Lina Viviana; Ardila, Carlos Martin; Duque, Andrés; Novoa, Camilo; López, Diego; Contreras, Adolfo

    2013-01-01

    Objective: untreated periodontal disease seems to cause low grade systemic inflammation and blood lipid alteration leading to increased cardiovascular disease risk. To start testing this hypothesis in colombian patients, a multicentre study was conducted including the three main state capitals: bogota, medellin and cali. Methods: in this study 192 (28.4%) advanced and 256 (37.8%) moderate periodontitis patients were investigated for socio-demographic variables, city of precedence, periodontal parameters, smoking, red complex periodontopathic bacteria, serum antibodies against porphyromonas gingivalis and aggregatibacter actinomycetemcomitans and blood lipids including total cholesterol, hdl, ldl and triglycerides (tg). Those parameters were compared to 229 (33.8%) controls having periodontal health or gingivitis. Results: advanced periodontitis had worst periodontal indexes, than moderate periodontitis and controls. Interestingly, higher hdl and tg levels were present in periodontitis. Bmi <30 and smoking were associated with increased hdl, hdl-35, ldl and tg, while glycemia >100 mg/dl associated with hdl, hdl-35 and tg. Tannerella forsythia showed a significant association with hdl-35 in bivariate analysis and serum igg1 against p. Gingivalis associated with hdl-35 and serum igg1 against t. Forsythia associated with tg and serum igg2 against a. Actinomycetemcomitans correlated with levels of hdl y hdl-35. In logistic regression the periodontitis patients from cali presented reduced hdl levels as compared to bogota and medellin patients. Presence of igg1 antibodies against p. Gingivalis and a. Actinomycetemcomitans correlated with reduced hdl levels. Conclusion: this study confirmed that untreated periodontitis generates alteration in serum lipid levels and systemic bacterial exposure against important periodontopathic bacteria could be the biological link. PMID:24892452

  10. A Modified Glycosaminoglycan, GM-0111, Inhibits Molecular Signaling Involved in Periodontitis

    PubMed Central

    Savage, Justin R.; Pulsipher, Abigail; Rao, Narayanam V.; Kennedy, Thomas P.; Prestwich, Glenn D.; Ryan, Maria E.; Lee, Won Yong

    2016-01-01

    Background Periodontitis is characterized by microbial infection, inflammation, tissue breakdown, and accelerated loss of alveolar bone matrix. Treatment targeting these multiple stages of the disease provides ways to treat or prevent periodontitis. Certain glycosaminoglycans (GAGs) block multiple inflammatory mediators as well as suppress bacterial growth, suggesting that these GAGs may be exploited as a therapeutic for periodontitis. Methods We investigated the effects of a synthetic GAG, GM-0111, on various molecular events associated with periodontitis: growth of Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) pathogenic bacteria associated with periodontitis; activation of pro-inflammatory signaling through TLR2 and TLR4 in mouse macrophage RAW 264.7 cells and heterologously expressed HEK 293 cells; osteoclast formation and bone matrix resorption in cultured mouse pre-osteoclasts. Results (1) GM-0111 suppressed the growth of P. gingivalis and A. actinomycetemcomitans even at 1% (w/v) solution. The antibacterial effects of GM-0111 were stronger than hyaluronic acid (HA) or xylitol in P. gingivalis at all concentrations and comparable to xylitol in A. actinomycetemcomitans at ≥2% (w/v) solution. We also observed that GM-0111 suppressed biofilm formation of P. gingivalis and these effects were much stronger than HA. (2) GM-0111 inhibited TLR-mediated pro-inflammatory cellular signaling both in macrophage and HEK 293 cells with higher selectivity for TLR2 than TLR4 (IC50 of 1–10 ng/mL vs. > 100 μg/mL, respectively). (3) GM-0111 blocked RANKL-induced osteoclast formation (as low as 300 ng/mL) and bone matrix resorption. While GM-0111 showed high affinity binding to RANKL, it did not interfere with RANKL/RANK/NF-κB signaling, suggesting that GM-0111 inhibits osteoclast formation by a RANKL-RANK-independent mechanism. Conclusions We report that GM-0111 inhibits multiple molecular events involved in

  11. Antimicrobial activity against periodontopathogenic bacteria, antioxidant and cytotoxic effects of various extracts from endemic Thermopsis turcica

    PubMed Central

    Bali, Elif Burcu; Açık, Leyla; Akca, Gülçin; Sarper, Meral; Elçi, Mualla Pınar; Avcu, Ferit; Vural, Mecit

    2014-01-01

    Objective To investigate the in vitro antimicrobial potential of Thermopsis turcica Kit Tan, Vural & Küçüködük against periodontopathogenic bacteria, its antioxidant activity and cytotoxic effect on various cancer cell lines. Methods In vitro antimicrobial activities of ethanol, methanol, ethyl acetate (EtAc), n-hexane and water extracts of Thermopsis turcica herb against periodontopathogenic bacteria, Aggregatibacter actinomycetemcomitans ATCC 29523 and Porphyromonas gingivalis ATCC 33277 were tested by agar well diffusion, minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Antioxidant properties of the extracts were evaluated by 1,1-diphenyl-2-picryl-hydrazyl radical scavenging activity and β-carotene bleaching methods. Amounts of phenolic contents of the extracts were also analysed by using the Folin-Ciocalteu reagent. Additionally, cytotoxic activity of the extracts on androgen-insensitive prostate cancer, androgen-sensitive prostate cancer, chronic myelogenous leukemia and acute promyelocytic leukemia human cancer cell lines were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Human gingival fibroblast cells were used as a control. Results Our data showed that EtAc extract had the highest antimicrobial effect on Aggregatibacter actinomycetemcomitans (MIC: 1.562 mg/mL, MBC: 3.124 mg/mL) and Porphyromonas gingivalis (MIC: 0.781 mg/mL, MBC: 1.562 mg/mL). In antioxidant assays, EtAc extract exhibited also the highest radical scavenging activity [IC50=(30.0±0.3) µg/mL] and the highest inhibition [(74.35±0.30)%] against lineloic acide oxidation. The amount of phenolic content of it was also the highest [(162.5±1.2) µg/mg gallic acid]. In cytotoxic assay, only ethanol [IC50=(80.00±1.21) µg/mL] and EtAc extract [IC50=(70.0±0.9) µg/mL] were toxic on acute promyelocytic leukemia cells at 20-100 µg/mL (P<0.05). However, no toxic effect was observed on human gingival fibroblast cells

  12. Bacterial inactivation/sterilization by argon plasma treatment on contaminated titanium implant surfaces:In vitro study

    PubMed Central

    Annunziata, Marco; Donnarumma, Giovanna; Caputo, Pina; Nastri, Livia; Guida, Luigi

    2016-01-01

    Background Surface treatment by argon plasma is widely used as the last step of the manufacturing process of titanium implant fixtures before their sterilization by gamma rays. The possibility of using such a technology in the daily clinical practice is particularly fascinating. The aim of the present study was to assess the effects of the argon plasma treatment on different titanium implant surfaces previously exposed In vitro to bacterial contamination. Material and Methods Sterile c.p. titanium implant discs with turned (T, Sa: 0.8 µm ), sandblasted/acid-etched (SAE, Sa: 1.3 µm) and titanium plasma sprayed (TPS, Sa: 3.0µm) surface were used in this study. A strain of Aggregatibacter actinomycetemcomitans ATCC3718 was grown at 37°C under anaerobic conditions for 24 h and then transferred on six discs for each of the three surface types. After 24 hours, a half of the contaminated discs (control group) were directly used to evaluate the colony forming units (CFUs). The other half of the contaminated discs (test group) were treated in an argon plasma chamber for 12 minutes at room temperature prior to be analyzed for CFU counting. All assays were performed using triplicate samples of each material in 3 different experiments. Results When the CFU counting was carried out on control discs, a total of 1.50x106±1.4x105, 1.55x106±7.07x104 and 3.15x106±2.12x105 CFU was respectively assessed for T, SAE and TPS discs, without statistically significant differences among the three surfaces. On the contrary, any trace of bacterial contamination was assessed for titanium discs treated in the argon plasma chamber prior to be analyzed, irrespectively to the implant surface tested. Conclusions Within the limit of this study, reported data suggested that the argon plasma technology could be efficiently used to decontaminate/sterilize previously infected titanium implant surfaces. Key words:Argon plasma, titanium implant surface, Aggregatibacter actinomycetemcomitans. PMID

  13. Increased levels of Porphyromonas gingivalis are associated with ischemic and hemorrhagic cerebrovascular disease in humans: an in vivo study

    PubMed Central

    GHIZONI, Janaina Salomon; TAVEIRA, Luís Antônio de Assis; GARLET, Gustavo Pompermaier; GHIZONI, Marcos Flávio; PEREIRA, Jefferson Ricardo; DIONÍSIO, Thiago José; BROZOSKI, Daniel Thomas; SANTOS, Carlos Ferreira; SANT'ANA, Adriana Campos Passanezi

    2012-01-01

    Objective: This study investigated the role of periodontal disease in the development of stroke or cerebral infarction in patients by evaluating the clinical periodontal conditions and the subgingival levels of periodontopathogens. Material and Methods: Twenty patients with ischemic (I-CVA) or hemorrhagic (H-CVA) cerebrovascular episodes (test group) and 60 systemically healthy patients (control group) were evaluated for: probing depth, clinical attachment level, bleeding on probing and plaque index. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were both identified and quantified in subgingival plaque samples by conventional and real-time PCR, respectively. Results: The test group showed a significant increase in each of the following parameters: pocket depth, clinical attachment loss, bleeding on probing, plaque index and number of missing teeth when compared to control values (p<0.05, unpaired t-test). Likewise, the test group had increased numbers of sites that were contaminated with P. gingivalis (60%x10%; p<0.001; chi-squared test) and displayed greater prevalence of periodontal disease, with an odds ratio of 48.06 (95% CI: 5.96-387.72; p<0.001). Notably, a positive correlation between probing depth and the levels of P. gingivalis in ischemic stroke was found (r=0.60; p=0.03; Spearman's rank correlation coefficient test). A. actinomycetemcomitans DNA was not detected in any of the groups by conventional or real-time PCR. Conclusions: Stroke patients had deeper pockets, more severe attachment loss, increased bleeding on probing, increased plaque indexes, and in their pockets harbored increased levels of P. gingivalis. These findings suggest that periodontal disease is a risk factor for the development of cerebral hemorrhage or infarction. Early treatment of periodontitis may counteract the development of cerebrovascular episodes. PMID:22437687

  14. In-vitro activity of taurolidine on single species and a multispecies population associated with periodontitis.

    PubMed

    Zollinger, Lilly; Schnyder, Simone; Nietzsche, Sandor; Sculean, Anton; Eick, Sigrun

    2015-04-01

    The antimicrobial activity of taurolidine was compared with minocycline against microbial species associated with periodontitis (four single strains and a 12-species mixture). Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs), killing as well as activities on established and forming single-species biofilms and a 12-species biofilm were determined. The MICs of taurolidine against single species were always 0.31 mg/ml, the MBCs were 0.64 mg/ml. The used mixed microbiota was less sensitive to taurolidine, MIC and the MBC was 2.5 mg/ml. The strains and the mixture were completely killed by 2.5 mg/ml taurolidine, whereas 256 μg/ml minocycline reduced the bacterial counts of the mixture by 5 log10 colony forming units (cfu). Coating the surface with 10 mg/ml taurolidine or 256 μg/ml minocycline prevented completely biofilm formation of Porphyromonas gingivalis ATCC 33277 but not of Aggregatibacter actinomycetemcomitans Y4 and the mixture. On 4.5 d old biofilms, taurolidine acted concentration dependent with a reduction by 5 log10 cfu (P. gingivalis ATCC 33277) and 7 log10 cfu (A. actinomycetemcomitans Y4) when applying 10 mg/ml. Minocycline decreased the cfu counts by 1-2 log10 cfu independent of the used concentration. The reduction of the cfu counts in the 4.5 d old multi-species biofilms was about 3 log10 cfu after application of any minocycline concentration and after using 10 mg/ml taurolidine. Taurolidine is active against species associated with periodontitis, even within biofilms. Nevertheless a complete elimination of complex biofilms by taurolidine seems to be impossible and underlines the importance of a mechanical removal of biofilms prior to application of taurolidine.

  15. Selected dietary (poly)phenols inhibit periodontal pathogen growth and biofilm formation.

    PubMed

    Shahzad, Muhammad; Millhouse, Emma; Culshaw, Shauna; Edwards, Christine A; Ramage, Gordon; Combet, Emilie

    2015-03-01

    Periodontitis (PD) is a chronic infectious disease mediated by bacteria in the oral cavity. (Poly)phenols (PPs), ubiquitous in plant foods, possess antimicrobial activities and may be useful in the prevention and management of periodontitis. The objective of this study was to test the antibacterial effects of selected PPs on periodontal pathogens, on both planktonic and biofilm modes of growth. Selected PPs (n = 48) were screened against Streptococcus mitis (S. mitis), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), Fusobacterium nucleatum (F. nucleatum) and Porphyromonas gingivalis (P. gingivalis). The antibacterial potential of each compound was evaluated in terms of planktonic minimum inhibitory concentration (PMIC) and planktonic minimum bactericidal concentration (PMBC) using standardized broth microdilution assays. The most active PPs were further tested for their effect on mono-species and multi-species biofilms using a colorimetric resazurin-based viability assay and scanning electron microscopy. Of the 48 PPs tested, 43 showed effective inhibition of planktonic growth of one or more test strains, of which curcumin was the most potent (PMIC range = 7.8-62.5 μg mL(-1)), followed by pyrogallol (PMIC range = 2.4-2500 μg mL(-1)), pyrocatechol (MIC range = 4.9-312.5 μg mL(-1)) and quercetin (PMIC range = 31.2-500 μg mL(-1)). At this concentration, adhesion of curcumin and quercetin to the substrate also inhibited adhesion of S. mitis, and biofilm formation and maturation. While both curcumin and quercetin were able to alter architecture of mature multi-species biofilms, only curcumin-treated biofilms displayed a significantly reduced metabolic activity. Overall, PPs possess antibacterial activities against periodontopathic bacteria in both planktonic and biofilm modes of growth. Further cellular and in vivo studies are necessary to confirm their beneficial activities and potential use in the prevention and or treatment of periodontal

  16. Prevalence of periodontopathogens and Candida spp. in smokers after nonsurgical periodontal therapy - a pilot study.

    PubMed

    Camargo, Gabriela Alessandra da Cruz Galhardo; Abreu, Mariana Gouvêa Latini; Cordeiro, Renata Dos Santos; Wenderoscky, Letícia de Farias; Duque, Cristiane

    2016-01-01

    This pilot study aimed to evaluate the influence of smoking on clinical and microbiological parameters after nonsurgical periodontal therapy. Forty-eight subjects were grouped into smokers (SM, n = 24) and nonsmokers (NS, n = 24) and paired according to gender, age, ethnicity, and periodontal status. Both groups received oral hygiene education and scaling and root planing. Clinical evaluation was performed using plaque index (PI), bleeding on probing (BOP), pocket probing depth (PPD), gingival recession (GR), and clinical attachment level (CAL) before instrumentation (baseline) and at 3 and 6 months. The prevalence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis in subgingival biofilm was determined by polymerase chain reaction. The data were statistically analyzed considering p < 0.05. Clinical conditions improved between baseline and 3 months after periodontal treatment. However, NS had a better clinical response, presenting greater PPD reduction and CAL increase in comparison to SM. Periodontal treatment reduced the levels of P. gingivalis, A. actinomycetemcomitans, and T. forsythia individually after 3 months for the NS group and after 6 months for both groups. The prevalence of Candida species was markedly higher in SM than in NS at all time points evaluated. Periodontopathogens associated or not with C. albicans or C. dubliniensis were more prevalent in SM than in NS at baseline and after 3 months. It was concluded that smoking impairs clinical and microbiological responses to periodontal therapy. Periodontopathogens combined or not with some Candida species are resistant to short-term periodontal therapy in SM. PMID:27556680

  17. Influence of topography and hydrophilicity on initial oral biofilm formation on microstructured titanium surfaces in vitro

    PubMed Central

    Almaguer-Flores, A.; Olivares-Navarrete, R.; Wieland, M.; Ximénez-Fyvie, L. A.; Schwartz, Z.; Boyan, B. D.

    2014-01-01

    Objectives The aim of this study was to analyse the influence of the microtopography and hydrophilicity of titanium (Ti) substrates on initial oral biofilm formation. Materials and methods Nine bacterial species belonging to the normal oral microbiota, including: Aggregatibacter actinomycetemcomitans, Actinomyces israelii, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Parvimonas micra, Porphyromonas gingivalis, Prevotella intermedia, and Streptococcus sanguinis were tested on Ti surfaces: pretreatment (PT [Ra<0.2 μm]), acid-etched (A [Ra<0.8 μm]), A modified to be hydrophilic (modA), sand-blasted/acid-etched (SLA [Ra = 4 μm]), and hydrophilic SLA (modSLA). Disks were incubated for 24 h in anaerobic conditions using a normal culture medium (CM) or human saliva (HS). The total counts of bacteria and the proportion of each bacterial species were analysed by checkerboard DNA–DNA hybridization. Results: Higher counts of bacteria were observed on all surfaces incubated with CM compared with the samples incubated with HS. PT, SLA, and modSLA exhibited higher numbers of attached bacteria in CM, whereas SLA and modSLA had a significant increase in bacterial adhesion in HS. The proportion of the species in the initial biofilms was also influenced by the surface properties and the media used: SLA and modSLA increased the proportion of species like A. actinomycetemcomitans and S. sanguinis in both media, while the adhesion of A. israelii and P. gingivalis on the same surfaces was affected in the presence of saliva. Conclusions The initial biofilm formation and composition were affected by the microtopography and hydrophilicity of the surface and by the media used. PMID:21492236

  18. A protein-repellent and antibacterial nanocomposite for Class-V restorations to inhibit periodontitis-related pathogens.

    PubMed

    Wang, Lin; Xie, Xianju; Imazato, Satoshi; Weir, Michael D; Reynolds, Mark A; Xu, Hockin H K

    2016-10-01

    The objectives of this study were to develop a bioactive dental composite and investigate the effects of 2-methacryloyloxyethyl phosphorylcholine (MPC) and dimethylaminohexadecyl methacrylate (DMAHDM) in Class V composite on mechanical properties, water sorption, protein adsorption, and inhibition of four species of periodontitis-related biofilms for the first time. The resin consisted of ethoxylated bisphenol A dimethacrylate (EBPADMA) and pyromellitic glycerol dimethacrylate (PMGDM). DMAHDM, MPC and nanoparticles of amorphous calcium phosphate (NACP) were incorporated into the resin. Four species (Porphyromonas gingivalis, Prevotella intermedia, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum) were tested for biofilm colony-forming units (CFU), live/dead, metabolic activity, and polysaccharide production. The results showed that adding DMAHDM and MPC to the composite did not compromise the mechanical properties (p>0.1), with acceptable water sorption values. Composite with 3% MPC reduced protein adsorption to 1/9 that of a commercial composite (p<0.05). For all four species, the composite with 3% DMAHDM+3% MPC had much greater reduction in biofilms than using DMAHDM or MPC alone (p<0.05). Biofilm CFU was reduced by about 4 orders of magnitude via 3% DMAHDM+3% MPC, compared to control. The inhibition efficacy for the four species was: P. gingivalis>P intermedia=A. actinomycetemcomitans>F. nucleatum. In conclusion, a novel bioactive composite with 3% DMAHDM and 3% MPC achieved the greatest reduction in biofilm growth, metabolic activity and polysaccharide of four periodontal pathogens. The new composite is promising for Class V restorations especially with subgingival margins to inhibit periodontal pathogens, combat periodontitis and protect the periodontium.

  19. Selected dietary (poly)phenols inhibit periodontal pathogen growth and biofilm formation.

    PubMed

    Shahzad, Muhammad; Millhouse, Emma; Culshaw, Shauna; Edwards, Christine A; Ramage, Gordon; Combet, Emilie

    2015-03-01

    Periodontitis (PD) is a chronic infectious disease mediated by bacteria in the oral cavity. (Poly)phenols (PPs), ubiquitous in plant foods, possess antimicrobial activities and may be useful in the prevention and management of periodontitis. The objective of this study was to test the antibacterial effects of selected PPs on periodontal pathogens, on both planktonic and biofilm modes of growth. Selected PPs (n = 48) were screened against Streptococcus mitis (S. mitis), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), Fusobacterium nucleatum (F. nucleatum) and Porphyromonas gingivalis (P. gingivalis). The antibacterial potential of each compound was evaluated in terms of planktonic minimum inhibitory concentration (PMIC) and planktonic minimum bactericidal concentration (PMBC) using standardized broth microdilution assays. The most active PPs were further tested for their effect on mono-species and multi-species biofilms using a colorimetric resazurin-based viability assay and scanning electron microscopy. Of the 48 PPs tested, 43 showed effective inhibition of planktonic growth of one or more test strains, of which curcumin was the most potent (PMIC range = 7.8-62.5 μg mL(-1)), followed by pyrogallol (PMIC range = 2.4-2500 μg mL(-1)), pyrocatechol (MIC range = 4.9-312.5 μg mL(-1)) and quercetin (PMIC range = 31.2-500 μg mL(-1)). At this concentration, adhesion of curcumin and quercetin to the substrate also inhibited adhesion of S. mitis, and biofilm formation and maturation. While both curcumin and quercetin were able to alter architecture of mature multi-species biofilms, only curcumin-treated biofilms displayed a significantly reduced metabolic activity. Overall, PPs possess antibacterial activities against periodontopathic bacteria in both planktonic and biofilm modes of growth. Further cellular and in vivo studies are necessary to confirm their beneficial activities and potential use in the prevention and or treatment of periodontal

  20. A protein-repellent and antibacterial nanocomposite for Class-V restorations to inhibit periodontitis-related pathogens.

    PubMed

    Wang, Lin; Xie, Xianju; Imazato, Satoshi; Weir, Michael D; Reynolds, Mark A; Xu, Hockin H K

    2016-10-01

    The objectives of this study were to develop a bioactive dental composite and investigate the effects of 2-methacryloyloxyethyl phosphorylcholine (MPC) and dimethylaminohexadecyl methacrylate (DMAHDM) in Class V composite on mechanical properties, water sorption, protein adsorption, and inhibition of four species of periodontitis-related biofilms for the first time. The resin consisted of ethoxylated bisphenol A dimethacrylate (EBPADMA) and pyromellitic glycerol dimethacrylate (PMGDM). DMAHDM, MPC and nanoparticles of amorphous calcium phosphate (NACP) were incorporated into the resin. Four species (Porphyromonas gingivalis, Prevotella intermedia, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum) were tested for biofilm colony-forming units (CFU), live/dead, metabolic activity, and polysaccharide production. The results showed that adding DMAHDM and MPC to the composite did not compromise the mechanical properties (p>0.1), with acceptable water sorption values. Composite with 3% MPC reduced protein adsorption to 1/9 that of a commercial composite (p<0.05). For all four species, the composite with 3% DMAHDM+3% MPC had much greater reduction in biofilms than using DMAHDM or MPC alone (p<0.05). Biofilm CFU was reduced by about 4 orders of magnitude via 3% DMAHDM+3% MPC, compared to control. The inhibition efficacy for the four species was: P. gingivalis>P intermedia=A. actinomycetemcomitans>F. nucleatum. In conclusion, a novel bioactive composite with 3% DMAHDM and 3% MPC achieved the greatest reduction in biofilm growth, metabolic activity and polysaccharide of four periodontal pathogens. The new composite is promising for Class V restorations especially with subgingival margins to inhibit periodontal pathogens, combat periodontitis and protect the periodontium. PMID:27287170

  1. Malva sylvestris Inhibits Inflammatory Response in Oral Human Cells. An In Vitro Infection Model

    PubMed Central

    Benso, Bruna; Rosalen, Pedro Luiz; Alencar, Severino Matias; Murata, Ramiro Mendonça

    2015-01-01

    The aim of this study was to investigate the in vitro anti-inflammatory activity of Malva sylvestris extract (MSE) and fractions in a co-culture model of cells infected by Aggregatibacter actinomycetemcomitans. In addition, we evaluated the phytochemical content in the extract and fractions of M. sylvestris and demonstrated that polyphenols were the most frequent group in all samples studied. An in vitro dual-chamber model to mimic the periodontal structure was developed using a monolayer of epithelial keratinocytes (OBA-9) and a subepithelial layer of fibroblasts (HGF-1). The invasive periodontopathogen A. actinomycetemcomitans (D7S-1) was applied to migrate through the cell layers and induce the synthesis of immune factors and cytokines in the host cells. In an attempt to analyze the antimicrobial properties of MSE and fractions, a susceptibility test was carried out. The extract (MIC 175 μg/mL, MBC 500μg/mL) and chloroform fraction (MIC 150 μg/mL, MBC 250 μg/mL) were found to have inhibitory activity. The extract and all fractions were assessed using a cytotoxicity test and results showed that concentrations under 100 μg/mL did not significantly reduce cell viability compared to the control group (p > 0.05, viability > 90%). In order to analyze the inflammatory response, transcriptional factors and cytokines were quantified in the supernatant released from the cells. The chloroform fraction was the most effective in reducing the bacterial colonization (p< 0.05) and controlling inflammatory mediators, and promoted the down-regulation of genes including IL-1beta, IL-6, IL-10, CD14, PTGS, MMP-1 and FOS as well as the reduction of the IL-1beta, IL-6, IL-8 and GM-CSF protein levels (p< 0.05). Malva sylvestris and its chloroform fraction minimized the A. actinomycetemcomitans infection and inflammation processes in oral human cells by a putative pathway that involves important cytokines and receptors. Therefore, this natural product may be considered as a

  2. FcγRIIB-nt645+25A/G gene polymorphism and periodontitis in Japanese women with preeclampsia.

    PubMed

    Wang, Y; Sugita, N; Kikuchi, A; Iwanaga, R; Hirano, E; Shimada, Y; Sasahara, J; Tanaka, K; Yoshie, H

    2012-12-01

    FcγRIIB contains a unique immunoreceptor tyrosine-based inhibition motif (ITIM) and functions as a negative feedback regulator of leucocyte activation and antibody production. We have previously reported FcγRIIB-nt645+25A/G gene polymorphism to be associated with prevalence and severity of periodontitis, FcγRIIB expression level on peripheral B lymphocytes and the serum IgG level against periodontopathic bacteria. Previous studies have reported maternal periodontal disease to be associated with an increased risk for preeclampsia. Therefore, FcγRIIB-nt645+25A/G gene polymorphism may be associated with preeclampsia by affecting immune response to periodontopathic bacteria in pregnant women. To elucidate whether FcγRIIB-nt645+25A/G gene polymorphism has associations with preeclampsia and/or periodontitis in pregnant Japanese women, a case-control study was carried out on women with preeclampsia (n = 13) and without preeclampsia (n = 106). Maternal periodontal parameters and bacterial data of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in subgingival plaque were collected within 5 days of delivery. FcγR genotypes of each woman were determined using the genomic DNA isolated from peripheral blood. Serum IgG levels specific for each bacteria were determined. There was a significant association between FcγRIIB-nt645+25A/G polymorphism and preeclampsia (P = 0.013). The frequency of the FcγRIIB-nt645+25AA genotype was higher in the preeclampsia group compared with the nonpreeclampsia group (P = 0.007). The DNA level of A. actinomycetemcomitans from subgingival plaque was shown to be higher in the preeclampsia group (P = 0.017). In conclusion, maternal FcγRIIB-nt645+25A/G polymorphism and subgingival DNA level of A. actinomycetemcomitans were significantly associated with the prevalence of preeclampsia in a limited number of Japanese women independently with periodontal infection. Further investigations should be

  3. Anti-inflammatory and wound healing potential of citrus auraptene.

    PubMed

    La, Vu Dang; Zhao, Lei; Epifano, Francesco; Genovese, Salvatore; Grenier, Daniel

    2013-10-01

    Auraptene is the most abundant naturally occurring geranyloxycoumarin. It is primarily isolated from plants in the Rutaceae family, many of which, like citrus fruits, are used as food in many countries. Auraptene is a biologically active secondary metabolite with valuable properties. The aim of our study was to identify novel properties of auraptene with potential for managing periodontal diseases, an inflammatory disease of bacterial origin affecting the tissues surrounding and supporting the teeth. In vitro assays showed that auraptene decreased, in a dose-dependent manner, the secretion of matrix metalloproteinase 2 as well as key inflammatory mediators, including interleukin-6 (IL-6), IL-8, and chemokine (C-C motif) ligand-5 secreted by Aggregatibacter actinomycetemcomitans lipopolysaccharide-stimulated oral epithelial cells. Using gingival fibroblasts, auraptene showed a significant (P<.05) wound healing effect by its capacity to increase cell migration. In conclusion, auraptene shows promise for promoting wound healing and controlling periodontal diseases through its capacity to interfere with inflammatory mediator secretion.

  4. Cytolethal Distending Toxin Family Members Are Differentially Affected by Alterations in Host Glycans and Membrane Cholesterol*

    PubMed Central

    Eshraghi, Aria; Maldonado-Arocho, Francisco J.; Gargi, Amandeep; Cardwell, Marissa M.; Prouty, Michael G.; Blanke, Steven R.; Bradley, Kenneth A.

    2010-01-01

    Cytolethal distending toxins (CDTs) are tripartite protein exotoxins produced by a diverse group of pathogenic Gram-negative bacteria. Based on their ability to induce DNA damage, cell cycle arrest, and apoptosis of cultured cells, CDTs are proposed to enhance virulence by blocking cellular division and/or directly killing epithelial and immune cells. Despite the widespread distribution of CDTs among several important human pathogens, our understanding of how these toxins interact with host cells is limited. Here we demonstrate that CDTs from Haemophilus ducreyi, Aggregatibacter actinomycetemcomitans, Escherichia coli, and Campylobacter jejuni differ in their abilities to intoxicate host cells with defined defects in host factors previously implicated in CDT binding, including glycoproteins, and glycosphingolipids. The absence of cell surface sialic acid sensitized cells to intoxication by three of the four CDTs tested. Surprisingly, fucosylated N-linked glycans and glycolipids, previously implicated in CDT-host interactions, were not required for intoxication by any of the CDTs tested. Finally, altering host-cellular cholesterol, also previously implicated in CDT binding, affected intoxication by only a subset of CDTs tested. The findings presented here provide insight into the molecular and cellular basis of CDT-host interactions. PMID:20385557

  5. Essential Oil from Berries of Lebanese Juniperus excelsa M. Bieb Displays Similar Antibacterial Activity to Chlorhexidine but Higher Cytocompatibility with Human Oral Primary Cells.

    PubMed

    Azzimonti, Barbara; Cochis, Andrea; Beyrouthy, Marc El; Iriti, Marcello; Uberti, Francesca; Sorrentino, Rita; Landini, Manuela Miriam; Rimondini, Lia; Varoni, Elena Maria

    2015-05-21

    Chlorhexidine (CHX), one of the most effective drugs administered for periodontal treatment, presents collateral effects including toxicity when used for prolonged periods; here, we have evaluated the bactericidal potency and the cytocompatibility of Juniperus excelsa M. Bieb essential oil (EO) in comparison with 0.05% CHX. The EO was extracted from berries by hydrodistillation and components identified by gas chromatography and mass spectrometry. Bacterial inhibition halo analysis, quantitative cell viability 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenyl amino) carbonyl]-2H-tetrazolium hydroxide assay (XTT), and colony forming unit (CFU) count were evaluated against the two biofilm formers Aggregatibacter actinomycetemcomitans and Streptococcus mutans. Finally, cytocompatibility was assessed with human primary gingival fibroblasts (HGF) and mucosal keratinocytes (HK). The resulting EO was mainly composed of monoterpene hydrocarbons and oxygenated monoterpenes. An inhibition halo test demonstrated that both bacteria were sensitive to the EO; XTT analysis and CFU counts confirmed that 10-fold-diluted EO determined a statistically significant (p < 0.05) reduction in bacteria count and viability towards both biofilm and planktonic forms in a comparable manner to those obtained with CHX. Moreover, EO displayed higher cytocompatibility than CHX (p < 0.05). In conclusion, EO exhibited bactericidal activity similar to CHX, but a superior cytocompatibility, making it a promising antiseptic alternative to CHX.

  6. Total Antioxidant Capacity and Total Oxidant Status in Saliva of Periodontitis Patients in Relation to Bacterial Load

    PubMed Central

    Zhang, Taowen; Andrukhov, Oleh; Haririan, Hady; Müller-Kern, Michael; Liu, Shutai; Liu, Zhonghao; Rausch-Fan, Xiaohui

    2016-01-01

    The detection of salivary biomarkers has a potential application in early diagnosis and monitoring of periodontal inflammation. However, searching sensitive salivary biomarkers for periodontitis is still ongoing. Oxidative stress is supposed to play an important role in periodontitis progression and tissue destruction. In this cross-sectional study, we investigated total antioxidant capacity (TAC) and total oxidant status (TOS) in saliva of periodontitis patients compared to healthy controls and their relationship with periodontopathic bacteria and periodontal disease severity. Unstimulated saliva was collected from 45 patients with generalized severe periodontitis and 37 healthy individuals and the TAC/TOS were measured. In addition, salivary levels of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Fusobacterium nucleatum in saliva were measured. Salivary TAC was lower in periodontitis patients compared to healthy controls. Moreover, a significant negative correlation of salivary TAC with clinical attachment loss was observed in periodontitis patients. No significant difference in the salivary TOS was observed between periodontitis patients and healthy controls. Bacterial load was enhanced in periodontitis patients and exhibited correlation with periodontal disease severity but not with salivary TAC/TOS. Our data suggest that changes in antioxidant capacity in periodontitis patients are not associated with increased bacterial load and are probably due to a dysregulated immune response. PMID:26779448

  7. Detection of five potentially periodontal pathogenic bacteria in peri-implant disease: A comparison of PCR and real-time PCR.

    PubMed

    Schmalz, Gerhard; Tsigaras, Sandra; Rinke, Sven; Kottmann, Tanja; Haak, Rainer; Ziebolz, Dirk

    2016-07-01

    The aim of this study was to compare the microbial analysis methods of polymerase chain reaction (PCR) and real-time PCR (RT-PCR) in terms of detection of five selected potentially periodontal pathogenic bacteria in peri-implant disease. Therefore 45 samples of healthy, mucositis and peri-implantitis (n = 15 each) were assessed according to presence of the following bacteria using PCR (DNA-strip technology) and RT-PCR (fluorescent dye SYBR green-system): Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tanerella forsythia (Tf), and Fusobacterium nucleatum (Fn). There were no significant correlations between the bacterial and disease patterns, so the benefit of using microbiological tests for the diagnosis of peri-implant diseases is questionable. Correlations between the methods were highest for Tf (Kendall's Tau: 0.65, Spearman: 0.78), Fn (0.49, 0.61) and Td (0.49, 0.59). For Aa (0.38, 0.42) and Pg (0.04, 0.04), lower correlation values were detected. Accordingly, conventional semi-quantitative PCR seems to be sufficient for analyzing potentially periodontal pathogenic bacterial species.

  8. Implants with internal hexagon and conical implant-abutment connections: an in vitro study of the bacterial contamination.

    PubMed

    D'Ercole, Simonetta; Scarano, Antonio; Perrotti, Vittoria; Mulatinho, Jorge; Piattelli, Adriano; Iezzi, Giovanna; Tripodi, Domenico

    2014-02-01

    Prevention of microbial leakage at the implant-abutment junction is a major challenge for the construction of 2-stage implants in order to minimize inflammatory reactions and to maximize bone stability at the implant neck. The aim of the present in vitro study was an evaluation of the leakage observed over a period of 28 days in Cone Morse taper internal connections and in screwed-abutments connections. In the present study 10 specimens of Cone Morse (Group 1) and 10 of internal hexagon (Group 2) implants were used. The inner parts of 5 implants per group were inoculated with Pseudomonas aeruginosa (PS) suspension and 5 implants per group with Aggregatibacter actinomycetemcomitans (AA). The possible penetration of bacterial suspension into the surrounding solution was determined by the observation of turbidity of the broth. In Group 1, bacterial contamination was found in 3 out of 5 implant-abutment assemblies seeded with the PS and in 2 samples out of 5 in the assemblies seeded with AA, with a total of leaked assemblies in this group of 5 out of 10. In Group 2, bacterial contamination was found in 4 out of 5 implant-abutment assemblies seeded with the PS, and in 4 out of 5 samples seeded with AA, with a total of leaked assemblies of 8 out of 10. The present data confirm the reported high permeability to bacterial leakage of screw-retained abutment connections, and the lower infiltration rates-although not significantly-of Cone Morse taper internal connections.

  9. Relationship of Clinical and Microbiological Variables in Patients with Type 1 Diabetes Mellitus and Periodontitis

    PubMed Central

    Sakalauskiene, Jurgina; Kubilius, Ricardas; Gleiznys, Alvydas; Vitkauskiene, Astra; Ivanauskiene, Egle; Šaferis, Viktoras

    2014-01-01

    Background The aim of the study was to analyze how metabolic control of type 1 diabetes is related to clinical and microbiological periodontal parameters. Material/Methods The study involved 56 subjects aged from 19 to 50 years divided into 2 groups: healthy subjects (the H group), and diabetic (type 1 diabetes) patients with chronic untreated generalized periodontitis (the DM group). The glycosylated hemoglobin value (HbA1c) was determined using the UniCel DxC 800 SYNCHRON System (Beckman Coulter, USA), and the concentration in blood was measured by the turbidimetric immunoinhibition method. A molecular genetic assay (Micro-IDent plus, Germany) was used to detect periodontopathogenic bacteria in plaque samples. Periodontitis was confirmed by clinical and radiological examination. Results Fusobacterium nucleatum, Capnocytophaga species, and Eikenella corrodens were the most frequently found bacteria in dental plaque samples (77.8%, 66.7%, and 33.4%, respectively), whereas Aggregatibacter actinomycetemcomitans was identified 40.7% less frequently in the DM group than in the H group. The strongest relationship was observed between the presence of 2 periodontal pathogens – F. nucleatum and Capnocytophaga spp. – and poorer metabolic control in type 1 diabetes patients (HbA1c) and all clinical parameters of periodontal pathology. Conclusions Periodontal disease was more evident in type 1 diabetic patients, and the prevalence of periodontitis was greatly increased in subjects with poorer metabolic control. PMID:25294115

  10. In vitro antimicrobial activity of Brazilian medicinal plant extracts against pathogenic microorganisms of interest to dentistry.

    PubMed

    Pereira, Elizete Maria; Gomes, Rafael Tomaz; Freire, Natália Ribeiro; Aguiar, Evandro Guimarães; Brandão, Maria das Graças Lins; Santos, Vagner Rodrigues

    2011-03-01

    This study evaluated the susceptibility of oral pathogenic microorganisms Candida albicans, Streptococcus mutans, Staphylococcus aureus, and Aggregatibacter actinomycetemcomitans to Brazilian medicinal plant extracts of Schinus terebinthifolius (aroeira), Croton campestris (velame), Lafoensia pacari (pacari), Centaurium erythraea (centáurea), Stryphnodendron adstringens (barbatimão), and Anacardium humile (cajuzinho-docerrado), as compared to standardized antimicrobial agents (nystatin, chloramphenicol and tetracycline hydrochloride). Ethanol, hexane and butane fractions from stem barks, rinds, leaves, and/or roots were extracted and tested. Antimicrobial diffusion agar test and MIC were performed according to CLSI. After 24 h of incubation at 37 °C, the diameter of inhibition zones and spectrophotometer readings were measured and compared. The results were reported as means ± standard deviation (M ± SD). With the exception of five extracts that showed no antimicrobial activity, all the extracts tested showed antimicrobial activity, in different levels. This study suggests that extracts from the plants tested could be an alternative therapeutic option for infectious conditions of the oral cavity, such as denture stomatitis, dental caries, and periodontitis.

  11. Differentiation of oral bacteria in in vitro cultures and human saliva by secondary electrospray ionization - mass spectrometry

    NASA Astrophysics Data System (ADS)

    Bregy, Lukas; Müggler, Annick R.; Martinez-Lozano Sinues, Pablo; García-Gómez, Diego; Suter, Yannick; Belibasakis, Georgios N.; Kohler, Malcolm; Schmidlin, Patrick R.; Zenobi, Renato

    2015-10-01

    The detection of bacterial-specific volatile metabolites may be a valuable tool to predict infection. Here we applied a real-time mass spectrometric technique to investigate differences in volatile metabolic profiles of oral bacteria that cause periodontitis. We coupled a secondary electrospray ionization (SESI) source to a commercial high-resolution mass spectrometer to interrogate the headspace from bacterial cultures and human saliva. We identified 120 potential markers characteristic for periodontal pathogens Aggregatibacter actinomycetemcomitans (n = 13), Porphyromonas gingivalis (n = 70), Tanerella forsythia (n = 30) and Treponema denticola (n = 7) in in vitro cultures. In a second proof-of-principle phase, we found 18 (P. gingivalis, T. forsythia and T. denticola) of the 120 in vitro compounds in the saliva from a periodontitis patient with confirmed infection with P. gingivalis, T. forsythia and T. denticola with enhanced ion intensity compared to two healthy controls. In conclusion, this method has the ability to identify individual metabolites of microbial pathogens in a complex medium such as saliva.

  12. Total Antioxidant Capacity and Total Oxidant Status in Saliva of Periodontitis Patients in Relation to Bacterial Load.

    PubMed

    Zhang, Taowen; Andrukhov, Oleh; Haririan, Hady; Müller-Kern, Michael; Liu, Shutai; Liu, Zhonghao; Rausch-Fan, Xiaohui

    2015-01-01

    The detection of salivary biomarkers has a potential application in early diagnosis and monitoring of periodontal inflammation. However, searching sensitive salivary biomarkers for periodontitis is still ongoing. Oxidative stress is supposed to play an important role in periodontitis progression and tissue destruction. In this cross-sectional study, we investigated total antioxidant capacity (TAC) and total oxidant status (TOS) in saliva of periodontitis patients compared to healthy controls and their relationship with periodontopathic bacteria and periodontal disease severity. Unstimulated saliva was collected from 45 patients with generalized severe periodontitis and 37 healthy individuals and the TAC/TOS were measured. In addition, salivary levels of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Fusobacterium nucleatum in saliva were measured. Salivary TAC was lower in periodontitis patients compared to healthy controls. Moreover, a significant negative correlation of salivary TAC with clinical attachment loss was observed in periodontitis patients. No significant difference in the salivary TOS was observed between periodontitis patients and healthy controls. Bacterial load was enhanced in periodontitis patients and exhibited correlation with periodontal disease severity but not with salivary TAC/TOS. Our data suggest that changes in antioxidant capacity in periodontitis patients are not associated with increased bacterial load and are probably due to a dysregulated immune response.

  13. Microarray Analysis of Microbiota of Gingival Lesions in Noma Patients

    PubMed Central

    Huyghe, Antoine; François, Patrice; Mombelli, Andrea; Tangomo, Manuela; Girard, Myriam; Baratti-Mayer, Denise; Bolivar, Ignacio; Pittet, Didier; Schrenzel, Jacques

    2013-01-01

    Noma (cancrum oris) is a gangrenous disease of unknown etiology affecting the maxillo-facial region of young children in extremely limited resource countries. In an attempt to better understand the microbiological events occurring during this disease, we used phylogenetic and low-density microarrays targeting the 16S rRNA gene to characterize the gingival flora of acute noma and acute necrotizing gingivitis (ANG) lesions, and compared them to healthy control subjects of the same geographical and social background. Our observations raise doubts about Fusobacterium necrophorum, a previously suspected causative agent of noma, as this species was not associated with noma lesions. Various oral pathogens were more abundant in noma lesions, notably Atopobium spp., Prevotella intermedia, Peptostreptococcus spp., Streptococcus pyogenes and Streptococcus anginosus. On the other hand, pathogens associated with periodontal diseases such as Aggregatibacter actinomycetemcomitans, Capnocytophaga spp., Porphyromonas spp. and Fusobacteriales were more abundant in healthy controls. Importantly, the overall loss of bacterial diversity observed in noma samples as well as its homology to that of ANG microbiota supports the hypothesis that ANG might be the immediate step preceding noma. PMID:24086784

  14. Staphylococcus epidermidis Biofilms: Functional Molecules, Relation to Virulence, and Vaccine Potential

    NASA Astrophysics Data System (ADS)

    Mack, Dietrich; Davies, Angharad P.; Harris, Llinos G.; Knobloch, Johannes K. M.; Rohde, Holger

    Medical device-associated infections, most frequently caused by Staphylococcus epidermidis and Staphylococcus aureus, are of increasing importance in modern medicine. The formation of adherent, multilayered bacterial biofilms is crucial in the pathogenesis of these infections. Polysaccharide intercellular adhesin (PIA), a homoglycan of β-1,6-linked 2-acetamido-2-deoxy-d-glucopyranosyl residues, of which about 15% are non-N-acetylated, is central to biofilm accumulation in staphylococci. It transpires that polysaccharides - structurally very similar to PIA - are also key to biofilm formation in a number of other organisms including the important human pathogens Escherichia coli, Aggregatibacter (Actinobacillus) actinomycetemcomitans, Yersinia pestis, and Bordetella spp. Apparently, synthesis of PIA and related polysaccharides is a general feature important for biofilm formation in diverse bacterial genera. Current knowledge about the structure and biosynthesis of PIA and related polysaccharides is reviewed. Additionally, information on their role in pathogenesis of biomaterial-related and other type of infections and the potential use of PIA and related compounds for prevention of infection is evaluated.

  15. Comparison of the detection of periodontal pathogens in bacteraemia after tooth brushing by culture and molecular techniques

    PubMed Central

    Figuero, Elena; González, Itziar; O´Connor, Ana; Diz, Pedro; Álvarez, Maximiliano; Herrera, David; Sanz, Mariano

    2016-01-01

    Background The prevalence and amounts of periodontal pathogens detected in bacteraemia samples after tooth brushing-induced by means of four diagnostic technique, three based on culture and one in a molecular-based technique, have been compared in this study. Material and Methods Blood samples were collected from thirty-six subjects with different periodontal status (17 were healthy, 10 with gingivitis and 9 with periodontitis) at baseline and 2 minutes after tooth brushing. Each sample was analyzed by three culture-based methods [direct anaerobic culturing (DAC), hemo-culture (BACTEC), and lysis-centrifugation (LC)] and one molecular-based technique [quantitative polymerase chain reaction (qPCR)]. With culture any bacterial isolate was detected and quantified, while with qPCR only Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were detected and quantified. Descriptive analyses, ANOVA and Chi-squared tests, were performed. Results Neither BACTEC nor qPCR detected any type of bacteria in the blood samples. Only LC (2.7%) and DAC (8.3%) detected bacteraemia, although not in the same patients. Fusobacterium nucleatum was the most frequently detected bacterial species. Conclusions The disparity in the results when the same samples were analyzed with four different microbiological detection methods highlights the need for a proper validation of the methodology to detect periodontal pathogens in bacteraemia samples, mainly when the presence of periodontal pathogens in blood samples after tooth brushing was very seldom. Key words:Bacteraemia, periodontitis, culture, PCR, tooth brushing. PMID:26946197

  16. Antibiofilm activity of Actinobacillus pleuropneumoniae serotype 5 capsular polysaccharide.

    PubMed

    Karwacki, Michael T; Kadouri, Daniel E; Bendaoud, Meriem; Izano, Era A; Sampathkumar, Vandana; Inzana, Thomas J; Kaplan, Jeffrey B

    2013-01-01

    Cell-free extracts isolated from colony biofilms of Actinobacillus pleuropneumoniae serotype 5 were found to inhibit biofilm formation by Staphylococcus aureus, S. epidermidis and Aggregatibacter actinomycetemcomitans, but not by A. pleuropneumoniae serotype 5 itself, in a 96-well microtiter plate assay. Physical and chemical analyses indicated that the antibiofilm activity in the extract was due to high-molecular-weight polysaccharide. Extracts isolated from a mutant strain deficient in the production of serotype 5 capsular polysaccharide did not exhibit antibiofilm activity. A plasmid harboring the serotype 5 capsule genes restored the antibiofilm activity in the mutant extract. Purified serotype 5 capsular polysaccharide also exhibited antibiofilm activity against S. aureus. A. pleuropneumoniae wild-type extracts did not inhibit S. aureus growth, but did inhibit S. aureus intercellular adhesion and binding of S. aureus cells to stainless steel surfaces. Furthermore, polystyrene surfaces coated with A. pleuropneumoniae wild-type extracts, but not with capsule-mutant extracts, resisted S. aureus biofilm formation. Our findings suggest that the A. pleuropneumoniae serotype 5 capsule inhibits cell-to-cell and cell-to-surface interactions of other bacteria. A. pleuropneumoniae serotype 5 capsular polysaccharide is one of a growing number of bacterial polysaccharides that exhibit broad-spectrum, nonbiocidal antibiofilm activity. Future studies on these antibiofilm polysaccharides may uncover novel functions for bacterial polysaccharides in nature, and may lead to the development of new classes of antibiofilm agents for industrial and clinical applications. PMID:23691104

  17. Antibiofilm Activity of Actinobacillus pleuropneumoniae Serotype 5 Capsular Polysaccharide

    PubMed Central

    Karwacki, Michael T.; Kadouri, Daniel E.; Bendaoud, Meriem; Izano, Era A.; Sampathkumar, Vandana; Inzana, Thomas J.; Kaplan, Jeffrey B.

    2013-01-01

    Cell-free extracts isolated from colony biofilms of Actinobacillus pleuropneumoniae serotype 5 were found to inhibit biofilm formation by Staphylococcus aureus, S. epidermidis and Aggregatibacter actinomycetemcomitans, but not by A. pleuropneumoniae serotype 5 itself, in a 96-well microtiter plate assay. Physical and chemical analyses indicated that the antibiofilm activity in the extract was due to high-molecular-weight polysaccharide. Extracts isolated from a mutant strain deficient in the production of serotype 5 capsular polysaccharide did not exhibit antibiofilm activity. A plasmid harboring the serotype 5 capsule genes restored the antibiofilm activity in the mutant extract. Purified serotype 5 capsular polysaccharide also exhibited antibiofilm activity against S. aureus. A. pleuropneumoniae wild-type extracts did not inhibit S. aureus growth, but did inhibit S. aureus intercellular adhesion and binding of S. aureus cells to stainless steel surfaces. Furthermore, polystyrene surfaces coated with A. pleuropneumoniae wild-type extracts, but not with capsule-mutant extracts, resisted S. aureus biofilm formation. Our findings suggest that the A. pleuropneumoniae serotype 5 capsule inhibits cell-to-cell and cell-to-surface interactions of other bacteria. A. pleuropneumoniae serotype 5 capsular polysaccharide is one of a growing number of bacterial polysaccharides that exhibit broad-spectrum, nonbiocidal antibiofilm activity. Future studies on these antibiofilm polysaccharides may uncover novel functions for bacterial polysaccharides in nature, and may lead to the development of new classes of antibiofilm agents for industrial and clinical applications. PMID:23691104

  18. Prevalence of fimA genotypes of Porphyromonas gingivalis and other periodontal bacteria in a Spanish population with chronic periodontitis

    PubMed Central

    Puig-Silla, Miriam; Dasí-Fernánde, Francisco; Montiel-Company, José-María

    2012-01-01

    Objectives: The aim of this study was to determine the prevalence of the different fimA genotypes of Porphyromonas gingivalis in adult Spanish patients with chronic periodontitis, patients with gingivitis and periodontally healthy subjects, and the relationship between these genotypes and other periodontopathogenic bacteria. Study design: Samples of subgingival plaque were taken from 86 patients (33 with chronic periodontitis, 16 with gingivitis, and 37 periodontally healthy) in the course of a full periodontal examination. PCR was employed to determine the presence of the 6 fimA genotypes of Porphyromonas gingivalis (I-V and Ib) and of Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola. Results: Porphyromonas gingivalis fimA genotypes II and Ib were present in significantly higher percentages in periodontal patients (39.4% and 12.1% respectively) than in healthy or gingivitis subjects. The prevalence of Tannerella forsythia, Treponema denticola, and Porphyromonas gingivalis fimA genotype IV was significantly higher in the group that presented bleeding greater than 30%. A positive correlation was found between Porphyromonas gingivalis fimA genotype IV and Treponema denticola. Conclusions: A strong association between Porphyromonas gingivalis fimA genotypes II and Ib and chronic periodontitis exists in the Spanish population. The most prevalent genotype in periodontal patients is II. Key words:Periodontitis, Porphyromonas gingivalis, fimA genotype, periodontal bacteria, polymerase chain reaction. PMID:22549664

  19. Scaling and root planning, and locally delivered minocycline reduces the load of Prevotella intermedia in an interdependent pattern, correlating with symptomatic improvements of chronic periodontitis: a short-term randomized clinical trial

    PubMed Central

    Deng, Shuli; Wang, Ying; Sun, Wei; Chen, Hui; Wu, Gang

    2015-01-01

    Background To evaluate the respective or combinatory efficacy of locally delivered 2% minocycline (MO), and scaling and root planning (SRP) by assessing both clinical parameters and the loads of four main periodontal pathogens in treating chronic periodontitis (CP). Methods Seventy adults with CP were randomly assigned to the three treatment groups: 1) SRP alone; 2) MO alone; and 3) combinatory use of SRP and MO (SRP + MO). Before and 7 days after the treatments, we evaluated both clinical parameters (pocket depth [PD] and sulcus bleeding index [SBI]) and the gene load of four main periodontal pathogens (Aggregatibacter actinomycetemcomitans [Aa], Fusobacterium nucleatum [Fn], Porphyromonas gingivalis [Pg], and Prevotella intermedia [Pi]). Results The bacterial prevalence per patient was: Aa, 31.25%; Fn, 100%; Pg, 95.31%; and Pi, 98.44%. Seven days after treatment, the three treatments significantly reduced both PD and SBI, but not detection frequencies of the four pathogens. For PD, the reduction efficacy of SRP + MO was significantly higher than that of either MO or SRP. Only Pg responded significantly to SRP. Pg and Fn were significantly reduced in the presence of MO. Only SRP + MO showed a significant reduction effect on the gene load of Pi. The reduction of PD significantly correlated with the gene load of Pi (r=0.26; P=0.042) but not of the other bacteria. Conclusion SRP and MO reduced the load of Pi in an interdependent pattern, which correlated with symptomatic improvements of CP. PMID:26676022

  20. Bacterial fight-and-flight responses enhance virulence in a polymicrobial infection.

    PubMed

    Stacy, Apollo; Everett, Jake; Jorth, Peter; Trivedi, Urvish; Rumbaugh, Kendra P; Whiteley, Marvin

    2014-05-27

    The oral pathogen Aggregatibacter actinomycetemcomitans (Aa) resides in infection sites with many microbes, including commensal streptococci such as Streptococcus gordonii (Sg). During infection, Sg promotes the virulence of Aa by producing its preferred carbon source, l-lactate, a phenomenon referred to as cross-feeding. However, as with many streptococci, Sg also produces high levels of the antimicrobial hydrogen peroxide (H2O2), leading to the question of how Aa deals with this potent antimicrobial during coinfection. Here, we show that Aa possesses two complementary responses to H2O2: a detoxification or fight response mediated by catalase (KatA) and a dispersion or flight response mediated by Dispersin B (DspB), an enzyme that dissolves Aa biofilms. Using a murine abscess infection model, we show that both of these responses are required for Sg to promote Aa virulence. Although the role of KatA is to detoxify H2O2 during coinfection, 3D spatial analysis of mixed infections revealed that DspB is required for Aa to spatially organize itself at an optimal distance (>4 µm) from Sg, which we propose allows cross-feeding but reduces exposure to inhibitory levels of H2O2. In addition, these behaviors benefit not only Aa but also Sg, suggesting that fight and flight stimulate the fitness of the community. These results reveal that an antimicrobial produced by a human commensal bacterium enhances the virulence of a pathogenic bacterium by modulating its spatial location in the infection site.

  1. Efficacy of taurolidine against periodontopathic species--an in vitro study.

    PubMed

    Eick, Sigrun; Radakovic, Sabrina; Pfister, Wolfgang; Nietzsche, Sandor; Sculean, Anton

    2012-06-01

    The antimicrobial effect of taurolidine was tested against periodontopathic species in comparison to chlorhexidine digluconate in the presence or absence of serum. Minimal inhibitory concentrations (MIC), microbiocidal concentrations (MBC), as well as killing were determined against 32 different microbial strains including 3 Porphyromonas gingivalis, 3 Aggregatibacter actinomycetemcomitans, and 15 potentially superinfecting species with and without 25% v/v human serum. The MIC(50) of taurolidine against the tested microbial strains was 0.025% and the MIC(90) 0.05%. The respective values for the MBCs were 0.05% and 0.1%. Addition of 25% serum (heat-inactivated) did not change the MIC and MBC values of taurolidine. In contrast, MICs and MBCs of chlorhexidine (CHX) increased by two steps after addition of serum. Taurolidine killed microorganisms in a concentration and time-dependent manner, the killing rate of 1.6% taurolidine was 99.08% ± 2.27% in mean after 2 h. Again, killing activity of taurolidine was not affected if serum was added, whereas addition of inactivated serum clearly reduced the killing rate of all selected bacterial strains by CHX. Therefore, taurolidine possesses antimicrobial properties which are not reduced in the presence of serum as a main component in gingival crevicular fluid and wound fluid. Taurolidine may have potential as an antimicrobial agent in non-surgical and surgical periodontal treatment.

  2. Longitudinal study on clinical and microbial analysis of periodontal status in pregnancy.

    PubMed

    Machado, Fernanda Campos; Cesar, Dionéia Evangelista; Apolônio, Ana Carolina Morais; Ribeiro, Luiz Claudio; Ribeiro, Rosangela Almeida

    2016-01-01

    This study was aimed to provide a longitudinal overview of the subgingival bacterial microbiome using fluorescence in situ hybridization (FISH) technique, in women in the second trimester of pregnancy (between 14 and 24 weeks), and 48 h and 8 weeks postpartum. Of 31 women evaluated during pregnancy, 24 returned for the 48-h and 18 for their 8-week exams postpartum. Probing depth (PD), bleeding on probing, clinical attachment level, and presence of calculus were recorded. Subgingival plaque samples were collected, and FISH was used to identify the numbers of eight periodontal pathogens. Friedman test was used to compare differences between follow-up examinations, followed by a multiple comparison test for a post hoc pairwise comparison. Clinically, a significantly greater number of teeth with PD = 4-5 mm were found during pregnancy than on postpartum examinations. Microbial analysis showed a statistically significant decrease in cell count over the study period for Prevotella nigrescens. P. intermedia, Campylobacter rectus, and Porphyromonas gingivalis also decrease, although not significantly, and Aggregatibacter actinomycetemcomitans increased. No significant changes were found for Fusobacterium nucleatum, Treponema denticola, or Tannerella forsythia. Our data demonstrate a change in the subgingival microbiota during pregnancy, at least for P. nigrescens. PMID:27556678

  3. Evaluation of the efficacy of a new oral gel containing carvacrol and thymol for home oral care in the management of chronic periodontitis using PCR analysis: a microbiological pilot study.

    PubMed

    Lauritano, D; Pazzi, D; Iapichino, A; Gaudio, R M; Di Muzio, M; Lo Russo, L; Pezzetti, F

    2016-01-01

    The use of chemical devices for domestic oral hygiene in periodontal patients has led to new treatment strategies aiming primarily at a control of infection. Over the last few years, carvacrol and thymol (CT) have been subjected to many scientific and medical studies. The purpose of the present study was to assess the effect of CT on the red complex bacteria using Polymerase Chain Reaction (PCR) for microbiological analysis. Five patients with a diagnosis of chronic periodontitis in the age group >25 years, were selected. None of these patients had received any surgical or non-surgical periodontal therapy and demonstrated radiographic evidence of moderate bone loss. After scaling and root planning, patients received a CT gel to be used at home. Four non-adjacent sites in separate quadrants were selected in each patient for monitoring, based on criteria that the sites localize chronic periodontitis. Microbial analysis (MA) was analyzed at baseline and at day 15. SPSS program was used for statistical purposes and a paired samples correlation was performed at the end of the observation period. Although an absolute reduction was observed among the studied bacteria (i.e. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Fusobacterium nucleatum, Campylobacter rectus and Total bacteria loading) none reach a statistical significant value. The present study demonstrated that CT gel has a small impact on oral biofilm. Additional studies are needed to detect the efficacy of CT gel. PMID:27469559

  4. Bacterial load of periodontal pathogens among italian patients with chronic periodontitis: a comparative study of three different areas.

    PubMed

    Lauritano, D; Martinelli, M; Mucchi, D; Palmieri, A; Lo Muzio, L; Carinci, F

    2016-01-01

    The aim of the present study was to evaluate the mean bacterial load of some periodontal pathogenic bacteria in Italian patients affected by chronic periodontitis. The sample consisted of 1,762 patients with a clinical diagnosis of chronic periodontitis based on the criteria of the American Academy of Periodontology sampled in the period 2013-2015; 1,323 patients were from Northern Italy, 317 from Central Italy and 122 from Southern Italy. Samples for microbiological analysis were collected from the four sites of the greatest probing depth in each patient and then processed by quantitative polymerase chain reaction. Periodontal pathogens have the following percentage respect to total bacteria load: Aggregatibacter actinomycetemcomitans 0.1%, Campylobacter rectus 2%, Fusobacterium nucleatum 8%, Porphyromonas gingivalis 6%, Treponema denticola 2% and Tannerella forsythia 1.5%. There are significant differences in bacterial load among the different geographical areas both for the total bacterial and for the single species. The results of our study in this Italian population showed that a different geographic distribution exists among periodontal pathogens. We hypothesize that these differences in bacterial load could be related to genetic and environmental factors. Additional studies are necessary to confirm these data and to get more insight on additional factors, which may play a role in periodontal pathogens in different geographic areas. PMID:27469562

  5. Comparison of Riboflavin and Toluidine Blue O as Photosensitizers for Photoactivated Disinfection on Endodontic and Periodontal Pathogens In Vitro.

    PubMed

    Nielsen, Henrik Krarup; Garcia, Javier; Væth, Michael; Schlafer, Sebastian

    2015-01-01

    Photoactivated disinfection has a strong local antimicrobial effect. In the field of dentistry it is an emerging adjunct to mechanical debridement during endodontic and periodontal treatment. In the present study, we investigate the effect of photoactivated disinfection using riboflavin as a photosensitizer and blue LED light for activation, and compare it to photoactivated disinfection with the widely used combination of toluidine blue O and red light. Riboflavin is highly biocompatible and can be activated with LED lamps at hand in the dental office. To date, no reports are available on the antimicrobial effect of photoactivated disinfection using riboflavin/blue light on oral microorganisms. Planktonic cultures of eight organisms frequently isolated from periodontal and/or endodontic lesions (Aggregatibacter actinomycetemcomitans, Candida albicans, Enterococcus faecalis, Escherischia coli, Lactobacillus paracasei, Porphyromonas gingivalis, Prevotella intermedia and Propionibacterium acnes) were subjected to photoactivated disinfection with riboflavin/blue light and toluidine blue O/red light, and survival rates were determined by CFU counts. Within the limited irradiation time of one minute, photoactivated disinfection with riboflavin/blue light only resulted in minor reductions in CFU counts, whereas full kills were achieved for all organisms when using toluidine blue O/red light. The black pigmented anaerobes P. gingivalis and P. intermedia were eradicated completely by riboflavin/blue light, but also by blue light treatment alone, suggesting that endogenous chromophores acted as photosensitizers in these bacteria. On the basis of our results, riboflavin cannot be recommended as a photosensitizer used for photoactivated disinfection of periodontal or endodontic infections.

  6. A Journey of Cytolethal Distending Toxins through Cell Membranes

    PubMed Central

    Boesze-Battaglia, Kathleen; Alexander, Desiree; Dlakić, Mensur; Shenker, Bruce J.

    2016-01-01

    The multifunctional role of lipids as structural components of membranes, signaling molecules, and metabolic substrates makes them an ideal partner for pathogens to hijack host cell processes for their own survival. The properties and composition of unique membrane micro-domains such as membrane rafts make these regions a natural target for pathogens as it affords them an opportunity to hijack cell signaling and intracellular trafficking pathways. Cytolethal distending toxins (Cdts), members of the AB2 family of toxins are comprised of three subunits, the active, CdtB unit, and the binding, CdtA-CdtC unit. Cdts are cyclomodulins leading to cell cycle arrest and apoptosis in a wide variety of cell types. Cdts from several species share a requirement for membrane rafts, and often cholesterol specifically for cell binding and CdtB mediated cytotoxicity. In this review we focus on how host–cell membrane bilayer organization contributes to the cell surface association, internalization, and action of bacteria derived cytolethal distending toxins (Cdts), with an emphasis on Aggregatibacter actinomycetemcomitans Cdt. PMID:27559534

  7. Oxidative Stress Parameters in Saliva and Its Association with Periodontal Disease and Types of Bacteria

    PubMed Central

    Almerich-Silla, Jose Manuel; Montiel-Company, Jose María; Pastor, Sara; Serrano, Felipe; Puig-Silla, Miriam; Dasí, Francisco

    2015-01-01

    Objective. To determine the association between oxidative stress parameters with periodontal disease, bleeding, and the presence of different periodontal bacteria. Methods. A cross-sectional study in a sample of eighty-six patients, divided into three groups depending on their periodontal status. Thirty-three with chronic periodontitis, sixteen with gingivitis, and thirty-seven with periodontal healthy as control. Oxidative stress biomarkers (8-OHdG and MDA), total antioxidant capacity (TAOC), and the activity of two antioxidant enzymes (GPx and SOD) were determined in saliva. Subgingival plaque samples were obtained from the deepest periodontal pocket and PCR was used to determine the presence of the 6 fimA genotypes of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, and Treponema denticola. Results. Periodontal disease was found to be associated with increased oxidative stress parameter levels. These levels rose according to the number and type of different periodontal bacteria found in the periodontal pockets. The presence of different types of periodontal bacteria is predictive independent variables in linear regresion models of oxidative stress parameters as dependent variable, above all 8-OHdG. Conclusions. Oxidative stress parameter levels are correlated with the presence of different types of bacteria. Determination of these levels and periodontal bacteria could be a potent tool for controlling periodontal disease development. PMID:26494938

  8. Kinetic-dependent Killing of Oral Pathogens with Nitric Oxide

    PubMed Central

    Backlund, C.J.; Worley, B.V.; Sergesketter, A.R.

    2015-01-01

    Nitric oxide (NO)–releasing silica nanoparticles were synthesized via the co-condensation of tetramethyl orthosilicate with aminosilanes and subsequent conversion of secondary amines to N-diazeniumdiolate NO donors. A series of ~150 nm NO-releasing particles with different NO totals and release kinetics (i.e., half-lives) were achieved by altering both the identity and mol% composition of the aminosilane precursors. Independent of identical 2 h NO-release totals, enhanced antibacterial action was observed against the periodontopathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis with extended NO-release kinetics at pH 7.4. Negligible bactericidal effect was observed against cariogenic Streptococcus mutans at pH 7.4, even when using NO-releasing silica particles with greater NO-release totals. However, antibacterial activity was observed against S. mutans at lower pH (6.4). This result was attributed to more rapid proton-initiated decomposition of the N-diazeniumdiolate NO donors and greater NO-release payloads. The data suggest a differential sensitivity to NO between cariogenic and periodontopathogenic bacteria with implications for the future development of NO-releasing oral care therapeutics. PMID:26078424

  9. Quantification of periodontopathic bacteria in saliva using the invader assay.

    PubMed

    Tada, Akio; Takeuchi, Hiroaki; Shimizu, Hajime; Tadokoro, Kenichi; Tanaka, Kazuya; Kawamura, Katsumi; Yamaguchi, Toshikazu; Egashira, Toru; Nomura, Yoshiaki; Hanada, Nobuhiro

    2012-01-01

    When quantifying periodontopathic bacteria, it is important to use a convenient method that does not produce false negative results. The Invader assay is a convenient method because it does not involve gene amplification. The purpose of this study was to evaluate the validity of the Invader assay to quantify periodontopathic bacteria. The Invader technology was applied in quantifying five periodontopathic bacteria (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Treponema denticola). The Invader assay produced a linear quantitative detection range over concentrations spanning seven exponential values, with a detection limit of 10(3.7) copies/tube and intra-day and inter-day variance of 0.1% to 4.7% and 0.1% to 3.4%, respectively, in quantifying five periodontopathic bacteria. We compared the results of the Invader assay with those of real-time polymerase chain reaction (PCR) performed for quantifying five periodontopathic bacteria in 22 patients with periodontitis. Among the Invader-detectable bacterial strains of each species, significant correlations were observed in the counts of concerned bacterial species between these two methods, with correlation coefficients ranging from 0.757 to 0.996. This study validated repeatability and reproducibility of the Invader assay in quantifying periodontopathic bacteria and demonstrated consistent agreement between the Invader assay and real-time PCR in quantifying periodontopathic bacteria.

  10. Detection and enumeration of periodontopathogenic bacteria in subgingival biofilm of pregnant women.

    PubMed

    Machado, Fernanda Campos; Cesar, Dionéia Evangelista; Assis, Amanda Vervloet Dutra Agostinho; Diniz, Cláudio Galuppo; Ribeiro, Rosangela Almeida

    2012-01-01

    The aim of this study was to use the fluorescence in situ hybridization (FISH) technique to test the hypothesis of qualitative and quantitative differences of 8 periodontopathogens between pregnant and non-pregnant women. This cross-sectional study included 20 pregnant women in their second trimester of pregnancy and 20 non-pregnant women. Probing depth, bleeding on probing, clinical attachment level, and presence of calculus were recorded. Subgingival plaque samples were collected and the FISH technique identified the presence and numbers of Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Campylobacter rectus, Porphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatum, Prevotella intermedia and Prevotella nigrescens. The Mann-Whitney U-test was applied to compare the data between the two groups. The mean age, ethnicity, marital status, education, and economic level in both groups were similar. The clinical parameters showed no significant differences between pregnant and non-pregnant women. The numbers of subgingival periodontopathogens were not found to be significantly different between groups, despite the higher mean counts of P. intermedia in pregnant women. Colonization patterns of the different bacteria most commonly associated with periodontal disease were not different in the subgingival plaque of pregnant and non-pregnant women.

  11. A natural therapeutic approach for the treatment of periodontitis by MK615.

    PubMed

    Morimoto-Yamashita, Yoko; Kawakami, Yoshiko; Tatsuyama, Syoko; Miyashita, Keiko; Emoto, Makiko; Kikuchi, Kiyoshi; Kawahara, Ko-ichi; Tokuda, Masayuki

    2015-11-01

    Periodontitis is a chronic inflammatory disease that affects the tooth-supporting tissues. Gingival fibroblasts are the most abundant cells in periodontal tissues and they participate actively in the host inflammatory response to periodontal pathogens that is known to mediate local tissue destruction in periodontitis. The Japanese apricot, known as Ume in Japanese, has been a traditional Japanese medicine for centuries and is a familiar and commonly consumed food. The health benefits of Ume are widely recognized and have been confirmed in recent studies showing that MK615, an extract of compounds from Ume, has strong anticancer and anti-inflammatory effects. However, the potential role of MK615 in oral health is unknown. We hypothesized that the anti-inflammatory activities of MK615 could be exploited to inhibit the effects of lipopolysaccharide (LPS) produced by periodontal bacterial pathogens, such as Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Here, we show that LPS-induced interleukin (IL)-6 and IL-8 production by gingival fibroblasts was dose-dependently inhibited by MK615. As a potent inhibitor of the inflammatory responses induced by periodontal pathogens, MK615 merits further testing as a therapeutic agent in inflammatory diseases such as periodontitis. PMID:26305447

  12. Microarray analysis of microbiota of gingival lesions in noma patients.

    PubMed

    Huyghe, Antoine; François, Patrice; Mombelli, Andrea; Tangomo, Manuela; Girard, Myriam; Baratti-Mayer, Denise; Bolivar, Ignacio; Pittet, Didier; Schrenzel, Jacques

    2013-01-01

    Noma (cancrum oris) is a gangrenous disease of unknown etiology affecting the maxillo-facial region of young children in extremely limited resource countries. In an attempt to better understand the microbiological events occurring during this disease, we used phylogenetic and low-density microarrays targeting the 16S rRNA gene to characterize the gingival flora of acute noma and acute necrotizing gingivitis (ANG) lesions, and compared them to healthy control subjects of the same geographical and social background. Our observations raise doubts about Fusobacterium necrophorum, a previously suspected causative agent of noma, as this species was not associated with noma lesions. Various oral pathogens were more abundant in noma lesions, notably Atopobium spp., Prevotella intermedia, Peptostreptococcus spp., Streptococcus pyogenes and Streptococcus anginosus. On the other hand, pathogens associated with periodontal diseases such as Aggregatibacter actinomycetemcomitans, Capnocytophaga spp., Porphyromonas spp. and Fusobacteriales were more abundant in healthy controls. Importantly, the overall loss of bacterial diversity observed in noma samples as well as its homology to that of ANG microbiota supports the hypothesis that ANG might be the immediate step preceding noma.

  13. Broad-Spectrum Biofilm Inhibition by Kingella kingae Exopolysaccharide▿

    PubMed Central

    Bendaoud, Meriem; Vinogradov, Evgeny; Balashova, Nataliya V.; Kadouri, Daniel E.; Kachlany, Scott C.; Kaplan, Jeffrey B.

    2011-01-01

    Cell-free extracts prepared from Kingella kingae colony biofilms were found to inhibit biofilm formation by Aggregatibacter actinomycetemcomitans, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Candida albicans, and K. kingae. The extracts evidently inhibited biofilm formation by modifying the physicochemical properties of the cell surface, the biofilm matrix, and the substrate. Chemical and biochemical analyses indicated that the biofilm inhibition activity in the K. kingae extract was due to polysaccharide. Structural analyses showed that the extract contained two major polysaccharides. One was a linear polysaccharide with the structure →6)-α-d-GlcNAcp-(1→5)-β-d-OclAp-(2→, which was identical to a capsular polysaccharide produced by Actinobacillus pleuropneumoniae serotype 5. The second was a novel linear polysaccharide, designated PAM galactan, with the structure →3)-β-d-Galf-(1→6)-β-d-Galf-(1→. Purified PAM galactan exhibited broad-spectrum biofilm inhibition activity. A cluster of three K. kingae genes encoding UDP-galactopyranose mutase (ugm) and two putative galactofuranosyl transferases was sufficient for the synthesis of PAM galactan in Escherichia coli. PAM galactan is one of a growing number of bacterial polysaccharides that exhibit antibiofilm activity. The biological roles and potential technological applications of these molecules remain unknown. PMID:21602333

  14. Antimicrobial Activity of Chemokine CXCL10 for Dermal and Oral Microorganisms.

    PubMed

    Holdren, Grant O; Rosenthal, David J; Yang, Jianyi; Bates, Amber M; Fischer, Carol L; Zhang, Yang; Brogden, Nicole K; Brogden, Kim A

    2014-10-23

    CXCL10 (IP-10) is a small 10 kDa chemokine with antimicrobial activity. It is induced by IFN-γ, chemoattracts mononuclear cells, and promotes adhesion of T cells. Recently, we detected CXCL10 on the surface of the skin and in the oral cavity. In the current study, we used broth microdilution and radial diffusion assays to show that CXCL10 inhibits the growth of Escherichia coli, Staphylococcus aureus, Corynebacterium jeikeium, Corynebacterium striatum, and Candida albicans HMV4C, but not Corynebacterium bovis, Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, Poryphromonas gingivalis, or C. albicans ATCC 64124. The reason for the selective antimicrobial activity is not yet known. However, antimicrobial activity of CXCL10 may be related to its composition and structure, as a cationic 98 amino acid residue molecule with 10 lysine residues, 7 arginine residues, a total net charge of +11, and a theoretical pI of 9.93. Modeling studies revealed that CXCL10 contains an α-helix at the N-terminal, three anti-parallel β-strands in the middle, and an α-helix at the C-terminal. Thus, CXCL10, when produced on the surface of the skin or in the oral cavity, likely has antimicrobial activity and may enhance innate antimicrobial and cellular responses to the presence of select commensal or opportunistic microorganisms.

  15. In vitro evaluation of DispersinB on methicillin-resistant Staphylococcus pseudintermedius biofilm.

    PubMed

    Turk, Ryen; Singh, Ameet; Rousseau, Joyce; Weese, J Scott

    2013-10-25

    Methicillin resistant Staphylococcus pseudintermedius (MRSP) is an important canine pathogen that has been shown to produce biofilm in vitro. Biofilm production may be an important virulence factor and methods to eliminate biofilm-associated infections are required. An enzyme of Aggregatibacter actinomycetemcomitans, DispersinB, has been shown to degrade the extracellular matrix in the biofilm of a variety of bacteria including Staphylococcus aureus and Staphylococcus epidermidis. The objective of this study was to determine the effect of DispersinB on MRSP biofilm production and eradication in vitro. A quantitative microtitre plate assay was used to assess the impact of DispersinB on 30 MRSP isolates from dogs. While DispersinB did not have any effect on MRSP growth (P=0.98), it reduced biofilm formation (P=0.0002) and degraded established biofilm (P=0.0001). These data indicate that in vivo study of the effect of this enzyme in indicated to determine if it may be a useful treatment option for MRSP biofilm-associated infections.

  16. In Vitro Antimicrobial and Antiproliferative Activity of Amphipterygium adstringens

    PubMed Central

    Rodriguez-Garcia, A.; Peixoto, I. T. A.; Verde-Star, M. J.; De la Torre-Zavala, S.; Aviles-Arnaut, H.; Ruiz, A. L. T. G.

    2015-01-01

    Amphipterygium adstringens is a plant widely used in Mexican traditional medicine for its known anti-inflammatory and antiulcer properties. In this work, we evaluated the in vitro antimicrobial and antiproliferative activities of the methanolic extract of A. adstringens against oral pathogens such as Streptococcus mutans, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Candida albicans, and Candida dubliniensis, using microdilution (MIC) and agar diffusion methods (MBC), and the antiproliferative activity evaluating total growth inhibition (TGI) by staining the protein content with sulforhodamine B (SRB), using nine human cancer cell lines. Crude extract (CE) of A. adstringens showed some degree of activity against one or more of the strains with a MIC from 0.125 mg/mL to 63 mg/mL and MBC from 1.6 to 6.3 mg/mL and cytotoxic activity, particularly against NCI-ADR/RES, an ovarian cell line expressing multiple resistance drugs phenotype. The CE is a complex mixture of possible multitarget metabolites that could be responsible for both antimicrobial and antiproliferative activities, and further investigation is required to elucidate the identity of active compounds. Nevertheless the CE itself is useful in the development of new antimicrobial treatment based on natural products to prevent oral diseases and as alternative natural source for cancer treatment and prevention. PMID:26451151

  17. Differentiation of oral bacteria in in vitro cultures and human saliva by secondary electrospray ionization - mass spectrometry.

    PubMed

    Bregy, Lukas; Müggler, Annick R; Martinez-Lozano Sinues, Pablo; García-Gómez, Diego; Suter, Yannick; Belibasakis, Georgios N; Kohler, Malcolm; Schmidlin, Patrick R; Zenobi, Renato

    2015-01-01

    The detection of bacterial-specific volatile metabolites may be a valuable tool to predict infection. Here we applied a real-time mass spectrometric technique to investigate differences in volatile metabolic profiles of oral bacteria that cause periodontitis. We coupled a secondary electrospray ionization (SESI) source to a commercial high-resolution mass spectrometer to interrogate the headspace from bacterial cultures and human saliva. We identified 120 potential markers characteristic for periodontal pathogens Aggregatibacter actinomycetemcomitans (n = 13), Porphyromonas gingivalis (n = 70), Tanerella forsythia (n = 30) and Treponema denticola (n = 7) in in vitro cultures. In a second proof-of-principle phase, we found 18 (P. gingivalis, T. forsythia and T. denticola) of the 120 in vitro compounds in the saliva from a periodontitis patient with confirmed infection with P. gingivalis, T. forsythia and T. denticola with enhanced ion intensity compared to two healthy controls. In conclusion, this method has the ability to identify individual metabolites of microbial pathogens in a complex medium such as saliva.

  18. Quantitative Molecular Detection of 19 Major Pathogens in the Interdental Biofilm of Periodontally Healthy Young Adults

    PubMed Central

    Carrouel, Florence; Viennot, Stéphane; Santamaria, Julie; Veber, Philippe; Bourgeois, Denis

    2016-01-01

    In oral health, the interdental spaces are a real ecological niche for which the body has few or no alternative defenses and where the traditional daily methods for control by disrupting biofilm are not adequate. The interdental spaces are the source of many hypotheses regarding their potential associations with and/or causes of cardiovascular disease, diabetes, chronic kidney disease, degenerative disease, and depression. This PCR study is the first to describe the interdental microbiota in healthy adults aged 18–35 years-old with reference to the Socransky complexes. The complexes tended to reflect microbial succession events in developing dental biofilms. Early colonizers included members of the yellow, green, and purple complexes. The orange complex bacteria generally appear after the early colonizers and include many putative periodontal pathogens, such as Fusobacterium nucleatum. The red complex (Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola) was considered the climax community and is on the list of putative periodontal pathogens. The 19 major periodontal pathogens tested were expressed at various levels. F. nucleatum was the most abundant species, and the least abundant were Actinomyces viscosus, P. gingivalis, and Aggregatibacter actinomycetemcomitans. The genome counts for Eikenella corrodens, Campylobacter concisus, Campylobacter rectus, T. denticola, and Tannerella forsythensis increased significantly with subject age. The study highlights the observation that bacteria from the yellow complex (Streptococcus spp., S. mitis), the green complex (E. corrodens, Campylobacter gracilis, Capnocytophaga ochracea, Capnocytophaga sputigena, A. actinomycetemcomitans), the purple complex (Veillonella parvula, Actinomyces odontolyticus) and the blue complex (A. viscosus) are correlated. Concerning the orange complex, F. nucleatum is the most abundant species in interdental biofilm. The red complex, which is recognized as the most important

  19. Quantitative PCR analysis of salivary pathogen burden in periodontitis.

    PubMed

    Salminen, Aino; Kopra, K A Elisa; Hyvärinen, Kati; Paju, Susanna; Mäntylä, Päivi; Buhlin, Kåre; Nieminen, Markku S; Sinisalo, Juha; Pussinen, Pirkko J

    2015-01-01

    Our aim was to investigate the value of salivary concentrations of four major periodontal pathogens and their combination in diagnostics of periodontitis. The Parogene study included 462 dentate subjects (mean age 62.9 ± 9.2 years) with coronary artery disease (CAD) diagnosis who underwent an extensive clinical and radiographic oral examination. Salivary levels of four major periodontal bacteria were measured by quantitative real-time PCR (qPCR). Median salivary concentrations of Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia, as well as the sum of the concentrations of the four bacteria, were higher in subjects with moderate to severe periodontitis compared to subjects with no to mild periodontitis. Median salivary Aggregatibacter actinomycetemcomitans concentrations did not differ significantly between the subjects with no to mild periodontitis and subjects with moderate to severe periodontitis. In logistic regression analysis adjusted for age, gender, diabetes, and the number of teeth and implants, high salivary concentrations of P. gingivalis, T. forsythia, and P. intermedia were significantly associated with moderate to severe periodontitis. When looking at different clinical and radiographic parameters of periodontitis, high concentrations of P. gingivalis and T. forsythia were significantly associated with the number of 4-5 mm periodontal pockets, ≥6 mm pockets, and alveolar bone loss (ABL). High level of T. forsythia was associated also with bleeding on probing (BOP). The combination of the four bacteria, i.e., the bacterial burden index, was associated with moderate to severe periodontitis with an odds ratio (OR) of 2.40 (95% CI 1.39-4.13). When A. actinomycetemcomitans was excluded from the combination of the bacteria, the OR was improved to 2.61 (95% CI 1.51-4.52). The highest OR 3.59 (95% CI 1.94-6.63) was achieved when P. intermedia was further excluded from the combination and only the levels of P. gingivalis and T

  20. Synthesis of new antibacterial composite coating for titanium based on highly ordered nanoporous silica and silver nanoparticles.

    PubMed

    Massa, Miguel A; Covarrubias, Cristian; Bittner, Mauricio; Fuentevilla, Ignacio Andrés; Capetillo, Pavel; Von Marttens, Alfredo; Carvajal, Juan Carlos

    2014-12-01

    Infection is the most common factor that leads to dental titanium implant failure. Antibacterial implant surfaces based on nano-scale modifications of the titanium appear as an attractive strategy for control of peri-implantitis. In the present work, the preparation and antibacterial properties of a novel composite coating for titanium based on nanoporous silica and silver nanoparticles are presented. Starch-capped silver nanoparticles (AgNPs) were synthesized and then incorporated into sol-gel based solution system. The AgNP-doped nanoporous silica coatings were prepared on titanium surface using a combined sol-gel and evaporation-induced self-assembly (EISA) method. The coating nanostructure was characterized by XRD, SEM-EDX, and HR-TEM. Antibacterial activity was evaluated against Aggregatibacter actinomycetemcomitans, a representative pathogen of dental peri-implantitis. Colony-forming units (CFUs) were counted within the biofilm and at the planktonic state. Biofilm development was quantified using crystal violet staining and viability of adherent bacteria was confirmed with the Live/Dead fluorescence assay. Silica-based composite coating containing AgNPs (AgNP/NSC) was prepared on titanium surface by direct incorporation of AgNP suspension into the sol-gel system. The self-assembly technique enabled the spontaneous formation of a highly ordered nanoporosity in the coating structure, which is a desired property for osseointegration aspects of titanium implant surface. AgNP/NSC coating produces a strong antibacterial effect on titanium surface by not only killing the adherent bacteria but also reducing the extent of biofilm formation. Biofilm survival is reduced by more than 70% on the AgNP/NSC-modified titanium surface, compared to the control. This antibacterial effect was verified for up to 7 days of incubation. The long-term antibacterial activity exhibited by the nanostructured AgNP/NSC-titanium surface against A. actinomycetemcomitans suggests that this

  1. Quantitative Molecular Detection of 19 Major Pathogens in the Interdental Biofilm of Periodontally Healthy Young Adults.

    PubMed

    Carrouel, Florence; Viennot, Stéphane; Santamaria, Julie; Veber, Philippe; Bourgeois, Denis

    2016-01-01

    In oral health, the interdental spaces are a real ecological niche for which the body has few or no alternative defenses and where the traditional daily methods for control by disrupting biofilm are not adequate. The interdental spaces are the source of many hypotheses regarding their potential associations with and/or causes of cardiovascular disease, diabetes, chronic kidney disease, degenerative disease, and depression. This PCR study is the first to describe the interdental microbiota in healthy adults aged 18-35 years-old with reference to the Socransky complexes. The complexes tended to reflect microbial succession events in developing dental biofilms. Early colonizers included members of the yellow, green, and purple complexes. The orange complex bacteria generally appear after the early colonizers and include many putative periodontal pathogens, such as Fusobacterium nucleatum. The red complex (Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola) was considered the climax community and is on the list of putative periodontal pathogens. The 19 major periodontal pathogens tested were expressed at various levels. F. nucleatum was the most abundant species, and the least abundant were Actinomyces viscosus, P. gingivalis, and Aggregatibacter actinomycetemcomitans. The genome counts for Eikenella corrodens, Campylobacter concisus, Campylobacter rectus, T. denticola, and Tannerella forsythensis increased significantly with subject age. The study highlights the observation that bacteria from the yellow complex (Streptococcus spp., S. mitis), the green complex (E. corrodens, Campylobacter gracilis, Capnocytophaga ochracea, Capnocytophaga sputigena, A. actinomycetemcomitans), the purple complex (Veillonella parvula, Actinomyces odontolyticus) and the blue complex (A. viscosus) are correlated. Concerning the orange complex, F. nucleatum is the most abundant species in interdental biofilm. The red complex, which is recognized as the most important

  2. Transcriptome Analysis of B Cell Immune Functions in Periodontitis: Mucosal Tissue Responses to the Oral Microbiome in Aging

    PubMed Central

    Ebersole, Jeffrey L.; Kirakodu, Sreenatha S.; Novak, M. John; Orraca, Luis; Martinez, Janis Gonzalez; Cunningham, Larry L.; Thomas, Mark V.; Stromberg, Arnold; Pandruvada, Subramanya N.; Gonzalez, Octavio A.

    2016-01-01

    Evidence has shown activation of T and B cells in gingival tissues in experimental models and in humans diagnosed with periodontitis. The results of this adaptive immune response are noted both locally and systemically with antigenic specificity for an array of oral bacteria, including periodontopathic species, e.g., Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. It has been recognized through epidemiological studies and clinical observations that the prevalence of periodontitis increases with age. This report describes our studies evaluating gingival tissue transcriptomes in humans and specifically exploiting the use of a non-human primate model of naturally occurring periodontitis to delineate gingival mucosal tissue gene expression profiles focusing on cells/genes critical for the development of humoral adaptive immune responses. Patterns of B cell and plasmacyte genes were altered in aging healthy gingival tissues. Substantial increases in a large number of genes reflecting antigen-dependent activation, B cell activation, B cell proliferation, and B cell differentiation/maturation were observed in periodontitis in adults and aged animals. Finally, evaluation of the relationship of these gene expression patterns with those of various tissue destructive molecules (MMP2, MMP9, CTSK, TNFα, and RANKL) showed a greater frequency of positive correlations in healthy tissues versus periodontitis tissues, with only MMP9 correlations similar between the two tissue types. These results are consistent with B cell response activities in healthy tissues potentially contributing to muting the effects of the tissue destructive biomolecules, whereas with periodontitis this relationship is adversely affected and enabling a progression of tissue destructive events. PMID:27486459

  3. Transcriptome Analysis of B Cell Immune Functions in Periodontitis: Mucosal Tissue Responses to the Oral Microbiome in Aging.

    PubMed

    Ebersole, Jeffrey L; Kirakodu, Sreenatha S; Novak, M John; Orraca, Luis; Martinez, Janis Gonzalez; Cunningham, Larry L; Thomas, Mark V; Stromberg, Arnold; Pandruvada, Subramanya N; Gonzalez, Octavio A

    2016-01-01

    Evidence has shown activation of T and B cells in gingival tissues in experimental models and in humans diagnosed with periodontitis. The results of this adaptive immune response are noted both locally and systemically with antigenic specificity for an array of oral bacteria, including periodontopathic species, e.g., Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. It has been recognized through epidemiological studies and clinical observations that the prevalence of periodontitis increases with age. This report describes our studies evaluating gingival tissue transcriptomes in humans and specifically exploiting the use of a non-human primate model of naturally occurring periodontitis to delineate gingival mucosal tissue gene expression profiles focusing on cells/genes critical for the development of humoral adaptive immune responses. Patterns of B cell and plasmacyte genes were altered in aging healthy gingival tissues. Substantial increases in a large number of genes reflecting antigen-dependent activation, B cell activation, B cell proliferation, and B cell differentiation/maturation were observed in periodontitis in adults and aged animals. Finally, evaluation of the relationship of these gene expression patterns with those of various tissue destructive molecules (MMP2, MMP9, CTSK, TNFα, and RANKL) showed a greater frequency of positive correlations in healthy tissues versus periodontitis tissues, with only MMP9 correlations similar between the two tissue types. These results are consistent with B cell response activities in healthy tissues potentially contributing to muting the effects of the tissue destructive biomolecules, whereas with periodontitis this relationship is adversely affected and enabling a progression of tissue destructive events. PMID:27486459

  4. D-Galactose as an autoinducer 2 inhibitor to control the biofilm formation of periodontopathogens.

    PubMed

    Ryu, Eun-Ju; Sim, Jaehyun; Sim, Jun; Lee, Julian; Choi, Bong-Kyu

    2016-09-01

    Autoinducer 2 (AI-2) is a quorum sensing molecule to which bacteria respond to regulate various phenotypes, including virulence and biofilm formation. AI-2 plays an important role in the formation of a subgingival biofilm composed mostly of Gram-negative anaerobes, by which periodontitis is initiated. The aim of this study was to evaluate D-galactose as an inhibitor of AI-2 activity and thus of the biofilm formation of periodontopathogens. In a search for an AI-2 receptor of Fusobacterium nucleatum, D-galactose binding protein (Gbp, Gene ID FN1165) showed high sequence similarity with the ribose binding protein (RbsB), a known AI-2 receptor of Aggregatibacter actinomycetemcomitans. D-Galactose was evaluated for its inhibitory effect on the AI-2 activity of Vibrio harveyi BB152 and F. nucleatum, the major coaggregation bridge organism, which connects early colonizing commensals and late pathogenic colonizers in dental biofilms. The inhibitory effect of D-galactose on the biofilm formation of periodontopathogens was assessed by crystal violet staining and confocal laser scanning microscopy in the absence or presence of AI-2 and secreted molecules of F. nucleatum. D-Galactose significantly inhibited the AI-2 activity of V. harveyi and F. nucleatum. In addition, D-galactose markedly inhibited the biofilm formation of F. nucleatum, Porphyromonas gingivalis, and Tannerella forsythia induced by the AI-2 of F. nucleatum without affecting bacterial growth. Our results demonstrate that the Gbp may function as an AI-2 receptor and that galactose may be used for prevention of the biofilm formation of periodontopathogens by targeting AI-2 activity. PMID:27572513

  5. The periodontal health of lead-exposed children living in a shipyard industrial area.

    PubMed

    Youravong, Nattaporn; Teanpaisan, Rawee

    2015-05-01

    In a cross-sectional design, 292 schoolchildren living around a shipyard area, known to be contaminated with lead from shipyard industry, were examined to verify the association between lead exposure and periodontal health. The probing pocket depth (PD), bleeding on probing, plaque and calculus, and the presence of Aggregatibacter actinomycetemcomitans (Aa) in subgingival crevices were recorded. Gingival inflammation was the most common (98%) among children in the area. No significant difference in gingival inflammation was observed between high blood lead (PbB) and low PbB children. The prevalence rate of probing PD of ≥5 mm was 14%. The high PbB group showed more deep pockets at tooth 16 (upper right first permanent molar) and tooth 46 (lower right first permanent molar) than the low PbB group. The odds ratios (ORs) for having probing PD ≥5 mm after adjusting for other factors were 3.63 (95% confidence interval (CI), 1.24-10.61; p = 0.02) for tooth 16 and 3.93 (95% CI, 1.18-13.00; p = 0.02) for tooth 46. The presence of Aa was observed in 17% of the children and it significantly increased in high PbB compared with low PbB children at tooth 46 (OR = 5.53, 95% CI: 1.68-18.15; p = 0.005). This study may suggest no association between lead exposure and gingival inflammation, yet there was the involvement of deeper periodontal tissue in lead-exposed children.

  6. Evaluation of the Antibacterial Potential of Liquid and Vapor Phase Phenolic Essential Oil Compounds against Oral Microorganisms

    PubMed Central

    Wu, Chi-Hao; Ko, Shun-Yao; Chen, Michael Yuanchien; Shih, Yin-Hua; Shieh, Tzong-Ming; Chuang, Li-Chuan; Wu, Ching-Yi

    2016-01-01

    The aim of the present study was to determine the antibacterial activities of the phenolic essential oil (EO) compounds hinokitiol, carvacrol, thymol, and menthol against oral pathogens. Aggregatibacter actinomycetemcomitans, Streptococcus mutans, Methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia. coli were used in this study. The minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), bacterial growth curves, temperature and pH stabilities, and synergistic effects of the liquid and vapor EO compounds were tested. The MIC/MBC of the EO compounds, ranging from the strongest to weakest, were hinokitiol (40–60 μg/mL/40-100 μg/mL), thymol (100–200 μg/mL/200-400 μg/mL), carvacrol (200–400 μg/mL/200-600 μg/mL), and menthol (500-more than 2500 μg/mL/1000-more than 2500 μg/mL). The antibacterial activities of the four EO phenolic compound based on the agar diffusion test and bacterial growth curves showed that the four EO phenolic compounds were stable under different temperatures for 24 h, but the thymol activity decreased when the temperature was higher than 80°C. The combination of liquid carvacrol with thymol did not show any synergistic effects. The activities of the vaporous carvacrol and thymol were inhibited by the presence of water. Continual violent shaking during culture enhanced the activity of menthol. Both liquid and vaporous hinokitiol were stable at different temperatures and pH conditions. The combination of vaporous hinokitiol with zinc oxide did not show synergistic effects. These results showed that the liquid and vapor phases of hinokitiol have strong anti-oral bacteria abilities. Hinokitiol has the potential to be applied in oral health care products, dental materials, and infection controls to exert antimicrobial activity. PMID:27681039

  7. Antibacterial Activity of Myristica fragrans against Oral Pathogens.

    PubMed

    Shafiei, Zaleha; Shuhairi, Nadia Najwa; Md Fazly Shah Yap, Nordiyana; Harry Sibungkil, Carrie-Anne; Latip, Jalifah

    2012-01-01

    Myristica fragrans Houtt is mostly cultivated for spices in Penang Island, Malaysia. The ethyl acetate and ethanol extracts of flesh, mace and seed of Myristica fragrans was evaluated the bactericidal potential against three Gram-positive cariogenic bacteria (Streptococcus mutans ATCC 25175, Streptococcus mitis ATCC 6249, and Streptococcus salivarius ATCC 13419) and three Gram-negative periodontopathic bacteria (Aggregatibacter actinomycetemcomitans ATCC 29522, Porphyromonas gingivalis ATCC 33277, and Fusobacterium nucleatum ATCC 25586). Antibacterial activities of the extracts was determined by twofold serial microdilution, with minimum inhibitory concentrations (MIC) ranging from 1.25 to 640 mg/mL and 0.075 to 40 mg/mL. The minimum bactericidal concentration (MBC) was obtained by subculturing method. Among all extracts tested, ethyl acetate extract of flesh has the highest significant inhibitory effects against Gram-positive and Gram-negative bacteria with mean MIC value ranging from 0.625 to 1.25 ± 0.00 (SD) mg/mL; P = 0.017) and highest bactericidal effects at mean MBC value ranging from 0.625 mg/mL to 20 ± 0.00 (SD) mg/mL. While for seed and mace of Myristica fragrans, their ethanol extracts exhibited good antibacterial activity against both groups of test pathogens compared to its ethyl acetate extracts. All of the extracts of Myristica fragrans did not show any antibacterial activities against Fusobacterium nucleatum ATCC 25586. Thus, our study showed the potential effect of ethyl acetate and ethanol extracts from flesh, seed and mace of Myristica fragrans to be new natural agent that can be incorporated in oral care products.

  8. Subgingival bacterial colonization profiles correlate with gingival tissue gene expression

    PubMed Central

    2009-01-01

    Background Periodontitis is a chronic inflammatory disease caused by the microbiota of the periodontal pocket. We investigated the association between subgingival bacterial profiles and gene expression patterns in gingival tissues of patients with periodontitis. A total of 120 patients undergoing periodontal surgery contributed with a minimum of two interproximal gingival papillae (range 2-4) from a maxillary posterior region. Prior to tissue harvesting, subgingival plaque samples were collected from the mesial and distal aspects of each tissue sample. Gingival tissue RNA was extracted, reverse-transcribed, labeled, and hybridized with whole-genome microarrays (310 in total). Plaque samples were analyzed using checkerboard DNA-DNA hybridizations with respect to 11 bacterial species. Random effects linear regression models considered bacterial levels as exposure and expression profiles as outcome variables. Gene Ontology analyses summarized the expression patterns into biologically relevant categories. Results Wide inter-species variation was noted in the number of differentially expressed gingival tissue genes according to subgingival bacterial levels: Using a Bonferroni correction (p < 9.15 × 10-7), 9,392 probe sets were differentially associated with levels of Tannerella forsythia, 8,537 with Porphyromonas gingivalis, 6,460 with Aggregatibacter actinomycetemcomitans, 506 with Eikenella corrodens and only 8 with Actinomyces naeslundii. Cluster analysis identified commonalities and differences among tissue gene expression patterns differentially regulated according to bacterial levels. Conclusion Our findings suggest that the microbial content of the periodontal pocket is a determinant of gene expression in the gingival tissues and provide new insights into the differential ability of periodontal species to elicit a local host response. PMID:19835625

  9. Genome-wide association study of biologically informed periodontal complex traits offers novel insights into the genetic basis of periodontal disease

    PubMed Central

    Offenbacher, Steven; Divaris, Kimon; Barros, Silvana P.; Moss, Kevin L.; Marchesan, Julie T.; Morelli, Thiago; Zhang, Shaoping; Kim, Steven; Sun, Lu; Beck, James D.; Laudes, Matthias; Munz, Matthias; Schaefer, Arne S.; North, Kari E.

    2016-01-01

    Genome-wide association studies (GWAS) of chronic periodontitis (CP) defined by clinical criteria alone have had modest success to-date. Here, we refine the CP phenotype by supplementing clinical data with biological intermediates of microbial burden (levels of eight periodontal pathogens) and local inflammatory response (gingival crevicular fluid IL-1β) and derive periodontal complex traits (PCTs) via principal component analysis. PCTs were carried forward to GWAS (∼2.5 million markers) to identify PCT-associated loci among 975 European American adult participants of the Dental ARIC study. We sought to validate these findings for CP in the larger ARIC cohort (n = 821 participants with severe CP, 2031—moderate CP, 1914—healthy/mild disease) and an independent German sample including 717 aggressive periodontitis cases and 4210 controls. We identified six PCTs with distinct microbial community/IL-1β structures, although with overlapping clinical presentations. PCT1 was characterized by a uniformly high pathogen load, whereas PCT3 and PCT5 were dominated by Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, respectively. We detected genome-wide significant signals for PCT1 (CLEC19A, TRA, GGTA2P, TM9SF2, IFI16, RBMS3), PCT4 (HPVC1) and PCT5 (SLC15A4, PKP2, SNRPN). Overall, the highlighted loci included genes associated with immune response and epithelial barrier function. With the exception of associations of BEGAIN with severe and UBE3D with moderate CP, no other loci were associated with CP in ARIC or aggressive periodontitis in the German sample. Although not associated with current clinically determined periodontal disease taxonomies, upon replication and mechanistic validation these candidate loci may highlight dysbiotic microbial community structures and altered inflammatory/immune responses underlying biological sub-types of CP. PMID:26962152

  10. Periowave demonstrates bactericidal activity against periopathogens and leads to improved clinical outcomes in the treatment of adult periodontitis

    NASA Astrophysics Data System (ADS)

    Street, Cale N.; Andersen, Roger; Loebel, Nicolas G.

    2009-02-01

    Periodontitis affects half of the U.S. population over 50, and is the leading cause of tooth loss after 35. It is believed to be caused by growth of complex bacterial biofilms on the tooth surface below the gumline. Photodynamic therapy, a technology used commonly in antitumor applications, has more recently been shown to exhibit antimicrobial efficacy. We have demonstrated eradication of the periopathogens Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans in vitro using PeriowaveTM; a commercial photodisinfection system. In addition, several clinical studies have now demonstrated the efficacy of this treatment. A pilot study in the U.S. showed that 68% of patients treated with PeriowaveTM adjunctively to scaling and root planing (SRP) showed clinical attachment level increase of >1 mm, as opposed to 30% with SRP alone. In a subsequent larger study, a second PeriowaveTM treatment 6 weeks after initial treatment led to pocket depth improvements of >1.5 mm in 89% of patients. Finally, in the most recent multicenter, randomized, examiner-blinded study conducted on 121 subjects in Canada, PeriowaveTM treatment produced highly significant gains in attachment level (0.88 mm vs. 0.57 mm; p=0.003) and pocket depth (0.87 mm vs. 0.63 mm; p=0.01) as compared to SRP alone. In summary, PeriowaveTM demonstrated strong bactericidal activity against known periopathogens, and treatment of periodontitis using this system produced significantly better clinical outcomes than SRP alone. This, along with the absence of any adverse events in patients treated to date demonstrates that PDT is a safe and effective treatment for adult chronic periodontitis.

  11. Transcriptional Activation of the tad Type IVb Pilus Operon by PypB in Yersinia enterocolitica▿

    PubMed Central

    Schilling, Jennifer; Wagner, Karin; Seekircher, Stephanie; Greune, Lilo; Humberg, Verena; Schmidt, M. Alexander; Heusipp, Gerhard

    2010-01-01

    Type IV pili are virulence factors in various bacteria and mediate, among other functions, the colonization of diverse surfaces. Various subclasses of type IV pili have been identified, but information on pilus expression, biogenesis, and the associated phenotypes is sparse for the genus Yersinia. We recently described the identification of PypB as a transcriptional regulator in Yersinia enterocolitica. Here we show that the pypB gene is associated with the tad locus, a genomic island that is widespread among bacterial and archaeal species. The genetic linkage of pypB with the tad locus is conserved throughout the yersiniae but is not found among other bacteria carrying the tad locus. We show that the genes of the tad locus form an operon in Y. enterocolitica that is controlled by PypB and that pypB is part of this operon. The tad genes encode functions necessary for the biogenesis of the Flp subfamily of type IVb pili initially described for Aggregatibacter actinomycetemcomitans to mediate a tight-adherence phenotype. In Y. enterocolitica, the Flp pilin protein shows some peculiarities in its amino acid sequence that imply similarities as well as differences compared to typical motifs found in the Flp subtype of type IVb pili. Flp is expressed and processed after PypB overproduction, resulting in microcolony formation but not in increased adherence to biotic or abiotic surfaces. Our data describe the transcriptional regulation of the tad type IVb pilus operon by PypB in Y. enterocolitica but fail to show most previously described phenotypes associated with this type of pilus in other bacteria. PMID:20472801

  12. FAM5C Contributes to Aggressive Periodontitis

    PubMed Central

    Carvalho, Flavia M.; Tinoco, Eduardo M. B.; Deeley, Kathleen; Duarte, Poliana M.; Faveri, Marcelo; Marques, Marcelo R.; Mendonça, Adriana C.; Wang, Xiaojing; Cuenco, Karen; Menezes, Renato; Garlet, Gustavo P.; Vieira, Alexandre R.

    2010-01-01

    Aggressive periodontitis is characterized by a rapid and severe periodontal destruction in young systemically healthy subjects. A greater prevalence is reported in Africans and African descendent groups than in Caucasians and Hispanics. We first fine mapped the interval 1q24.2 to 1q31.3 suggested as containing an aggressive periodontitis locus. Three hundred and eighty-nine subjects from 55 pedigrees were studied. Saliva samples were collected from all subjects, and DNA was extracted. Twenty-one single nucleotide polymorphisms were selected and analyzed by standard polymerase chain reaction using TaqMan chemistry. Non-parametric linkage and transmission distortion analyses were performed. Although linkage results were negative, statistically significant association between two markers, rs1935881 and rs1342913, in the FAM5C gene and aggressive periodontitis (p = 0.03) was found. Haplotype analysis showed an association between aggressive periodontitis and the haplotype A-G (rs1935881-rs1342913; p = 0.009). Sequence analysis of FAM5C coding regions did not disclose any mutations, but two variants in conserved intronic regions of FAM5C, rs57694932 and rs10494634, were found. However, these two variants are not associated with aggressive periodontitis. Secondly, we investigated the pattern of FAM5C expression in aggressive periodontitis lesions and its possible correlations with inflammatory/immunological factors and pathogens commonly associated with periodontal diseases. FAM5C mRNA expression was significantly higher in diseased versus healthy sites, and was found to be correlated to the IL-1β, IL-17A, IL-4 and RANKL mRNA levels. No correlations were found between FAM5C levels and the presence and load of red complex periodontopathogens or Aggregatibacter actinomycetemcomitans. This study provides evidence that FAM5C contributes to aggressive periodontitis. PMID:20383335

  13. Comparison of Riboflavin and Toluidine Blue O as Photosensitizers for Photoactivated Disinfection on Endodontic and Periodontal Pathogens In Vitro

    PubMed Central

    Nielsen, Henrik Krarup; Garcia, Javier; Væth, Michael; Schlafer, Sebastian

    2015-01-01

    Photoactivated disinfection has a strong local antimicrobial effect. In the field of dentistry it is an emerging adjunct to mechanical debridement during endodontic and periodontal treatment. In the present study, we investigate the effect of photoactivated disinfection using riboflavin as a photosensitizer and blue LED light for activation, and compare it to photoactivated disinfection with the widely used combination of toluidine blue O and red light. Riboflavin is highly biocompatible and can be activated with LED lamps at hand in the dental office. To date, no reports are available on the antimicrobial effect of photoactivated disinfection using riboflavin/blue light on oral microorganisms. Planktonic cultures of eight organisms frequently isolated from periodontal and/or endodontic lesions (Aggregatibacter actinomycetemcomitans, Candida albicans, Enterococcus faecalis, Escherischia coli, Lactobacillus paracasei, Porphyromonas gingivalis, Prevotella intermedia and Propionibacterium acnes) were subjected to photoactivated disinfection with riboflavin/blue light and toluidine blue O/red light, and survival rates were determined by CFU counts. Within the limited irradiation time of one minute, photoactivated disinfection with riboflavin/blue light only resulted in minor reductions in CFU counts, whereas full kills were achieved for all organisms when using toluidine blue O/red light. The black pigmented anaerobes P. gingivalis and P. intermedia were eradicated completely by riboflavin/blue light, but also by blue light treatment alone, suggesting that endogenous chromophores acted as photosensitizers in these bacteria. On the basis of our results, riboflavin cannot be recommended as a photosensitizer used for photoactivated disinfection of periodontal or endodontic infections. PMID:26469348

  14. Gingival Mesenchymal Stem Cell (GMSC) Delivery System Based on RGD-Coupled Alginate Hydrogel with Antimicrobial Properties: A Novel Treatment Modality for Peri-Implantitis

    PubMed Central

    Diniz, Ivana M. A.; Chen, Chider; Ansari, Sahar; Zadeh, Homayoun H.; Moshaverinia, Maryam; Chee, Daniel; Marques, Márcia M.; Shi, Songtao; Moshaverinia, Alireza

    2015-01-01

    Purpose Peri-implantitis is one of the most common inflammatory complications in dental implantology. Similar to periodontitis, in peri-implantitis, destructive inflammatory changes take place in the tissues surrounding a dental implant. Bacterial flora at the failing implant sites resemble the pathogens in periodontal disease and consist of Gram-negative anaerobic bacteria including Aggregatibacter actinomycetemcomitans (Aa). Here we demonstrate the effectiveness of a silver lactate (SL)-containing RGD-coupled alginate hydrogel scaffold as a promising stem cell delivery vehicle with antimicrobial properties. Materials and Methods Gingival mesenchymal stem cells (GMSCs) or human bone marrow mesenchymal stem cells (hBMMSCs) were encapsulated in SL-loaded alginate hydrogel microspheres. Stem cell viability, proliferation, and osteo-differentiation capacity were analyzed. Results Our results showed that SL exhibited antimicrobial properties against Aa in a dose-dependent manner, with 0.50 mg/ml showing the greatest antimicrobial properties while still maintaining cell viability. At this concentration, SL-containing alginate hydrogel was able to inhibit Aa on the surface of Ti discs and significantly reduce the bacterial load in Aa suspensions. Silver ions were effectively released from the SL-loaded alginate microspheres for up to 2 weeks. Osteogenic differentiation of GMSCs and hBMMSCs encapsulated in the SL-loaded alginate microspheres were confirmed by the intense mineral matrix deposition and high expression of osteogenesis-related genes. Conclusion Taken together, our findings confirm that GMSCs encapsulated in RGD-modified alginate hydrogel containing SL show promise for bone tissue engineering with antimicrobial properties against Aa bacteria in vitro. PMID:26216081

  15. Short- and medium-chain fatty acids exhibit antimicrobial activity for oral microorganisms

    PubMed Central

    Huang, Chifu B.; Altimova, Yelena; Myers, Taylor M.; Ebersole, Jeffrey L.

    2011-01-01

    Objectives This study assessed the antibacterial activity of short-, medium-, and long-chain fatty acids against various oral microorganisms. Methods The short-chain fatty acids [formic acid (C1), acetic acid (C2), propionic acid (C3), butyric acid (C4), isobutyric acid (C4), isovaleric acid (C5), hexanoic acid (C6)], medium-chain fatty acids [octanoic acid (C8), capric acid (C10), lauric acid (12)], and long-chain fatty acids [myristic acid (C14), palmitic acid (C16)], were investigated for antimicrobial activity against Streptococcus mutans, S. gordonii, S. sanguis, Candida albicans, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis. Results The data demonstrated that the fatty acids exhibited patterns of inhibition against oral bacteria with some specificity that appeared related more to the bacterial species that the general structural characteristics of the microorganism. As a group the fatty acids were much less effective against C. albicans than the oral bacteria, with effectiveness limited to hexanoic, octanoic, and lauric acids. Formic acid, capric, and lauric acids were broadly inhibitory for the bacteria. Interestingly, fatty acids that are produced at metabolic end-products by a number of these bacteria, were specifically inactive against the producing species, while substantially inhibiting the growth of other oral microorganisms. Conclusions The results indicate that the antimicrobial activity of short-chain fatty acids (SCFAs), medium-chain fatty acids (MCFAs), long-chain fatty acids (LCFAs) could influence the microbial ecology in the oral cavity via at least 2 potential pathways. First, the agents delivered exogenously as therapeutic adjuncts could be packaged to enhance a microbial-regulatory environment in the subgingival sulcus. Second, it would be the intrinsic nature of these fatty acid inhibitors in contributing to the characteristics of the microbial biofilms, their evolution, and emergence of

  16. Antimicrobial peptides and nitric oxide production by neutrophils from periodontitis subjects.

    PubMed

    Mariano, F S; Campanelli, A P; Nociti Jr, F H; Mattos-Graner, R O; Gonçalves, R B

    2012-11-01

    Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens.

  17. Early colonization of the oral cavity in 6- and 12-month-old infants by cariogenic and periodontal pathogens: a case-control study.

    PubMed

    Merglova, Vlasta; Polenik, Pavel

    2016-09-01

    The colonization of the oral cavity by cariogenic and periodontal pathogens occurs earlier than previously thought. This study aimed to identify the presence and quantity of representative cariogenic and periodontal pathogens in the oral cavities of 6- and 12-month olds and to evaluate the influence of C-section delivery on early Streptococcus mutans (Sm) colonization of the oral cavity. The research cohort was composed of 59 infants (35 infants were delivered vaginally and 24 via C-section) and their mothers. At 6 months of age, the infants were examined, and unstimulated saliva samples were collected. Variables concerning mothers were DMF index and salivary levels of Sm. Repeated saliva samples were taken 6 months later. The representative cariogenic and periodontal microorganisms were identified, and their quantities were measured using a polymerase chain reaction-based method. The relationships between the presence of detected microbes, the mode of delivery, and maternal variables were evaluated using paired t tests, chi-squared test, and ANOVAs. High rates of cariogenic bacteria, Aggregatibacter actinomycetemcomitans (Aa) and Fusobacterium nucleatum (Fn), were found in both infant cohorts. An analysis of the differences between delivery methods revealed that the group of 6-month-old vaginally delivered infants had a significantly higher amount of Sm. We conclude that the cariogenic bacteria, Aa and Fn, are present in edentulous infants. This presence increases in the months following the eruption of the deciduous teeth. Results did not confirm the influence of C-section delivery on the early Sm colonization of the oral cavity. PMID:26914065

  18. Suppression of LPS-induced matrix-metalloproteinase responses in macrophages exposed to phenytoin and its metabolite, 5-(p-hydroxyphenyl-), 5-phenylhydantoin

    PubMed Central

    2010-01-01

    Background Phenytoin (PHT) has been reported to induce gingival (gum) overgrowth (GO) in approximately 50% of patients taking this medication. While most studies have focused on the effects of PHT on the fibroblast in the pathophysiology underlying GO, few studies have investigated the potential regulatory role of macrophages in extracellular matrix (ECM) turnover and secretion of proinflammatory mediators. The aim of this study was to evaluate the effects of PHT and its metabolite, 5-(p-hydroxyphenyl-), 5-phenylhydantoin (HPPH) on LPS-elicited MMP, TIMP, TNF-α and IL-6 levels in macrophages. Methods Human primary monocyte-derived macrophages (n = 6 independent donors) were pretreated with 15-50 μg/mL PHT-Na+ or 15-50 μg/mL HPPH for 1 hour. Cells were then challenged with 100 ng/ml purified LPS from the periodontal pathogen, Aggregatibacter actinomycetemcomitans. Supernatants were collected after 24 hours and levels of MMP-1, MMP-2, MMP-3, MMP-9, MMP-12, TIMP-1, TIMP-2, TIMP-3, TIMP-4, TNF-α and IL-6 determined by multiplex analysis or enzyme-linked immunoadsorbent assay. Results A dose-dependent inhibition of MMP-1, MMP-3, MMP-9, TIMP-1 but not MMP-2 was noted in culture supernatants pretreated with PHT or HPPH prior to LPS challenge. MMP-12, TIMP-2, TIMP-3 and TIMP-2 were not detected in culture supernatants. High concentrations of PHT but not HPPH, blunted LPS-induced TNF-α production although neither significantly affected IL-6 levels. Conclusion The ability of macrophages to mediate turnover of ECM via the production of metalloproteinases is compromised not only by PHT, but its metabolite, HPPH in a dose-dependent fashion. Further, the preferential dysregulation of macrophage-derived TNF-α but not IL-6 in response to bacterial challenge may provide an inflammatory environment facilitating collagen accumulation without the counteracting production of MMPs. PMID:20843335

  19. Oxygen high level laser therapy is efficient in treatment of chronic periodontitis: a clinical and microbiological study using PCR analysis.

    PubMed

    Caccianiga, G; Rey, G; Paiusco, A; Lauritano, D; Cura, F; Ormianer, Z; Carinci, F

    2016-01-01

    In periodontology, lasers have been suggested for the photodynamic therapy (PDT). Such therapy can be defined as the inactivation of cells, microorganisms or molecules induced by light and not by heat. The aim of our study is to assess the effect of Oxygen high-level laser therapy (OHLLT) in removing all bacterial deposits on root or implant surface by means of mechanical instrumentation and laser irradiation. OHLLT has two effects on targeted bacteria and tissues, decontamination and biostimulation. A total of 33 patients were randomly selected with a diagnosis of chronic periodontitis. The patients enrolled were 16 females and 17 males, six smokers and 4 diabetic patients. For each patient a periodontal charting was performed, assessing probing depth, plaque index and bleeding on probing at baseline and after 6 months. Microbiological analysis were performed with PCR Real Time, using paper tips to withdraw gingival fluid in periodontal pockets before and after treatment, at baseline and after 6 months. All patients were treated with OHLLT at baseline, after 1 week, after 2 weeks and every month for 6 months. After 6 months, all periodontal pockets were treated successfully, without complications and no significant differences in results. All clinical parameters showed an improvement, with a decrease both of plaque index (average decrease of 75%), bleeding on probing (average decrease of 62%) and probing depth (average decrease of 1.8 mm). After the treatment, a remarkable decrease in bacteria amount, both for each species and for total bacteria was observed except for Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis demonstrating that this laser protocol is effective on periodontitis treatment. OHLLT is efficient in treatment of chronic periodontitis as demonstrated by clinical and microbiological parameters, going beyond the traditional periodontal therapy.

  20. Role of the NK Cell-Activating Receptor CRACC in Periodontitis

    PubMed Central

    Krämer, Benjamin; Kebschull, Moritz; Nowak, Michael; Demmer, Ryan T.; Haupt, Manuela; Körner, Christian; Perner, Sven; Jepsen, Søren

    2013-01-01

    Periodontitis is a highly prevalent, biofilm-mediated chronic inflammatory disease that results in the loss of the tooth-supporting tissues. It features two major clinical entities: chronic periodontitis, which is more common, and aggressive periodontitis, which usually has an early onset and a rapid progression. Natural killer (NK) cells are a distinct subgroup of lymphocytes that play a major role in the ability of the innate immune system to steer immune responses. NK cells are abundant in periodontitis lesions, and NK cell activation has been causally linked to periodontal tissue destruction. However, the exact mechanisms of their activation and their role in the pathophysiology of periodontitis are elusive. Here, we show that the predominant NK cell-activating molecule in periodontitis is CD2-like receptor activating cytotoxic cells (CRACC). We show that CRACC induction was significantly more pronounced in aggressive than chronic periodontitis and correlated positively with periodontal disease severity, subgingival levels of specific periodontal pathogens, and NK cell activation in vivo. We delineate how Aggregatibacter actinomycetemcomitans, an oral pathogen that is causally associated with aggressive periodontitis, indirectly induces CRACC on NK cells via activation of dendritic cells and subsequent interleukin 12 (IL-12) signaling. In contrast, we demonstrate that fimbriae from Porphyromonas gingivalis, a principal pathogen in chronic periodontitis, actively attenuate CRACC induction on NK cells. Our data suggest an involvement of CRACC-mediated NK cell activation in periodontal tissue destruction and point to a plausible distinction in the pathobiology of aggressive and chronic periodontitis that may help explain the accelerated tissue destruction in aggressive periodontitis. PMID:23250953

  1. Oxygen high level laser therapy is efficient in treatment of chronic periodontitis: a clinical and microbiological study using PCR analysis.

    PubMed

    Caccianiga, G; Rey, G; Paiusco, A; Lauritano, D; Cura, F; Ormianer, Z; Carinci, F

    2016-01-01

    In periodontology, lasers have been suggested for the photodynamic therapy (PDT). Such therapy can be defined as the inactivation of cells, microorganisms or molecules induced by light and not by heat. The aim of our study is to assess the effect of Oxygen high-level laser therapy (OHLLT) in removing all bacterial deposits on root or implant surface by means of mechanical instrumentation and laser irradiation. OHLLT has two effects on targeted bacteria and tissues, decontamination and biostimulation. A total of 33 patients were randomly selected with a diagnosis of chronic periodontitis. The patients enrolled were 16 females and 17 males, six smokers and 4 diabetic patients. For each patient a periodontal charting was performed, assessing probing depth, plaque index and bleeding on probing at baseline and after 6 months. Microbiological analysis were performed with PCR Real Time, using paper tips to withdraw gingival fluid in periodontal pockets before and after treatment, at baseline and after 6 months. All patients were treated with OHLLT at baseline, after 1 week, after 2 weeks and every month for 6 months. After 6 months, all periodontal pockets were treated successfully, without complications and no significant differences in results. All clinical parameters showed an improvement, with a decrease both of plaque index (average decrease of 75%), bleeding on probing (average decrease of 62%) and probing depth (average decrease of 1.8 mm). After the treatment, a remarkable decrease in bacteria amount, both for each species and for total bacteria was observed except for Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis demonstrating that this laser protocol is effective on periodontitis treatment. OHLLT is efficient in treatment of chronic periodontitis as demonstrated by clinical and microbiological parameters, going beyond the traditional periodontal therapy. PMID:27469554

  2. Microbial Signature Profiles of Periodontally Healthy and Diseased Patients

    PubMed Central

    Lourenço, Talita Gomes Baêta; Heller, Débora; da Silva-Boghossian, Carina Maciel; Cotton, Sean L.; Paster, Bruce J.; Colombo, Ana Paula Vieira

    2014-01-01

    Aim To determine microbial profiles that discriminate periodontal health from different forms of periodontal diseases. Methods Subgingival biofilm was obtained from patients with periodontal health (27), gingivitis (11), chronic periodontitis (35) and aggressive periodontitis (24), and analyzed for the presence of >250 species/phylotypes using HOMIM. Microbial differences among groups were examined by Mann-Whitney. Regression analyses were performed to determine microbial risk indicators of disease. Results Putative and potential new periodontal pathogens were more prevalent in subjects with periodontal diseases than periodontal health. Detection of Porphyromonas endodontalis/Porphyromonas spp. (OR 9.5 [1.2–73.1]) and Tannerella forsythia (OR 38.2 [3.2–450.6]), and absence of Neisseria polysaccharea (OR 0.004 [0–0.15]) and Prevotella denticola (OR 0.014 [0–0.49], p<0.05) were risk indicators of periodontal disease. Presence of Aggregatibacter actinomycetemcomitans (OR 29.4 [3.4–176.5]), Cardiobacterium hominis (OR 14.9 [2.3–98.7]), Peptostreptococcaceae sp. (OR 35.9 [2.7–483.9]), P. alactolyticus (OR 31.3 [2.1–477.2]), and absence of Fretibacterium spp. (OR 0.024 [0.002–0.357]), Fusobacterium naviforme/Fusobacterium nucleatum ss vincentii (OR 0.015 [0.001–0.223]), Granulicatella adiacens/Granulicatella elegans (OR 0.013 [0.001–0.233], p<0.05) were associated with aggressive periodontitis. Conclusion There were specific microbial signatures of the subgingival biofilm that were able to distinguish between microbiomes of periodontal health and diseases. Such profiles may be used to establish risk of disease. PMID:25139407

  3. Distribution of Systemic Clarithromycin to Gingiva

    PubMed Central

    Burrell, Renita C.; Walters, John D.

    2008-01-01

    Background Aggressive and recurrent forms of periodontitis are associated with infections by Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Since these pathogens invade tissue, they are difficult to eliminate by root planing alone. Use of systemic antibiotics in conjunction with root planing significantly enhances clinical and microbiological treatment outcomes. While it is not widely prescribed by periodontists, clarithromycin is potentially useful because it is taken up by host cells and has favorable antimicrobial activity. Methods Experimental gingivitis was induced in eight healthy subjects at one randomly selected maxillary posterior site. The contralateral maxillary site served as the healthy control. Thereafter, subjects were administered 6 doses of 500 mg clarithromycin every 12 hours. Blood was then drawn and samples of gingiva were harvested from both sites. The samples were extracted and clarithromycin content was analyzed by liquid chromatography. Results Mean clarithromycin concentrations in healthy control and inflamed gingiva (2.4 μg/g and 3.0 μg/g, respectively) were significantly higher than in serum (0.5 μg/ml, P <0.05). Clarithromycin levels at control and gingivitis sites were higher than serum by 5.7 and 7.0-fold, respectively (difference between sites significant, P = 0.02). At control sites, a significant decrease in gingival crevicular fluid flow rate was evident at the conclusion of the clarithromycin regimen (P = 0.018). Conclusions Clarithromycin can attain higher levels in gingiva than serum and reaches higher levels in inflamed gingiva than in healthy gingiva. Its distribution profile appears to be suitable for treatment of periodontitis. The reduction in crevicular fluid flow at control sites suggests that clarithromycin may produce anti-inflammatory effects. PMID:18771373

  4. Host response mechanisms in periodontal diseases

    PubMed Central

    SILVA, Nora; ABUSLEME, Loreto; BRAVO, Denisse; DUTZAN, Nicolás; GARCIA-SESNICH, Jocelyn; VERNAL, Rolando; HERNÁNDEZ, Marcela; GAMONAL, Jorge

    2015-01-01

    Periodontal diseases usually refer to common inflammatory disorders known as gingivitis and periodontitis, which are caused by a pathogenic microbiota in the subgingival biofilm, including Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola that trigger innate, inflammatory, and adaptive immune responses. These processes result in the destruction of the tissues surrounding and supporting the teeth, and eventually in tissue, bone and finally, tooth loss. The innate immune response constitutes a homeostatic system, which is the first line of defense, and is able to recognize invading microorganisms as non-self, triggering immune responses to eliminate them. In addition to the innate immunity, adaptive immunity cells and characteristic cytokines have been described as important players in the periodontal disease pathogenesis scenario, with a special attention to CD4+ T-cells (T-helper cells). Interestingly, the T cell-mediated adaptive immunity development is highly dependent on innate immunity-associated antigen presenting cells, which after antigen capture undergo into a maturation process and migrate towards the lymph nodes, where they produce distinct patterns of cytokines that will contribute to the subsequent polarization and activation of specific T CD4+ lymphocytes. Skeletal homeostasis depends on a dynamic balance between the activities of the bone-forming osteoblasts (OBLs) and bone-resorbing osteoclasts (OCLs). This balance is tightly controlled by various regulatory systems, such as the endocrine system, and is influenced by the immune system, an osteoimmunological regulation depending on lymphocyte- and macrophage-derived cytokines. All these cytokines and inflammatory mediators are capable of acting alone or in concert, to stimulate periodontal breakdown and collagen destruction via tissue-derived matrix metalloproteinases, a characterization of the progression of periodontitis as a stage that

  5. A Five-Species Transcriptome Array for Oral Mixed-Biofilm Studies

    PubMed Central

    Podbielski, Andreas; Kreikemeyer, Bernd

    2011-01-01

    Background Oral polymicrobial interactions and biofilm formation are associated with initiation and progression of caries, gingivitis, and periodontitis. Transcriptome studies of such interactions, allowing a first mechanistic insight, are hampered by current single-species array designs. Methodology/Principal Findings In this study we used 385 K NimbleGene™ technology for design and evaluation of an array covering the full genomes of 5 important physiological-, cariogenic-, and periodontitis-associated microorganisms (Streptococcus sanguinis, Streptococcus mutans, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis). Array hybridization was done with cDNA from cultures grown for 24 h anaerobically. Single species experiments identified cross-species hybridizing array probes. These probes could be neglected in a mixed-species experimental setting without the need to exclude the whole genes from the analysis. Between 69% and almost 99% of the genomes were actively transcribed under the mono-species planktonic, monolayer, and biofilm conditions. The influence of Streptococcus mitis (not represented on the array) on S. mutans gene transcription was determined as a test for a dual-species mixed biofilm setup. Phenotypically, under the influence of S. mitis an increase in S. mutans biofilm mass and a decrease in media pH-value were noticed, thereby confirming previously published data. Employing a stringent cut-off (2-fold, p<0.05), 19 S. mutans transcripts were identified with increased abundance, and 11 with decreased abundance compared to a S. mutans mono-species biofilm. Several of these genes have previously been found differentially regulated under general and acid stress, thereby confirming the value of this array. Conclusions/Significance This new array allows transcriptome studies on multi-species oral biofilm interactions. It may become an important asset in future oral biofilm and inhibitor/therapy studies. PMID

  6. Frequency of periodontal pathogens and Helicobacter pylori in the mouths and stomachs of obese individuals submitted to bariatric surgery: a cross-sectional study

    PubMed Central

    PATARO, André Luiz; CORTELLI, Sheila Cavalca; ABREU, Mauro Henrique Nogueira Guimarães; CORTELLI, José Roberto; FRANCO, Gilson Cesar Nobre; AQUINO, Davi Romeiro; COTA, Luis Otavio Miranda; COSTA, Fernando Oliveira

    2016-01-01

    ABSTRACT Objectives This cross-sectional study compared the frequency of oral periodontopathogens and H. pylori in the mouths and stomachs of obese individuals with or without periodontitis submitted to bariatric surgery. Material and Methods One hundred and fifty-four men and women aged 18-65 were conveniently distributed into four groups. Two groups were composed of individuals who underwent bariatric surgery with (BP) (n=40) and without (BNP) (n=39) periodontitis and two obese control groups with (CP) (n=35) and without (CNP) (n=40) periodontitis. The oral pathogens Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Parvimonas micra, Treponema denticola, Tannerella forsythia, Campylobacter rectus, and Helicobacter pylori were detected by a polymerase chain reaction technique using saliva, tongue and stomach biopsy samples. Results Statistical analysis demonstrated that periodontopathogens were highly frequent in the mouth (up to 91.4%). In the bariatric surgically treated group, orally, P. gingivalis, T. denticola and T. forsythia were more frequent in periodontitis, while C. rectus was more frequent in non-periodontitis subjects. Stomach biopsies also revealed the high frequency of five oral species in both candidates for bariatric surgery (91.6%) and the bariatric (83.3%) groups. H. pylori was frequently detected in the mouth (50.0%) and stomach (83.3%). In the stomach, oral species and H. pylori appeared in lower frequency in the bariatric group. Conclusions Obese individuals showed high frequencies of periodontopathogens and H. pylori in their mouths and stomachs. Bariatric surgery showed an inverse microbial effect on oral and stomach environments by revealing higher oral and lower stomach bacterial frequencies. PMID:27383704

  7. Anaerobic Co-Culture of Mesenchymal Stem Cells and Anaerobic Pathogens - A New In Vitro Model System

    PubMed Central

    Kriebel, Katja; Biedermann, Anne; Kreikemeyer, Bernd; Lang, Hermann

    2013-01-01

    Background Human mesenchymal stem cells (hMSCs) are multipotent by nature and are originally isolated from bone marrow. In light of a future application of hMSCs in the oral cavity, a body compartment with varying oxygen partial pressures and an omnipresence of different bacterial species i.e. periodontitis pathogens, we performed this study to gain information about the behavior of hMSC in an anaerobic system and the response in interaction with oral bacterial pathogens. Methodology/Principal Findings We established a model system with oral pathogenic bacterial species and eukaryotic cells cultured in anaerobic conditions. The facultative anaerobe bacteria Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were studied. Their effects on hMSCs and primary as well as permanent gingival epithelial cells (Ca9-22, HGPEC) were comparatively analyzed. We show that hMSCs cope with anoxic conditions, since 40% vital cells remain after 72 h of anaerobic culture. The Ca9-22 and HGPEC cells are significantly more sensitive to lack of oxygen. All bacterial species reveal a comparatively low adherence to and internalization into hMSCs (0.2% and 0.01% of the initial inoculum, respectively). In comparison, the Ca9-22 and HGPEC cells present better targets for bacterial adherence and internalization. The production of the pro-inflammatory chemokine IL-8 is higher in both gingival epithelial cell lines compared to hMSCs and Fusobacterium nucleatum induce a time-dependent cytokine secretion in both cell lines. Porphyromonas gingivalis is less effective in stimulating secretion of IL-8 in the co-cultivation experiments. Conclusions/significance HMSCs are suitable for use in anoxic regions of the oral cavity. The interaction with local pathogenic bacteria does not result in massive pro-inflammatory cytokine responses. The test system established in this study allowed further investigation of parameters prior to set up of oral hMSC in vivo

  8. Quantification of key periodontal pathogens in insulin-dependent type 2 diabetic and non-diabetic patients with generalized chronic periodontitis.

    PubMed

    Aemaimanan, Piyamas; Amimanan, Piyawan; Taweechaisupapong, Suwimol

    2013-08-01

    Periodontitis is a common problem in patients with diabetes mellitus (DM), however, differences in the putative periodontal pathogens in subjects with DM compared to non-DM subjects are still inconclusive. The red complex, which includes Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, encompasses the most important pathogens in adult periodontal disease. The aim of the present study was to compare cell numbers of P. gingivalis, T. denticola, T. forsythia and Aggregatibacter actinomycetemcomitans in gingival sulcus of healthy, gingivitis and periodontitis sites of non-diabetes mellitus (NDM), controlled and poorly controlled insulin-dependent DM (CDM and PDM) patients with generalized chronic periodontitis. Subgingival plaque samples were collected from 19 CDM, 19 PDM and 19 NDM patients. Taqman real time-PCR was used to determine bacterial cell number. At subject level, the quantity of red complex bacteria was significantly higher in PDM than those of NDM and positively correlated with HbA1c. At site level (total 342 sites), cell numbers of T. denticola and T. forsythia in healthy sites of CDM and PDM were significantly higher than those of NDM. In gingivitis sites, the numbers of P. gingivalis in CDM and PDM and T. forsythia in PDM were significantly higher than those of NDM while in periodontitis sites, higher quantity of P. gingivalis in PDM was observed. Our study indicated that poor glycemic control is associated with increasing cell numbers of red complex bacteria in subgingival biofilm. PMID:23827459

  9. Adjunct Antimicrobial Therapy and Periodontal Surgery to Treat Generalized Aggressive Periodontitis: A Case Report.

    PubMed

    Irokawa, Daisuke; Makino-Oi, Asako; Fujita, Takahisa; Yamamoto, Shigeki; Tomita, Sachiyo; Saito, Atsushi

    2016-01-01

    Here we report a case of generalized aggressive periodontitis treated with periodontal therapy including adjunct antimicrobial therapy and periodontal surgery. The patient was a 22-year-old woman who presented with the chief complaint of gingival recession. Baseline examination revealed generalized plaque deposition and gingival inflammation. Thirty-nine percent of the sites had a probing depth (PD) of 4-6 mm and 2% a PD of ≥7 mm; 63% exhibited bleeding on probing (BOP). Radiographic examination revealed vertical bone loss in the molars and horizontal bone loss in other teeth. Microbiological examination of subgingival plaque revealed the presence of Aggregatibacter actinomycetemcomitans and Tannerella forsythia. Oral health-related quality of life was assessed as a measure of patient-reported outcome. Based on a clinical diagnosis of generalized aggressive periodontitis, initial periodontal therapy and adjunct antimicrobial therapy were implemented. After reducing inflammation and subgingival bacteria, open flap debridement was performed for teeth with a PD of ≥4 mm. Reevaluation showed no sites with a PD of ≥5 mm, a minimal level of BOP, and a marked reduction in the level of the targeted periodontal pathogens. The patient's oral health-related quality of life was slightly worsened during supportive periodontal therapy (SPT). Implementation of adjunct antimicrobial therapy targeting periodontal pathogens and subsequent periodontal surgery resulted in improvement in periodontal and microbiological parameters. This improvement has been adequately maintained over a 2-year period. However, additional care is necessary to further improve the patient's oral health-related quality of life during SPT. PMID:27320300

  10. Comparison of Riboflavin and Toluidine Blue O as Photosensitizers for Photoactivated Disinfection on Endodontic and Periodontal Pathogens In Vitro.

    PubMed

    Nielsen, Henrik Krarup; Garcia, Javier; Væth, Michael; Schlafer, Sebastian

    2015-01-01

    Photoactivated disinfection has a strong local antimicrobial effect. In the field of dentistry it is an emerging adjunct to mechanical debridement during endodontic and periodontal treatment. In the present study, we investigate the effect of photoactivated disinfection using riboflavin as a photosensitizer and blue LED light for activation, and compare it to photoactivated disinfection with the widely used combination of toluidine blue O and red light. Riboflavin is highly biocompatible and can be activated with LED lamps at hand in the dental office. To date, no reports are available on the antimicrobial effect of photoactivated disinfection using riboflavin/blue light on oral microorganisms. Planktonic cultures of eight organisms frequently isolated from periodontal and/or endodontic lesions (Aggregatibacter actinomycetemcomitans, Candida albicans, Enterococcus faecalis, Escherischia coli, Lactobacillus paracasei, Porphyromonas gingivalis, Prevotella intermedia and Propionibacterium acnes) were subjected to photoactivated disinfection with riboflavin/blue light and toluidine blue O/red light, and survival rates were determined by CFU counts. Within the limited irradiation time of one minute, photoactivated disinfection with riboflavin/blue light only resulted in minor reductions in CFU counts, whereas full kills were achieved for all organisms when using toluidine blue O/red light. The black pigmented anaerobes P. gingivalis and P. intermedia were eradicated completely by riboflavin/blue light, but also by blue light treatment alone, suggesting that endogenous chromophores acted as photosensitizers in these bacteria. On the basis of our results, riboflavin cannot be recommended as a photosensitizer used for photoactivated disinfection of periodontal or endodontic infections. PMID:26469348

  11. Comparison of Gingival Crevicular Fluid Sampling Methods in Patients with Severe Chronic Periodontitis

    PubMed Central

    Guentsch, Arndt; Kramesberger, Martin; Sroka, Aneta; Pfister, Wolfgang; Potempa, Jan; Eick, Sigrun

    2011-01-01

    Background Analysis of samplings from periodontal pockets is important in diagnosis and therapy control of periodontitis. In this study, three different sampling techniques were compared to determine if one method can yield samples suitable for reproducible and simultaneous determination of bacterial load, cytokines, neutrophil elastase, and Arg-specific gingipains. R-gingipains are an important virulence factor of Porphyromonas gingivalis, the exact concentration of which in gingival crevicular fluid (GCF) has not yet been quantified. Methods GCF was sampled from four sites per patient (each two sites one method) in 36 chronic periodontitis patients. One week later, the procedure was repeated with alternative methods. The variables that had been determined were: loads of Aggregatibacter actinomycetemcomitans and P. gingivalis, levels of interleukin-6 and interleukin-8, activity of neutrophil elastase and level of R-gingipains. Results The detected cytokine levels were higher using paper strips compared to paper points. Bacteria were found in similar loads from the paper strips and paper points. R-gingipains were detectable in high quantities only by washing of the periodontal pocket. The level of R-gingipains correlated with the load of P. gingivalis. Conclusion The use of paper strips is suitable for simultaneous determination of microbial and immunological parameters. Obtaining GCF by washing can be useful for special purposes. Gingipain concentration in periodontal pockets was directly determined to be up to 1.5 μM. This value indicates that most of so far identified substrates of these proteases by in vitro assays can be easily degraded in P. gingivalis infected sites. PMID:21235330

  12. Azithromycin Concentrations in Blood and Gingival Crevicular Fluid after Systemic Administration

    PubMed Central

    Lai, Pin-Chuang; Ho, Weiting; Jain, Nidhi; Walters, John D.

    2011-01-01

    Background Azithromycin is a macrolide antibiotic that is active against several periodontal pathogens. Macrolides are taken up and concentrated inside gingival fibroblasts, which could influence their pharmacokinetics. This study tested the hypothesis that steady-state levels of azithromycin are higher and more sustained in gingival crevicular fluid (GCF) than in serum. Methods Four healthy subjects received an initial dose of 500 mg azithromycin followed by 250 mg doses on each of the next 2 days. Serum and GCF samples were obtained 2 hours after the last dose (day 2) and on days 4 and 7. GCF samples were collected from maxillary posterior sites with paper strips. The strips were pooled and eluted with high purity water. After extraction, the azithromycin content of the serum samples and GCF eluates was determined with an agar diffusion bioassay. Results On days 2, 4 and 7, the concentrations of azithromycin in blood serum were 0.22 ± 0.02, 0.08 ± 0.02 and 0.04 ± 0.01 μg/ml, respectively. The concentrations in GCF were 8.82 ± 1.25, 7.90 ± 1.72 and 7.38 ± 1.15 μg/ml, respectively. Mean GCF levels were significantly higher than mean serum levels (P ≤ 0.02, paired t-test). Conclusion The findings demonstrate that the pharmacokinetic profiles of azithromycin are different in GCF and serum. At steady state, azithromycin concentrations in GCF were higher and more sustained than those in serum. Based on previous studies, the levels observed in GCF were above the MIC for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia. PMID:21417585

  13. Phototoxic effect of blue light on the planktonic and biofilm state of anaerobic periodontal pathogens

    PubMed Central

    Song, Hyun-Hwa; Lee, Jae-Kwan; Um, Heung-Sik; Lee, Si-Young; Lee, Min-Ku

    2013-01-01

    Purpose The purpose of this study was to compare the phototoxic effects of blue light exposure on periodontal pathogens in both planktonic and biofilm cultures. Methods Strains of Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis, in planktonic or biofilm states, were exposed to visible light at wavelengths of 400.520 nm. A quartz-tungsten-halogen lamp at a power density of 500 mW/cm2 was used for the light source. Each sample was exposed to 15, 30, 60, 90, or 120 seconds of each bacterial strain in the planktonic or biofilm state. Confocal scanning laser microscopy (CSLM) was used to observe the distribution of live/dead bacterial cells in biofilms. After light exposure, the bacterial killing rates were calculated from colony forming unit (CFU) counts. Results CLSM images that were obtained from biofilms showed a mixture of dead and live bacterial cells extending to a depth of 30-45 µm. Obvious differences in the live-to-dead bacterial cell ratio were found in P. gingivalis biofilm according to light exposure time. In the planktonic state, almost all bacteria were killed with 60 seconds of light exposure to F. nucleatum (99.1%) and with 15 seconds to P. gingivalis (100%). In the biofilm state, however, only the CFU of P. gingivalis demonstrated a decreasing tendency with increasing light exposure time, and there was a lower efficacy of phototoxicity to P. gingivalis as biofilm than in the planktonic state. Conclusions Blue light exposure using a dental halogen curing unit is effective in reducing periodontal pathogens in the planktonic state. It is recommended that an adjunctive exogenous photosensitizer be used and that pathogens be exposed to visible light for clinical antimicrobial periodontal therapy. PMID:23678390

  14. Fusobacterium necrophorum in North American Bighorn Sheep ( Ovis canadensis ) Pneumonia.

    PubMed

    Shanthalingam, Sudarvili; Narayanan, Sanjeevkumar; Batra, Sai Arun; Jegarubee, Bavananthasivam; Srikumaran, Subramaniam

    2016-07-01

    Fusobacterium necrophorum has been detected in pneumonic bighorn sheep (BHS; Ovis canadensis ) lungs, in addition to the aerobic respiratory pathogens Mannheimia haemolytica , Bibersteinia trehalosi , Pasteurella multocida , and Mycoplasma ovipneumoniae . Similar to M. haemolytica , F. necrophorum produces a leukotoxin. Leukotoxin-induced lysis and degranulation of polymorphonuclear leukocytes (PMNs) and macrophages are responsible for acute inflammation and lung tissue damage characteristic of M. haemolytica -caused pneumonia. As one approach in elucidating the role of F. necrophorum in BHS pneumonia, we determined the frequency of the presence of F. necrophorum in archived pneumonic BHS lung tissues, and susceptibility of BHS leukocytes to F. necrophorum leukotoxin. A species-specific PCR assay detected F. necrophorum in 37% of pneumonic BHS lung tissues (total tested n=70). Sequences of PCR amplicons were similar to the less virulent F. necrophorum subsp. funduliforme. Fusobacterium necrophorum leukotoxin exhibited cytotoxicity to BHS PMNs and peripheral blood mononuclear cells. As with the M. haemolytica leukotoxin, F. necrophorum leukotoxin was more toxic to BHS PMNs than domestic sheep PMNs. It is likely that F. necrophorum enters the lungs after M. haemolytica and other aerobic respiratory pathogens enter the lungs and initiate tissue damage, thereby creating a microenvironment that is conducive for anaerobic bacterial growth. In summary, Fusobacterium leukotoxin is highly toxic for BHS leukocytes; however, based on the PCR findings, it is unlikely to play a direct role in the development of BHS pneumonia.

  15. Fusobacterium necrophorum in North American Bighorn Sheep ( Ovis canadensis ) Pneumonia.

    PubMed

    Shanthalingam, Sudarvili; Narayanan, Sanjeevkumar; Batra, Sai Arun; Jegarubee, Bavananthasivam; Srikumaran, Subramaniam

    2016-07-01

    Fusobacterium necrophorum has been detected in pneumonic bighorn sheep (BHS; Ovis canadensis ) lungs, in addition to the aerobic respiratory pathogens Mannheimia haemolytica , Bibersteinia trehalosi , Pasteurella multocida , and Mycoplasma ovipneumoniae . Similar to M. haemolytica , F. necrophorum produces a leukotoxin. Leukotoxin-induced lysis and degranulation of polymorphonuclear leukocytes (PMNs) and macrophages are responsible for acute inflammation and lung tissue damage characteristic of M. haemolytica -caused pneumonia. As one approach in elucidating the role of F. necrophorum in BHS pneumonia, we determined the frequency of the presence of F. necrophorum in archived pneumonic BHS lung tissues, and susceptibility of BHS leukocytes to F. necrophorum leukotoxin. A species-specific PCR assay detected F. necrophorum in 37% of pneumonic BHS lung tissues (total tested n=70). Sequences of PCR amplicons were similar to the less virulent F. necrophorum subsp. funduliforme. Fusobacterium necrophorum leukotoxin exhibited cytotoxicity to BHS PMNs and peripheral blood mononuclear cells. As with the M. haemolytica leukotoxin, F. necrophorum leukotoxin was more toxic to BHS PMNs than domestic sheep PMNs. It is likely that F. necrophorum enters the lungs after M. haemolytica and other aerobic respiratory pathogens enter the lungs and initiate tissue damage, thereby creating a microenvironment that is conducive for anaerobic bacterial growth. In summary, Fusobacterium leukotoxin is highly toxic for BHS leukocytes; however, based on the PCR findings, it is unlikely to play a direct role in the development of BHS pneumonia. PMID:27224212

  16. Comparison of Passively Transferred Antibodies in Bighorn and Domestic Lambs Reveals One Factor in Differential Susceptibility of These Species to Mannheimia haemolytica-Induced Pneumonia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mannheimia haemolytica consistently causes fatal bronchopneumonia in bighorn sheep (BHS; Ovis canadensis) under natural and experimental conditions. Leukotoxin is the primary virulence factor of this organism. BHS are more susceptible to developing fatal pneumonia than the related species Ovis aries...

  17. Photodynamic therapy as adjunct to non-surgical periodontal treatment in patients on periodontal maintenance: a randomized controlled clinical trial.

    PubMed

    Chondros, Panos; Nikolidakis, Dimitris; Christodoulides, Nicos; Rössler, Ralf; Gutknecht, Norbert; Sculean, Anton

    2009-09-01

    Recent preclinical and clinical data have suggested the potential benefit of photodynamic therapy (PDT) in the treatment of periodontitis. However, currently, there are very limited data from controlled clinical trials evaluating the effect of PDT in the treatment of periodontitis. The aim of the present study was to evaluate the clinical and microbiological effects of the adjunctive use of PDT in non-surgical periodontal treatment in patients receiving supportive periodontal therapy. Twenty-four patients receiving regularly supportive periodontal therapy were randomly treated with either subgingival scaling and root planing followed by a single episode of PDT (test) or subgingival scaling and root planing alone (control). The following parameters were evaluated at baseline and at 3 months and 6 months after therapy: full mouth plaque score (FMPS), full mouth bleeding score (FMBS), bleeding on probing (BOP) at experimental sites, probing pocket depth (PPD), gingival recession (REC), and clinical attachment level (CAL). Primary outcome variables were changes in PPD and CAL. Microbiological evaluation of Aggregatibacter actinomycetemcomitans (A.a.), Porphyromonas gingivalis (P.g.), Prevotella intermedia (P.i.), Tannerella forsythensis (T.f.), Treponema denticola (T.d.), Peptostreptococcus micros (P.m.), Fusobacterium nucleatum (F.n.), Campylobacter rectus (C.r.), Eubacterium nodatum (E.n.), Eikenella corrodens (E.c.), and Capnocytophaga species (C.s.) was also performed at baseline and at 3 months and 6 months after therapy, using a commercially available polymerase chain reaction test. No differences in any of the investigated parameters were observed at baseline between the two groups. At 3 months and 6 months after treatment, there were no statistically significant differences between the groups in terms of PPD, CAL and FMPS. At 3 months and 6 months, a statistically significantly higher improvement of BOP was found in the test group. At 3 months after therapy

  18. Comparative Evaluation of Antimicrobial Activity of Pomegranate-Containing Mouthwash Against Oral-Biofilm Forming Organisms: An Invitro Microbial Study

    PubMed Central

    Dabholkar, Charuta Sadanand; Shah, Mona; Bajaj, Monika; Doshi, Yogesh

    2016-01-01

    Introduction Pomegranate is considered “A pharmacy unto itself”. Hydrolysable tannins called punicalagins which have free scavenging properties are the most abundant polyphenols found in pomegranate-containing mouthwash. Aim To evaluate antimicrobial effect of pomegranate- containing mouthwash on oral biofilm-forming bacteria. Materials and Methods The mouthwashes used were divided into three groups- Group A: Chlorhexidine mouthwash (Hexidine); Group B: Herbal Mouthwash (Hiora) and Group C: Pomegranate-containing Mouthwash (Life-extension). Each mouthwash was diluted to five different concentrations. Reference strains of Streptococcus mutans (S.mutans) (ATCC 25175), Streptococcus salivarius (S.salivarius) (ATCC 7073), and Aggregatibacter actinomycetemcomitans (A.a) (NCTC 9710) were selected as being colonizers in dental biofilm formation. On each culture plate, five wells of 5mm were prepared and mouthwashes with different concentrations were added, followed by incubation in a CO2 jar for 24 hours at 37°C. Inhibition zone diameters were measured using a digital caliper. Results Chlorhexidine (0.12%) presented a zone of inhibition between 38.46% to 96.15% for all the three organisms, while Hiora presented zone of inhibition ranging from 33.33% to 69.23% but was resistant at <10 ml of dilution. Pomegranate mouthwash presented a zone of inhibition ranging from 38.48 to 57.69%, but was resistant at <10ml for S.mutans, and <25ml for A.a and S.salivarius. ANOVA test was done to compare the dilution of mouthwashes for a particular organism and Tukey’s multiple comparison tests were done to find the exact difference. A significant difference was seen between all the three groups at 50ml and 75 ml of dilution. At 75 ml concentration, a statistical difference was found between Groups B & C and Groups A & B; and at 50 ml between Groups A&C. Conclusion All the three types of mouthwash exhibit anti-microbial activity against biofilm forming organisms but at varying

  19. Evaluation of moxifloxacin-hydroxyapatite composite graft in the regeneration of intrabony defects: A clinical, radiographic, and microbiological study

    PubMed Central

    Nagarjuna Reddy, Y. V.; Deepika, P. C.; Venkatesh, M. P.; Rajeshwari, K. G.

    2016-01-01

    Background: The formation of new connective periodontal attachment is contingent upon the elimination or marked reduction of pathogens at the treated periodontal site. An anti-microbial agent, i.e. moxifloxacin has been incorporated into the bone graft to control infection and facilitate healing during and after periodontal therapy. Materials and Methods: By purposive sampling, 15 patients with at least two contralateral vertical defect sites were selected. The selected sites in each individual were divided randomly into test and control sites according to split-mouth design. Test site received moxifloxacin-hydroxyapatite composite graft and control site received hydroxyapatite-placebo gel composite graft. Probing depth (PD) and Clinical attachment level (CAL) were assessed at baseline, 3, 6, 9, and 12 months. Bone probing depth (BPD) and hard tissue parameters such as amount of defect fill, percentage of defect fill, and changes in alveolar crest were assessed at baseline, 6, and 12 months. Changes in subgingival microflora were also assessed by culturing the subgingival plaque samples at baseline and at 3-month follow-up. The clinical, radiographic, and microbiological data obtained were subjected to statistical analysis using descriptive statistics, paired sample t-test, independent t-test, and contingency test. Results: On intragroup comparison at test and control sites, there was a significant improvement in all clinical and radiographic parameters. However, on intergroup comparison of the same, there was no statistically significant difference between test and control sites at any interval. Although test sites showed slightly higher amount of bone fill, it was not statistically significant. There was a significant reduction in the counts of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis at both sites from baseline to 3 months. In addition, there was a significant reduction at test sites as compared to control sites at 3-month follow-up (P

  20. Evaluation of moxifloxacin-hydroxyapatite composite graft in the regeneration of intrabony defects: A clinical, radiographic, and microbiological study

    PubMed Central

    Nagarjuna Reddy, Y. V.; Deepika, P. C.; Venkatesh, M. P.; Rajeshwari, K. G.

    2016-01-01

    Background: The formation of new connective periodontal attachment is contingent upon the elimination or marked reduction of pathogens at the treated periodontal site. An anti-microbial agent, i.e. moxifloxacin has been incorporated into the bone graft to control infection and facilitate healing during and after periodontal therapy. Materials and Methods: By purposive sampling, 15 patients with at least two contralateral vertical defect sites were selected. The selected sites in each individual were divided randomly into test and control sites according to split-mouth design. Test site received moxifloxacin-hydroxyapatite composite graft and control site received hydroxyapatite-placebo gel composite graft. Probing depth (PD) and Clinical attachment level (CAL) were assessed at baseline, 3, 6, 9, and 12 months. Bone probing depth (BPD) and hard tissue parameters such as amount of defect fill, percentage of defect fill, and changes in alveolar crest were assessed at baseline, 6, and 12 months. Changes in subgingival microflora were also assessed by culturing the subgingival plaque samples at baseline and at 3-month follow-up. The clinical, radiographic, and microbiological data obtained were subjected to statistical analysis using descriptive statistics, paired sample t-test, independent t-test, and contingency test. Results: On intragroup comparison at test and control sites, there was a significant improvement in all clinical and radiographic parameters. However, on intergroup comparison of the same, there was no statistically significant difference between test and control sites at any interval. Although test sites showed slightly higher amount of bone fill, it was not statistically significant. There was a significant reduction in the counts of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis at both sites from baseline to 3 months. In addition, there was a significant reduction at test sites as compared to control sites at 3-month follow-up (P

  1. PCR assay detects Mannheimia haemolytica in culture-negative pneumonic lung tissues of bighorn sheep (Ovis canadensis) from outbreaks in the western USA, 2009-2010.

    PubMed

    Shanthalingam, Sudarvili; Goldy, Andrea; Bavananthasivam, Jegarubee; Subramaniam, Renuka; Batra, Sai Arun; Kugadas, Abirami; Raghavan, Bindu; Dassanayake, Rohana P; Jennings-Gaines, Jessica E; Killion, Halcyon J; Edwards, William H; Ramsey, Jennifer M; Anderson, Neil J; Wolff, Peregrine L; Mansfield, Kristin; Bruning, Darren; Srikumaran, Subramaniam

    2014-01-01

    Mannheimia haemolytica consistently causes severe bronchopneumonia and rapid death of bighorn sheep (Ovis canadensis) under experimental conditions. However, Bibersteinia trehalosi and Pasteurella multocida have been isolated from pneumonic bighorn lung tissues more frequently than M. haemolytica by culture-based methods. We hypothesized that assays more sensitive than culture would detect M. haemolytica in pneumonic lung tissues more accurately. Therefore, our first objective was to develop a PCR assay specific for M. haemolytica and use it to determine if this organism was present in the pneumonic lungs of bighorns during the 2009-2010 outbreaks in Montana, Nevada, and Washington, USA. Mannheimia haemolytica was detected by the species-specific PCR assay in 77% of archived pneumonic lung tissues that were negative by culture. Leukotoxin-negative M. haemolytica does not cause fatal pneumonia in bighorns. Therefore, our second objective was to determine if the leukotoxin gene was also present in the lung tissues as a means of determining the leukotoxicity of M. haemolytica that were present in the lungs. The leukotoxin-specific PCR assay detected leukotoxin gene in 91% of lung tissues that were negative for M. haemolytica by culture. Mycoplasma ovipneumoniae, an organism associated with bighorn pneumonia, was detected in 65% of pneumonic bighorn lung tissues by PCR or culture. A PCR assessment of distribution of these pathogens in the nasopharynx of healthy bighorns from populations that did not experience an all-age die-off in the past 20 yr revealed that M. ovipneumoniae was present in 31% of the animals whereas leukotoxin-positive M. haemolytica was present in only 4%. Taken together, these results indicate that culture-based methods are not reliable for detection of M. haemolytica and that leukotoxin-positive M. haemolytica was a predominant etiologic agent of the pneumonia outbreaks of 2009-2010.

  2. Periodontitis associated with Papillon-Lefèvre syndrome.

    PubMed

    Bimstein, E; Lustmann, J; Sela, M N; Neriah, Z B; Soskolne, W A

    1990-06-01

    The predominant subgingival microflora, host immune response, and genetic history of a 14-year-old girl with Papillon-Lefèvre Syndrome (PLS) are reported. The patient had high counts of Actinobacillus actinomycetemcomitans and surface translocating bacteria. She had significantly raised levels of antibodies to five of the bacterial species studied with the levels to A. actinomycetemcomitans remaining high after antibiotic therapy. The polymorphonuclear leukocytes (PMN) also released significantly increased amounts of O2 compared to controls. The data presented support a role for A. actinomycetemcomitans and PMN dysfunction in the pathogenesis of PLS. PMID:2164081

  3. MHC class II DR allelic diversity in bighorn sheep

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We hypothesized that decreased diversity and/or unique polymorphisms in MHC class II alleles of bighorn sheep (BHS, Ovis canadensis) are responsible for lower titer of antibodies against Mannheimia haemolytica leukotoxin, in comparison to domestic sheep (DS, Ovis aries). To test this hypothesis, DRA...

  4. Occupation and the risk of chronic toxic leukoencephalopathy.

    PubMed

    Filley, Christopher M

    2015-01-01

    Among the hundreds of environmental insults capable of inducing nervous system injury, a small number can produce clinically significant damage to the brain white matter. The use of magnetic resonance imaging (MRI) in affected individuals has greatly illuminated this previously obscure area of neurotoxicology. Toxic leukoencephalopathy has acute and chronic forms, in both of which cognitive dysfunction is the major clinical manifestation. Chronic toxic leukoencephalopathy (CTL) has been most thoroughly described in individuals with intense and prolonged exposure to leukotoxins, but the consequences of lesser degrees of exposure are not well understood. Rare cases of CTL have been reported in workers exposed to culpable leukotoxins, but study of this syndrome is hindered by many confounds such as uncertain level of toxin exposure, the presence of multiple toxins, vague dose-response relationship, comorbid medical or neurologic disorders, psychiatric illness, and legal issues. The risk of CTL in workers is low, although it is not possible to determine quantitative risk estimates. More knowledge can be expected with the application of advanced MRI techniques to the assessment of workers who may have been exposed to known or potential leukotoxins. Preventive measures for avoiding workplace CTL will be informed by clinical assessment involving the use of advanced neuroimaging and neuropsychologic evaluation in combination with accurate measurement of leukotoxin exposure. PMID:26563784

  5. Characterization of Fusobacterium necrophorum isolated from llama and alpaca.

    PubMed

    Kumar, Amit; Anderson, David; Amachawadi, Raghavendra G; Nagaraja, Tiruvoor G; Narayanan, Sanjeev K

    2013-07-01

    Fusobacterium necrophorum, a Gram-negative, anaerobic bacterium, is an opportunistic animal and human pathogen that causes a variety of infections termed necrobacillosis. There are 2 subspecies of F. necrophorum (subsp. necrophorum and subsp. funduliforme) that differ morphologically and biochemically and in virulence. Leukotoxin, a secreted protein, is considered to be the major virulence factor. In camelids, F. necrophorum causes a variety of infections, generally involving the lips, tongue, pharynx, interdigital spaces, foot pad, larynx, mandible, or maxillary bones. The objective of the current study was to characterize the presumptive Fusobacterium isolates from a variety of necrotic infections in llama (Lama glama) and alpaca (Vicugna pacos) and determine whether the strains possess leukotoxin activities. A total of 7 isolates from alpaca and 2 isolates from llama were characterized. Based on growth characteristics in broth culture, and biochemical and polymerase chain reaction analyses, all 9 isolates belonged to subsp. necrophorum and possessed the putative hemagglutinin gene. Western blot analysis with antileukotoxin antibodies raised in rabbit showed the presence of leukotoxin protein in the culture supernatant of all isolates. Furthermore, flow cytometry of the culture supernatants demonstrated cytotoxicity to bovine and alpaca polymorphonuclear leukocytes (PMNs). The extent of cytotoxicity to either alpaca or bovine PMNs differed among camelid strains. The cytotoxicity of many of the camelid strains was higher (P < 0.05) toward alpaca PMNs compared to bovine PMNs. Fusobacterium necrophorum isolates from llama and alpaca are similar to bovine isolates, and leukotoxin may be a major virulence factor.

  6. Pulmonary immunity in calves following stimulation of the gut-associated lymphatic tissue by bacterial exotoxin.

    PubMed Central

    Bowersock, T L; Walker, R D; Samuels, M L; Moore, R N

    1992-01-01

    Antibodies in serum and pulmonary lavage fluids were measured in calves following stimulation of the gut-associated lymphatic tissue (GALT) by inoculation of crude leukotoxin of Pasteurella haemolytica into the duodenum through a surgically placed catheter. Nine calves free of P. haemolytica were divided into two groups. Group 1 received an intraduodenal (ID) inoculation of leukotoxin and group 2 received an ID inoculation of phosphate buffered saline. Serum and pulmonary lavage fluids were collected weekly and assayed for antibodies specific to P. haemolytica including immunoglobulin (Ig)G, leukotoxin neutralizing antibodies (LNA), and IgA (lavage fluids only). The multiplicative increase (over baseline) in each class of antibody titer following ID inoculation of leukotoxin, the composite geometric mean increase of all antibodies together, and the composite number of the five antibody titers which increased at least fourfold were computed. Results showed that the geometric mean of each antibody titer and the two composite indices was higher in the GALT-primed groups than in the sham-primed group. The differences were statistically significant (p less than 0.05) for serum IgG and for the two composite indices. This experiment demonstrates for the first time that GALT stimulation by bacterial exotoxins results in increased pulmonary antibody levels in calves. PMID:1591657

  7. S. aureus Toxins Join the DARC Side.

    PubMed

    Ratner, Adam J

    2015-09-01

    Staphylococcus aureus, like other bacterial pathogens, scavenges host iron for growth through incompletely understood mechanisms. In this issue of Cell Host & Microbe, Spaan et al. (2015) demonstrate that two Staphylococcus aureus leukotoxins, HlgAB and LukED, target the Duffy antigen receptor for chemokines on erythrocytes, resulting in lysis and iron release. PMID:26355213

  8. Staphylococcus aureus toxins.

    PubMed

    Otto, Michael

    2014-02-01

    Staphylococcus aureus is a dangerous pathogen that causes a variety of severe diseases. The virulence of S. aureus is defined by a large repertoire of virulence factors, among which secreted toxins play a preeminent role. Many S. aureus toxins damage biological membranes, leading to cell death. In particular, S. aureus produces potent hemolysins and leukotoxins. Among the latter, some were recently identified to lyse neutrophils after ingestion, representing an especially powerful weapon against bacterial elimination by innate host defense. Furthermore, S. aureus secretes many factors that inhibit the complement cascade or prevent recognition by host defenses. Several further toxins add to this multi-faceted program of S. aureus to evade elimination in the host. This review will give an overview over S. aureus toxins focusing on recent advances in our understanding of how leukotoxins work in receptor-mediated or receptor-independent fashions.

  9. Role of carriers in the transmission of pneumonia in bighorn sheep (Ovis canadensis)

    PubMed Central

    Raghavan, Bindu; Erickson, Kayla; Kugadas, Abirami; Batra, Sai A.; Call, Douglas R.; Davis, Margaret A.; Foreyt, William J.

    2016-01-01

    ABSTRACT In the absence of livestock contact, recurring lamb mortality in bighorn sheep (Ovis canadensis) populations previously exposed to pneumonia indicates the likely presence of carriers of pneumonia-causing pathogens, and possibly inadequate maternally derived immunity. To investigate this problem we commingled naïve, pregnant ewes (n=3) with previously exposed rams (n=2). Post-commingling, all ewes and lambs born to them acquired pneumonia-causing pathogens (leukotoxin-producing Pasteurellaceae and Mycoplasma ovipneumoniae), with subsequent lamb mortality between 4-9 weeks of age. Infected ewes became carriers for two subsequent years and lambs born to them succumbed to pneumonia. In another experiment, we attempted to suppress the carriage of leukotoxin-producing Pasteurellaceae by administering an antibiotic to carrier ewes, and evaluated lamb survival. Lambs born to both treatment and control ewes (n=4 each) acquired pneumonia and died. Antibody titers against leukotoxin-producing Pasteurellaceae in all eight ewes were ‘protective’ (>1:800 and no apparent respiratory disease); however their lambs were either born with comparatively low titers, or with high (but non-protective) titers that declined rapidly within 2-8 weeks of age, rendering them susceptible to fatal disease. Thus, exposure to pneumonia-causing pathogens from carrier ewes, and inadequate titers of maternally derived protective antibodies, are likely to render bighorn lambs susceptible to fatal pneumonia. PMID:27185269

  10. Role of carriers in the transmission of pneumonia in bighorn sheep (Ovis canadensis).

    PubMed

    Raghavan, Bindu; Erickson, Kayla; Kugadas, Abirami; Batra, Sai A; Call, Douglas R; Davis, Margaret A; Foreyt, William J; Srikumaran, Subramaniam

    2016-01-01

    In the absence of livestock contact, recurring lamb mortality in bighorn sheep (Ovis canadensis) populations previously exposed to pneumonia indicates the likely presence of carriers of pneumonia-causing pathogens, and possibly inadequate maternally derived immunity. To investigate this problem we commingled naïve, pregnant ewes (n=3) with previously exposed rams (n=2). Post-commingling, all ewes and lambs born to them acquired pneumonia-causing pathogens (leukotoxin-producing Pasteurellaceae and Mycoplasma ovipneumoniae), with subsequent lamb mortality between 4-9 weeks of age. Infected ewes became carriers for two subsequent years and lambs born to them succumbed to pneumonia. In another experiment, we attempted to suppress the carriage of leukotoxin-producing Pasteurellaceae by administering an antibiotic to carrier ewes, and evaluated lamb survival. Lambs born to both treatment and control ewes (n=4 each) acquired pneumonia and died. Antibody titers against leukotoxin-producing Pasteurellaceae in all eight ewes were 'protective' (>1:800 and no apparent respiratory disease); however their lambs were either born with comparatively low titers, or with high (but non-protective) titers that declined rapidly within 2-8 weeks of age, rendering them susceptible to fatal disease. Thus, exposure to pneumonia-causing pathogens from carrier ewes, and inadequate titers of maternally derived protective antibodies, are likely to render bighorn lambs susceptible to fatal pneumonia. PMID:27185269

  11. Characterization of Fusobacterium isolates from the respiratory tract of white-tailed deer (Odocoileus virginianus).

    PubMed

    Brooks, Jason W; Kumar, Amit; Narayanan, Sanjeev; Myers, Suzanne; Brown, Kayla; Nagaraja, T G; Jayarao, Bhushan M

    2014-03-01

    A total of 23 clinical isolates of Fusobacterium spp. were recovered at necropsy over a 2-year period from the respiratory tract of white-tailed deer (Odocoileus virginianus). Isolates were identified as Fusobacterium varium (18/23), Fusobacterium necrophorum subsp. funduliforme (3/23), and Fusobacterium necrophorum subsp. necrophorum (2/23). Using polymerase chain reaction-based detection of virulence genes, all F. necrophorum isolates were positive for the promoter region of the leukotoxin operon and the hemagglutinin-related protein gene, while all F. varium isolates were negative. The presence of the leukotoxin gene in F. necrophorum isolates and the absence of this gene in F. varium isolates were confirmed by Southern hybridization using 2 separate probes. Toxicity to bovine polymorphonuclear leukocytes was observed with all F. necrophorum isolates, but was not observed in any F. varium isolates. Susceptibility to antimicrobials was markedly different for F. varium as compared to F. necrophorum. In summary, no evidence of leukotoxin production was detected in any of the 23 F. varium isolates used in the current study. The data suggests that F. varium, the most common species isolated, may be a significant pathogen in deer with a different virulence mechanism than F. necrophorum.

  12. Pasteurella haemolytica antigens associated with resistance to pneumonic pasteurellosis.

    PubMed Central

    Mosier, D A; Simons, K R; Confer, A W; Panciera, R J; Clinkenbeard, K D

    1989-01-01

    Antigens associated with whole Pasteurella haemolytica biotype A serotype 1, a capsular carbohydrate-protein extract of the organism, and P. haemolytica leukotoxin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antigens of the electrophoresed preparations were detected by Western blotting (immunoblotting) with sera from cattle which were either nonvaccinated or vaccinated with live or killed P. haemolytica vaccines and had variable degrees of resistance to experimental pneumonic pasteurellosis. Distinct, easily recognizable antigens of these preparations were identified, and the antibody responses to these antigens were quantified by densitometry. To determine their importance to disease resistance, we then compared antibody responses with experimental lesion scores. Antibody reactivity to surface antigens which were significantly correlated with resistance and present in two or more of the preparations were detected at 86, 66, 51, 49, 34, 31, and 16 kilodaltons (kDa). Of these, antibody responses to antigens at 86, 49, and 31 kDa appeared most important based on their concentration and significance levels. Antibody reactivity to leukotoxin antigens which were significantly correlated with resistance and common with important surface antigens were detected at 86, 66, and 49 kDa. Antibody responses to unique leukotoxin antigens which were significantly correlated with resistance were present at 92 and 58 kDa. Images PMID:2917783

  13. Periodontal pathogens in erupting third molars of periodontally healthy subjects.

    PubMed

    Rajasuo, A; Sihvonen, O J; Peltola, M; Meurman, J H

    2007-09-01

    The presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythensis in bacteriologic samples of 5-7-mm deep mandibular third-molar pericoronal pockets was analysed by polymerase chain reaction, to test the hypothesis that these sites would harbour the bacteria. The patients were periodontally healthy 20-year-old Finnish male conscripts. Sixteen had acute pericoronitis, 28 chronic pericoronitis, and 15 were symptom-free controls. A. actinomycetemcomitans was detected in only 7% of the samples from chronic pericoronitis cases, whereas P. gingivalis was positive in 20% of the symptom-free versus 69% (P = 0.018) of the acute and 57% (P = 0.044) of the chronic cases. The percentages for P. intermedia were 93, 94 and 93%, and for T. forsythensis 47, 63 and 57%, respectively. These results confirm that, apart from A. actinomycetemcomitans, periodontopathogens are common in third-molar sites in periodontally healthy individuals.

  14. Eosinophilic granuloma with Splendore-Hoeppli material caused by Mannheimia granulomatis in a calf.

    PubMed

    Kawashima, Yuuto; Takahashi, Hiroyasu; Shimoo, Megumi; Tamamura, Yukino; Ishikawa, Yoshiharu; Kadota, Koichi

    2016-07-01

    A large subcutaneous mass, formed on the left lower jaw of a 4-month-old Japanese Black male calf, was partially excised for histological and bacteriological examinations. Antibiotic treatment resulted in a good prognosis. Bacteria isolated from the excised material were characterized by weak hemolysis and positive reactions for catalase and oxidase, and were 99% identical to Mannheimia granulomatis strains. The presence of the leukotoxin gene product was demonstrated by polymerase chain reaction amplification. Histological examination showed that the excised material was composed of dense fibrous connective tissue with sparsely distributed eosinophilic granulomas or abscesses. These foci frequently contained Splendore-Hoeppli material with rod-shaped Gram-negative bacteria. Except for the absence of lymphangitis and the presence of basophils and mast cells, the histology of this lesion resembled that of lechiguana associated with coinfection of M. granulomatis and Dermatobia hominis. Leukotoxin was demonstrated by immunohistochemistry within Splendore-Hoeppli material and was judged to be responsible for its formation. PMID:26947171

  15. Calcium-Driven Folding of RTX Domain β-Rolls Ratchets Translocation of RTX Proteins through Type I Secretion Ducts.

    PubMed

    Bumba, Ladislav; Masin, Jiri; Macek, Pavel; Wald, Tomas; Motlova, Lucia; Bibova, Ilona; Klimova, Nela; Bednarova, Lucie; Veverka, Vaclav; Kachala, Michael; Svergun, Dmitri I; Barinka, Cyril; Sebo, Peter

    2016-04-01

    Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca(2+)-depleted cytosol of Gram-negative bacteria directly into the Ca(2+)-rich external milieu, passing through the "channel-tunnel" ducts of type I secretion systems (T1SSs). Using Bordetella pertussis adenylate cyclase toxin, we solved the structure of an essential C-terminal assembly that caps the RTX domains of RTX family leukotoxins. This is shown to scaffold directional Ca(2+)-dependent folding of the carboxy-proximal RTX repeat blocks into β-rolls. The resulting intramolecular Brownian ratchets then prevent backsliding of translocating RTX proteins in the T1SS conduits and thereby accelerate excretion of very large RTX leukotoxins from bacterial cells by a vectorial "push-ratchet" mechanism. Successive Ca(2+)-dependent and cosecretional acquisition of a functional RTX toxin structure in the course of T1SS-mediated translocation, through RTX domain folding from the C-terminal cap toward the N terminus, sets a paradigm that opens for design of virulence inhibitors of major pathogens.

  16. Residues Essential for Panton-Valentine Leukocidin S Component Binding to Its Cell Receptor Suggest Both Plasticity and Adaptability in Its Interaction Surface

    PubMed Central

    Laventie, Benoit-Joseph; Guérin, Frédéric; Mourey, Lionel; Tawk, Mira Y.; Jover, Emmanuel; Maveyraud, Laurent; Prévost, Gilles

    2014-01-01

    Panton-Valentine leukocidin (PVL), a bicomponent staphylococcal leukotoxin, is involved in the poor prognosis of necrotizing pneumonia. The present study aimed to elucidate the binding mechanism of PVL and in particular its cell-binding domain. The class S component of PVL, LukS-PV, is known to ensure cell targeting and exhibits the highest affinity for the neutrophil membrane (Kd∼10−10 M) compared to the class F component of PVL, LukF-PV (Kd∼10−9 M). Alanine scanning mutagenesis was used to identify the residues involved in LukS-PV binding to the neutrophil surface. Nineteen single alanine mutations were performed in the rim domain previously described as implicated in cell membrane interactions. Positions were chosen in order to replace polar or exposed charged residues and according to conservation between leukotoxin class S components. Characterization studies enabled to identify a cluster of residues essential for LukS-PV binding, localized on two loops of the rim domain. The mutations R73A, Y184A, T244A, H245A and Y250A led to dramatically reduced binding affinities for both human leukocytes and undifferentiated U937 cells expressing the C5a receptor. The three-dimensional structure of five of the mutants was determined using X-ray crystallography. Structure analysis identified residues Y184 and Y250 as crucial in providing structural flexibility in the receptor-binding domain of LukS-PV. PMID:24643034

  17. Non-haemolytic Mannheimia haemolytica as a cause of pleuropneumonia and septicemia in a calf.

    PubMed

    Mahu, Maxime; Valgaeren, Bonnie; Pardon, Bart; Deprez, Piet; Haesebrouck, Freddy; Boyen, Filip

    2015-10-22

    Pure cultures of non-haemolytic Mannheimia haemolytica, were cultivated from pleural effusion fluid and blood from a 1-month old Belgian Blue bull calf that was presented with apathy and anorexia. The isolates were identified as M. haemolytica by 16S rRNA gene sequencing and MALDI-TOF-MS. Since haemolysis on blood agar plates is considered a hallmark of M. haemolytica we wanted to elucidate the unusual phenotype of the isolated strain. Therefore the leukotoxin operon (lktCABD), responsible for the haemolytic phenotype of M. haemolytica and regarded as the most important virulence factor, was completely sequenced. The leukotoxin operon of the isolated strain showed a deletion in the lktA gene, resulting in a truncated LktA protein. The absence of a complete LktA protein is responsible for the non-haemolytic phenotype of the strain. To the best of our knowledge, this is the first report of a well-characterized non-haemolytic M. haemolytica isolate causing disease in cattle. PMID:26344042

  18. Fusobacterium necrophorum infections: virulence factors, pathogenic mechanism and control measures.

    PubMed

    Tan, Z L; Nagaraja, T G; Chengappa, M M

    1996-01-01

    Fusobacterium necrophorum, a Gram-negative, non-spore-forming anaerobe, is a normal inhabitant of the alimentary tract of animals and humans. Two types of F. necrophorum, subspecies necrophorum (biotype A) and funduliforme (biotype B), have been recognized, which differ morphologically, biochemically, and biologically. The organism is an opportunistic pathogen that causes numerous necrotic conditions (necrobacillosis) such as bovine hepatic abscesses, ruminant foot abscesses and human oral infections. The pathogenic mechanism of F. necrophorum is complex and not well defined. Several toxins, such as leukotoxin, endotoxin, haemolysin, haemagglutinin and adhesin, have been implicated as virulence factors. Among these, leukotoxin and endotoxin are believed to be more important than other toxins in overcoming the host's defence mechanisms to establish the infection. F. necrophorum is encountered frequently in mixed infections and, therefore, synergisms between F. necrophorum and other pathogens may play an important role in infection. Several investigators have attempted to induce protective immunity against F. necrophorum using bacterins, toxoids, and other cytoplasmic components. Generally, none of the immunogens has afforded satisfactory protection against Fusobacterium infections. Because of the unavailability of suitable immunoprophylaxis, the control of F. necrophorum infection has depended mainly on the use of antimicrobial compounds.

  19. Eosinophilic granuloma with Splendore-Hoeppli material caused by Mannheimia granulomatis in a calf

    PubMed Central

    KAWASHIMA, Yuuto; TAKAHASHI, Hiroyasu; SHIMOO, Megumi; TAMAMURA, Yukino; ISHIKAWA, Yoshiharu; KADOTA, Koichi

    2016-01-01

    A large subcutaneous mass, formed on the left lower jaw of a 4-month-old Japanese Black male calf, was partially excised for histological and bacteriological examinations. Antibiotic treatment resulted in a good prognosis. Bacteria isolated from the excised material were characterized by weak hemolysis and positive reactions for catalase and oxidase, and were 99% identical to Mannheimia granulomatis strains. The presence of the leukotoxin gene product was demonstrated by polymerase chain reaction amplification. Histological examination showed that the excised material was composed of dense fibrous connective tissue with sparsely distributed eosinophilic granulomas or abscesses. These foci frequently contained Splendore-Hoeppli material with rod-shaped Gram-negative bacteria. Except for the absence of lymphangitis and the presence of basophils and mast cells, the histology of this lesion resembled that of lechiguana associated with coinfection of M. granulomatis and Dermatobia hominis. Leukotoxin was demonstrated by immunohistochemistry within Splendore-Hoeppli material and was judged to be responsible for its formation. PMID:26947171

  20. Bivalent vaccination against pneumonic pasteurellosis in domestic sheep and goats with modified-live in-frame lktA deletion mutants of Mannheimia haemolytica.

    PubMed

    Briggs, Robert E; Hauglund, Melissa J; Maheswaran, Samuel K; Tatum, Fred M

    2013-11-01

    A temperature-sensitive shuttle vector, pBB80C, was utilized to generate in-frame deletion mutants of the leukotoxin structural gene (lktA) of Mannheimia haemolytica serotypes 1, 2, 5, 6, 7, 8, 9, and 12. Culture supernatants from the mutants contained a truncated protein with an approximate molecular weight of 66 kDa which was reactive to anti-leukotoxin monoclonal antibody. No protein reactive to anti-LktA monoclonal antibody was detected at the molecular weight 100-105 kDa of native LktA. Sheep and goats vaccinated intramuscularly with a mixture of serotypes 5 and 6 mutants were resistant to virulent challenge with a mixture of the wild-type parent strains. These vaccinates responded serologically to both vaccine serotypes and exhibited markedly-reduced lung lesion volume and pulmonary infectious load compared to control animals. Control animals yielded a mixture of serotypes from lung lobes, but the proportion even within an individual animal varied widely from 95% serotype 5-95% serotype 6. Cultures recovered from liver were homogeneous, but two animals yielded serotype 5 and the other two yielded serotype 6 in pure culture.

  1. Calcium-Driven Folding of RTX Domain β-Rolls Ratchets Translocation of RTX Proteins through Type I Secretion Ducts.

    PubMed

    Bumba, Ladislav; Masin, Jiri; Macek, Pavel; Wald, Tomas; Motlova, Lucia; Bibova, Ilona; Klimova, Nela; Bednarova, Lucie; Veverka, Vaclav; Kachala, Michael; Svergun, Dmitri I; Barinka, Cyril; Sebo, Peter

    2016-04-01

    Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca(2+)-depleted cytosol of Gram-negative bacteria directly into the Ca(2+)-rich external milieu, passing through the "channel-tunnel" ducts of type I secretion systems (T1SSs). Using Bordetella pertussis adenylate cyclase toxin, we solved the structure of an essential C-terminal assembly that caps the RTX domains of RTX family leukotoxins. This is shown to scaffold directional Ca(2+)-dependent folding of the carboxy-proximal RTX repeat blocks into β-rolls. The resulting intramolecular Brownian ratchets then prevent backsliding of translocating RTX proteins in the T1SS conduits and thereby accelerate excretion of very large RTX leukotoxins from bacterial cells by a vectorial "push-ratchet" mechanism. Successive Ca(2+)-dependent and cosecretional acquisition of a functional RTX toxin structure in the course of T1SS-mediated translocation, through RTX domain folding from the C-terminal cap toward the N terminus, sets a paradigm that opens for design of virulence inhibitors of major pathogens. PMID:27058787

  2. Microbiological and serological investigations of oral lesions in Papillon-Lefèvre syndrome.

    PubMed Central

    Clerehugh, V; Drucker, D B; Seymour, G J; Bird, P S

    1996-01-01

    Microbiological and serological (enzyme linked immunosorbent assay) investigations were carried out, including karyotyping, on two Asian children with Papillon-Lefèvre syndrome. In case 1, a girl aged four years, the most prevalent putative periodontopathogens were Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia (deciduous dentition) and Bacteroides gracilis, E corrodens and F nucleatum (permanent dentition). In case 2, a boy aged nine years, they were F nucleatum, P intermedia and P loeschii and E corrodens. Serum from case 2 showed a raised specific IgG antibody response to Actinomyces actino-mycetemcomitans serotype b. Thus, a wider range of species than hitherto reported may be associated with Papillon-Lefèvre syndrome, including A actino-mycetemcomitans and F nucleatum. PMID:8675741

  3. Monocyte chemoattractant protein 1 and interleukin-8 production in mononuclear cells stimulated by oral microorganisms.

    PubMed Central

    Jiang, Y; Russell, T R; Graves, D T; Cheng, H; Nong, S H; Levitz, S M

    1996-01-01

    Chemokines are a family of low-molecular-weight proinflammatory cytokines that stimulate recruitment of leukocytes. The chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) are relatively specific chemoattractants for neutrophils and monocytes, respectively. Chemokine expression contributes to the presence of different leukocyte populations observed in normal and pathologic states. In the present studies, peripheral blood mononuclear cells (PBMC) were stimulated by microbes (Candida albicans, Streptococcus mutans, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans) selected based upon their importance as oral pathogens. IL-8 and MCP-1 gene expression and protein release were determined by Northern blot (RNA blot) analysis and enzyme-linked immunosorbent assay. C. albicans, P. gingivalis, and A. actinomycetemcomitans induced high levels of production of both MCP-1 and IL-8. S. mutans was a strong inducer of MCP-1, but it did not stimulate significant production of IL-8. C. albicans, S. mutans, and A. actinomycetemcomitans were 500 to 5,000 times more potent than P. gingivalis in terms of MCP-1 production. In general, the microbe-to-PBMC ratios required for maximum gene expression of MCP-1 were lower than those for IL-8. However, for actual protein release of MCP-1 versus IL-8, differences in the effects of various microbe concentrations were observed only for A. actinomycetemcomitans. These results demonstrate that different oral pathogens induce specific dose-dependent patterns of chemokine gene expression and release. Such patterns may help explain the immunopathology of oral infections, particularly with regard to inflammatory leukocyte recruitment. PMID:8890191

  4. In vitro sensitivity of oral, gram-negative, facultative bacteria to the bactericidal activity of human neutrophil defensins.

    PubMed Central

    Miyasaki, K T; Bodeau, A L; Ganz, T; Selsted, M E; Lehrer, R I

    1990-01-01

    Neutrophils play a major role in defending the periodontium against infection by oral, gram-negative, facultative bacteria, such as Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. We examined the sensitivity of these bacteria to a mixture of low-molecular-weight peptides and highly purified individual defensin peptides (HNP-1, HNP-2, and HNP-3) isolated from human neutrophils. Whereas the Capnocytophaga spp. strains were killed significantly by the mixed human neutrophil peptides, the A. actinomycetemcomitans and E. corrodens strains were resistant. Killing was attributable to the defensins. The bactericidal activities of purified defensins HNP-1 and HNP-2 were equal, and both of these activities were greater than HNP-3 activity against strains of Capnocytophaga sputigena and Capnocytophaga gingivalis. The strain of Capnocytophaga ochracea was more sensitive to defensin-mediated bactericidal activity than either C. sputigena or C. gingivalis was. The three human defensins were equipotent in killing C. ochracea. C. ochracea was killed under aerobic and anaerobic conditions and over a broad pH range. Killing was most effective under hypotonic conditions but also occurred at physiologic salt concentrations. We concluded that Capnocytophaga spp. are sensitive to oxygen-independent killing by human defensins. Additional studies will be required to identify other components that may equip human neutrophils to kill A. actinomycetemcomitans, E. corrodens, and other oral gram-negative bacteria. Images PMID:2254020

  5. Lethal photosensitization for decontamination of implant surfaces in the treatment of peri-implantitis.

    PubMed

    Dörtbudak, O; Haas, R; Bernhart, T; Mailath-Pokorny, G

    2001-04-01

    Peri-implantitis is considered to be a multifactorial process involving bacterial contamination of the implant surface. A previous study demonstrated that a combination of toluidine blue O (100 microgram/ml) and irradiation with a diode soft laser with a wavelength of 905 nm results in an elimination of Porphyromonas gingivalis (P. gingivalis), Prevotella intermedia (P. intermedia), and Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) on different implant surfaces (machined, plasma-flame-sprayed, etched, hydroxyapatite-coated). The aim of this study was to examine the laser effect in vivo. In 15 patients with IMZ implants who showed clinical and radiographic signs of peri-implantitis, toluidine blue O was applied to the implant surface for 1 min and the surface was then irradiated with a diode soft laser with a wavelength of 690 nm for 60 s. Bacterial samples were taken before and after application of the dye and after lasing. The cultures were evaluated semiquantitatively for A. actinomycetemcomitans, P. gingivalis, and P. intermedia. It was found that the combined treatment reduced the bacterial counts by 2 log steps on average. The application of TBO and laser resulted in a significant reduction (P<0.0001) of the initial values in all 3 groups of bacteria. Complete elimination of bacteria was not achieved.

  6. The tad locus: postcards from the widespread colonization island.

    PubMed

    Tomich, Mladen; Planet, Paul J; Figurski, David H

    2007-05-01

    The Tad (tight adherence) macromolecular transport system, which is present in many bacterial and archaeal species, represents an ancient and major new subtype of type II secretion. The tad genes are present on a genomic island named the widespread colonization island (WCI), and encode the machinery that is required for the assembly of adhesive Flp (fimbrial low-molecular-weight protein) pili. The tad genes are essential for biofilm formation, colonization and pathogenesis in the genera Aggregatibacter (Actinobacillus), Haemophilus, Pasteurella, Pseudomonas, Yersinia, Caulobacter and perhaps others. Here we review the structure, function and evolution of the Tad secretion system.

  7. The detection of Dichelobacter nodosus and Fusobacterium necrophorum from ovine footrot in Kashmir, India.

    PubMed

    Farooq, Shaheen; Wani, Shakil A; Hassan, Mir Nadeem; Nazir, Nazima; Nyrah, Qazi Javed

    2015-10-01

    In a study conducted, a total of 450 swab samples from footrot lesions of naturally infected sheep were collected in all the ten districts of the Kashmir valley and were examined for the presence of Dichelobacter nodosus (D. nodosus) and Fusobacterium necrophorum (F. necrophorum), in order to determine if F. necrophorum was associated with ovine footrot. The detection of F. necrophorum and D. nodosus was carried out by polymerase chain reaction targeting the leukotoxin (lktA) and 16S rRNA genes, respectively. In this study, only less than 50% of positive samples contained both the bacteria, so it is not possible to conclude with certainty that both bacteria are together required for the disease manifestation.

  8. Serum IgG response in calves to the putative pneumonic virulence factor Gs60 of Mannheimia haemolytica A1.

    PubMed

    Orouji, Shahriar; Hodgins, Douglas C; Lo, Reggie Y C; Shewen, Patricia E

    2012-10-01

    Bovine pneumonic pasteurellosis vaccines incorporate various antigens of Mannheimia haemolytica, including the acknowledged virulence factor leukotoxin (Lkt), and Gs60, a surface lipoprotein. To examine the role of antibodies to Gs60 in protection, an enzyme-linked immunosorbent assay (ELISA) was developed for retrospective analysis of serum samples from previous trials in which vaccines containing native or recombinant Gs60 were administered parenterally. The analysis revealed a positive correlation between the titer of antibodies to Gs60 and protection against experimental challenge in both vaccinates and naturally exposed controls. There was a strong correlation between production of IgG antibodies to Gs60 and Lkt neutralizing antibodies. Analysis of the relationship between the serum antibody titers and resistance to experimental challenge using linear statistical models revealed a significant association between prechallenge titers of serum antibodies to Lkt and protection. Further analysis suggested that antibodies against Gs60 were beneficial when Lkt neutralizing antibody titers were low.

  9. LKTA and PlpE small fragments fusion protein protect against Mannheimia haemolytica challenge.

    PubMed

    Guzmán-Brambila, Carolina; Quintero-Fabián, Saray; González-Castillo, Celia; de Obeso-Fernández del Valle, Álvaro; Flores-Samaniego, Beatriz; de la Mora, Germán; Rojas-Mayorquín, Argelia E; Ortuño-Sahagún, Daniel

    2012-12-01

    Bovine respiratory disease (BRD) complex is a major cause of economic losses for the cattle backgrounding and feedlot industries. Mannheimia haemolytica is considered the most important pathogen associated with this disease. Vaccines against M. haemolytica have been prepared and used for many decades, but traditional bacterins have failed to demonstrate effective protection and their use has often exacerbated disease in vaccinated animals. Thus, the BRD complex continues to exert a strong adverse effect on the health and wellbeing of stocker and feeder cattle. Therefore, generation of recombinant proteins has been helpful in formulating enhanced vaccines against M. haemolytica, which could confer better protection against BRD. In the present study, we formulated a vaccine preparation enriched with recombinant small fragments of leukotoxin A (LKTA) and outer-membrane lipoprotein (PlpE) proteins, and demonstrated its ability to generate high antibody titers in rabbits and sheep, which protected against M. haemolytica bacterial challenge in mice. PMID:22840333

  10. Staphylococcus aureus LukAB cytotoxin kills human neutrophils by targeting the CD11b subunit of the integrin Mac-1

    PubMed Central

    DuMont, Ashley L.; Yoong, Pauline; Day, Christopher J.; Alonzo, Francis; McDonald, W. Hayes; Jennings, Michael P.; Torres, Victor J.

    2013-01-01

    Staphylococcus aureus causes diseases ranging from superficial wound infections to more invasive manifestations like osteomyelitis and endocarditis. The evasion of host phagocytes recruited to the site of infection is essential to the success of S. aureus as a pathogen. A single S. aureus strain can produce up to five different bicomponent pore-forming leukotoxins that lyse immune cells by forming pores in the cellular plasma membrane. Although these leukotoxins have been considered redundant due to their cytotoxic activity toward human neutrophils, each toxin displays varied species and cell-type specificities. This suggests that cellular factors may influence which cells each toxin targets. Here we describe the identification of CD11b, the α subunit of the αM/β2 integrin (CD11b/CD18), macrophage-1 antigen, or complement receptor 3, as a cellular receptor for leukocidin A/B (LukAB), an important toxin that contributes to S. aureus killing of human neutrophils. We demonstrate that CD11b renders human neutrophils susceptible to LukAB-mediated killing by purified LukAB as well as during S. aureus infection ex vivo. LukAB directly interacts with human CD11b by binding to the I domain, a property that determines the species specificity exhibited by this toxin. Identification of a LukAB cellular target has broad implications for the use of animal models to study the role of LukAB in S. aureus pathogenesis, explains the toxin’s tropism toward human neutrophils and other phagocytes, and provides a cellular therapeutic target to block the effect of LukAB toward human neutrophils. PMID:23754403

  11. Bighorn sheep × domestic sheep hybrids survive Mannheimia haemolytica challenge in the absence of vaccination.

    PubMed

    Subramaniam, R; Shanthalingam, S; Bavananthasivam, J; Kugadas, A; Raghavan, B; Batra, S A; Herndon, C N; Rodriguez, J; Tibary, A; Nelson, D; Potter, K A; Foreyt, W J; Srikumaran, S

    2014-06-01

    Bighorn sheep (BHS, Ovis canadensis) are much more susceptible than domestic sheep (DS, Ovis aries) to pneumonia caused by leukotoxin (Lkt)-producing members of the Family Pasteurellaceae, particularly Mannheimia haemolytica and Bibersteinia trehalosi. Leukotoxin is widely accepted as the critical virulence factor of these bacteria since Lkt-negative mutants do not cause death of BHS. Typically, DS carry Lkt-positive M. haemolytica and/or B. trehalosi as commensal bacteria in their nasopharynx. In contrast, most BHS do not carry Lkt-positive M. haemolytica or B. trehalosi, or carry Lkt-negative strains in their nasopharynx. In previous studies, we demonstrated that unimmunized DS resist M. haemolytica challenge while BHS succumb to it. We hypothesized that Lkt-neutralizing antibodies, induced by Lkt-positive M. haemolytica and/or B. trehalosi innately carried by DS in their nasopharynx, render them less susceptible to infection by these bacteria. In this study we developed BHS×DS F1 hybrids by artificial insemination of domestic ewes with BHS semen. F1 hybrids were fertile, and produced F2 hybrids and back-crosses. The F1, F2, and back-crosses were raised together with domestic ewes. All these animals acquired Lkt-positive M. haemolytica and/or B. trehalosi, and developed high titers of Lkt-neutralizing antibodies in the absence of vaccination. Furthermore, all of these animals resisted challenge with lethal dose of M. haemolytica. These results suggest that lack of previous exposure to Lkt is at least partially responsible for fatal pneumonia in BHS when they acquire Lkt-positive M. haemolytica and/or B. trehalosi from DS when the two species commingle.

  12. Structurally designed attenuated subunit vaccines for S. aureus LukS-PV and LukF-PV confer protection in a mouse bacteremia model.

    PubMed

    Karauzum, Hatice; Adhikari, Rajan P; Sarwar, Jawad; Devi, V Sathya; Abaandou, Laura; Haudenschild, Christian; Mahmoudieh, Mahta; Boroun, Atefeh R; Vu, Hong; Nguyen, Tam; Warfield, Kelly L; Shulenin, Sergey; Aman, M Javad

    2013-01-01

    Previous efforts towards S. aureus vaccine development have largely focused on cell surface antigens to induce opsonophagocytic killing aimed at providing sterile immunity, a concept successfully applied to other Gram-positive pathogens such as Streptococcus pneumoniae. However, these approaches have largely failed, possibly in part due to the remarkable diversity of the staphylococcal virulence factors such as secreted immunosuppressive and tissue destructive toxins. S. aureus produces several pore-forming toxins including the single subunit alpha hemolysin as well as bicomponent leukotoxins such as Panton-Valentine leukocidin (PVL), gamma hemolysins (Hlg), and LukED. Here we report the generation of highly attenuated mutants of PVL subunits LukS-PV and LukF-PV that were rationally designed, based on an octameric structural model of the toxin, to be deficient in oligomerization. The attenuated subunit vaccines were highly immunogenic and showed significant protection in a mouse model of S. aureus USA300 sepsis. Protection against sepsis was also demonstrated by passive transfer of rabbit immunoglobulin raised against LukS-PV. Antibodies to LukS-PV inhibited the homologous oligomerization of LukS-PV with LukF-PV as well heterologous oligomerization with HlgB. Importantly, immune sera from mice vaccinated with the LukS mutant not only inhibited the PMN lytic activity produced by the PVL-positive USA300 but also blocked PMN lysis induced by supernatants of PVL-negative strains suggesting a broad protective activity towards other bicomponent toxins. These findings strongly support the novel concept of an anti-virulence, toxin-based vaccine intended for prevention of clinical S. aureus invasive disease, rather than achieving sterile immunity. Such a multivalent vaccine may include attenuated leukotoxins, alpha hemolysin, and superantigens. PMID:23762356

  13. Anti-microbial Activity of Tulsi {Ocimum Sanctum (Linn.)} Extract on a Periodontal Pathogen in Human Dental Plaque: An Invitro Study

    PubMed Central

    Devaraj, C.G.; Agarwal, Payal

    2016-01-01

    Introduction Tulsi is a popular healing herb in Ayurvedic medicine. It is widely used in the treatment of several systemic diseases because of its anti-microbial property. However, studies documenting the effect of Tulsi on oral disease causing organisms are rare. Hence, an attempt was made to determine the effect of Tulsi on a periodontal microorganism in human dental plaque. Aim To determine if Ocimum sanctum (Linn.) has an anti-microbial activity (Minimum Inhibitory Concentration and zone of inhibition) against Actinobacillus actinomycetemcomitans in human dental plaque and to compare the antimicrobial activity of Ocimum sanctum(Linn.) extract with 0.2% chlorhexidine as the positive control and dimethyl sulfoxide as the negative control. Materials and Methods A lab based invitro experimental study design was adopted. Ethanolic extract of Ocimum sanctum (Linn.) was prepared by the cold extraction method. The extract was diluted with an inert solvent, dimethyl sulfoxide, to obtain ten different concentrations (1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%) of extract. Plaque sample was collected from 05 subjects diagnosed with periodontal disease. Isolation of Actinobacillus actinomycetemcomitans from plaque samples was done using Tryptic Soy Serum Bacitracin Vancomycin agar (TSBV) medium. Identification of Actinobacillus actinomycetemcomitans was done based on cultural, microscopic, biochemical characterization and multiple drug resistance patterns. Anti-microbial activity of Ocimum sanctum (Linn.) extract was tested by agar well-diffusion method against 0.2% chlorhexidine as a positive control and dimethyl sulfoxide as a negative control. The zone of inhibition was measured in millimeters using Vernier callipers. Results At the 6% w/v concentration of Ocimum sanctum (Linn.) extract, a zone of inhibition of 22 mm was obtained. This was the widest zone of inhibition observed among all the 10 different concentrations tested. The zone of inhibition for positive control

  14. Project 1: Microbial Genomes: A Genomic Approach to Understanding the Evolution of Virulence. Project 2: From Genomes to Life: Drosophilia Development in Space and Time

    SciTech Connect

    Robert DeSalle

    2004-09-10

    This project seeks to use the genomes of two close relatives, A. actinomycetemcomitans and H. aphrophilus, to understand the evolutionary changes that take place in a genome to make it more or less virulent. Our primary specific aim of this project was to sequence, annotate, and analyze the genomes of Actinobacillus actinomycetemcomitans (CU1000, serotype f) and Haemophilus aphrophilus. With these genome sequences we have then compared the whole genome sequences to each other and to the current Aa (HK1651 www.genome.ou.edu) genome project sequence along with other fully sequenced Pasteurellaceae to determine inter and intra species differences that may account for the differences and similarities in disease. We also propose to create and curate a comprehensive database where sequence information and analysis for the Pasteurellaceae (family that includes the genera Actinobacillus and Haemophilus) are readily accessible. And finally we have proposed to develop phylogenetic techniques that can be used to efficiently and accurately examine the evolution of genomes. Below we report on progress we have made on these major specific aims. Progress on the specific aims is reported below under two major headings--experimental approaches and bioinformatics and systematic biology approaches.

  15. Immunoglobulin G response to subgingival gram-negative bacteria in human subjects.

    PubMed Central

    Naito, Y; Okuda, K; Takazoe, I

    1984-01-01

    Serum and gingival crevicular fluid from normal healthy adults and patients with periodontitis were screened for immunoglobulin G antibodies to antigens from Bacteroides gingivalis 381, Bacteroides intermedius 24, Bacteroides loescheii ATCC 15930, Fusobacterium nucleatum ATCC 25586, Eikenella corrodens 1073, Actinobacillus actinomycetemcomitans ATCC 29522, and Capnocytophaga sp. strain M-12. Immunoglobulin G antibody titers to the antigens were measured by an enzyme-linked immunosorbent assay. The antibody levels to B. gingivalis in serum and gingival crevicular fluid were significantly higher in the samples from patients with periodontitis than in samples from healthy individuals. Although there were individual differences within patient groups, a positive correlation (P less than 0.01) was found between the serum immunoglobulin G levels to B. gingivalis and the development of periodontitis. The antibodies to F. nucleatum (P less than 0.05), E. corrodens (P less than 0.05), and A. actinomycetemcomitans were slightly higher in patients with periodontitis than in normal subjects. There were no remarkable differences between the two groups in titers to B. intermedius, B. loescheii, and Capnocytophaga sp. PMID:6735473

  16. Natural occurrence of black-pigmented Bacteroides species in the gingival crevice of the squirrel monkey.

    PubMed Central

    Clark, W B; Magnusson, I; Abee, C; Collins, B; Beem, J E; McArthur, W P

    1988-01-01

    The objective of this study was to determine whether the squirrel monkey (Saimiri scuireus) is indigenously colonized with black-pigmented bacteroides (BPB) resembling human Bacteroides gingivalis and Bacteroides intermedius (suspected periodontal pathogens) and to determine the usefulness of the squirrel monkey as an in vivo model for studying colonization by putative pathogens. We assayed the subgingival plaques of 138 monkeys of various ages and in four different colonies for the presence of anaerobic BPB microorganisms. We also tested half the animals for the presence of Actinobacillus actinomycetemcomitans. Clinical indices and levels of serum antibody to B. gingivalis were recorded. We detected BPB in 50% of the animals and A. actinomycetemcomitans in 69% of the animals. The presence of BPB was generally associated with increased age, increased gingival index, presence of calculus, and increased levels of serum antibody. These data indicate that the squirrel monkey may be a good model for studying the parameters of natural infection of the gingival crevice with suspected periodontopathogenic BPB microorganisms. PMID:3410543

  17. Periodontal profile and presence of periodontal pathogens in young African-Americans from Salvador, Ba, Brazil

    PubMed Central

    Victor, Ligia Valéria; Cortelli, Sheila Cavalca; Aquino, Davi Romeiro; de Carvalho Filho, Jonas; Cortelli, José Roberto

    2008-01-01

    This cross-sectional study evaluated the periodontal status and the presence of periodontopathogens in 132 young, black ethnic subjects who live in Salvador/Bahia-Brazil and have never smoked. Periodontal Probing Depth (PPD), Clinical Attachment Level (CAL), Plaque Index (PI) and Gingival Index (GI) were measured and analyzed by ANOVA and Wilcoxon tests (p<0.05) according to gender and age. The presence of A.actinomycetemcomitans, P.gingivalis, E.corrodens and F.nucleatum was determined by PCR and was analyzed by ANOVA, Wilcoxon, Student-t tests (p<0.05). Mean values of PPD and CAL were 2.18 and 1.0mm, respectively. Clinical parameters did not show differences between subjects of varying gender and age. The microbial prevalence was observed to be 95.45% for E.corrodens followed by F.nucleatum with 68.18%, A.actinomycetemcomitans with 45.45% and P gingivalis with 40.9%. An association between the presence of pathogens and gender and age was not observed (p<0.05). PPD, CAL and PI were not associated with P.gingivalis; however, GI appeared in higher frequencies among subjects without P.gingivalis. In this young, black ethnic, Brazilian population, a high percentage (96.96%) of subjects harbored at least one selected periodontal pathogen, but most subjects showed a healthy periodontal status. Further investigations are required to evaluate the actual influence of the presence of these bacterial species. PMID:24031206

  18. Antibacterial potential of Manuka honey against three oral bacteria in vitro.

    PubMed

    Schmidlin, PatrickSchmidlin R; English, Helen; Duncan, Warwick; Belibasakis, Georgios N; Thurnheer, Thomas

    2014-01-01

    Honey is an ancient natural remedy for the treatment of infected wounds. It has regained attention in the medical profession, as it has recently been reported to have a broad-spectrum inhibitory effect against bacteria. Data concerning Manuka honey of New Zealand origin, which is claimed to provide additional non-peroxide antimicrobial activity (so-called standard NPA) against oral pathogens, is still scarce. Therefore, this study aimed to screen for the antibacterial efficacy of different Manuka honey products against S. mutans (OMZ 918), P. gingivalis (OMZ 925) and A. actinomycetemcomitans (OMZ 299). Chlorhexidine and saline served as positive and negative controls, respectively, whereas a Swiss multifloral honey served as control honey without intrinsic non-peroxide activity. Chlorhexidine showed the highest inhibiting potential against all specimens tested. Manuka honey below an NPA value of 15 showed the least bacterial growth-inhibiting potential, even less – although not significantly so – than multifloral Swiss honey. Manuka honey above an NPA value of 15 showed a significantly higher antibacterial effect compared to the other honeys tested. All Manuka honey preparations were more effective in inhibiting the growth of P. gingivalis and A. actinomycetemcomitans, rather than S. mutans. In conclusion, the study showed an NPA dose-dependent antibacterial efficacy of Manuka honey. Further investigations of this natural product are now open for scrutiny. PMID:25253413

  19. Uptake of photosensitizers by bacteria is influenced by the presence of cations

    NASA Astrophysics Data System (ADS)

    Kishen, A.; George, S.

    2007-05-01

    This investigation studies the influence of cations on photosensitizer uptake by Enterococcus faecalis (gram positive) and Actinobacillus actinomycetemcomitans (gram negative). Methods- The uptake of Methylene blue (MB) and Indocyanine Green (ICG), by bacteria were studied under the influence of divalent cations (CaCl II & MgCl II) and EDTA. Further, E. faecalis cells subjected to trypsinisation and calcium channel blocker (verapamil) were also analysed for MB and ICG uptake inorder to understand the mechanism of photosensitizer uptake. Results- Uptake of ICG was enhanced in the presence of divalent cations in E. faecalis and A. actinomycetemcomitans. Treating cells with EDTA had no significant effect on the photosensitizer uptake, although the highest concentration tested showed an enhancement of uptake. In contrast to ICG, MB showed a decreased uptake by bacterial cells on subjecting them to divalent cations and EDTA. Calcium channel blocker had no significant inhibitory effect on photosensitizers uptake. However, trypsin treatment resulted in significant reduction of ICG uptake. The result suggested that ICG uptake by bacteria is mediated through specific transporter protein while MB is associated with the outer surface structures of bacterial cells.

  20. Antibacterial Activity of As-Annealed TiO2 Nanotubes Doped with Ag Nanoparticles against Periodontal Pathogens.

    PubMed

    Yeniyol, Sinem; He, Zhiming; Yüksel, Behiye; Boylan, Robert Joseph; Urgen, Mustafa; Ozdemir, Tayfun; Ricci, John Lawrence

    2014-01-01

    It is important to develop functional transmucosal implant surfaces that reduce the number of initially adhering bacteria and they need to be modified to improve the anti-bacterial performance. Commercially pure Ti sheets were anodized in an electrolyte containing ethylene glycol, distilled water and ammonium fluoride at room temperature to produce TiO2 nanotubes. These structures were then annealed at 450°C to transform them to anatase. As-annealed TiO2 nanotubes were then treated in an electrolyte containing 80.7 g/L NiSO4 ·7H2O, 41 g/L MgSO4 ·7H2O, 45 g/L H3BO3, and 1.44 g/L Ag2SO4 at 20°C by the application of 9 V AC voltage for doping them with silver. As-annealed TiO2 nanotubes and as-annealed Ag doped TiO2 nanotubes were evaluated by SEM, FESEM, and XRD. Antibacterial activity was assessed by determining the adherence of A. actinomycetemcomitans, T. forsythia, and C. rectus to the surface of the nanotubes. Bacterial morphology was examined using an SEM. As-annealed Ag doped TiO2 nanotubes revealed intense peak of Ag. Bacterial death against the as-annealed Ag doped TiO2 nanotubes were detected against A. actinomycetemcomitans, T. forsythia, and C. rectus indicating antibacterial efficacy.

  1. [Markers of periodontal diseases and sensitivity to taromentine in patients with aggressive periodontitis].

    PubMed

    Iverieli, M V; Abashidze, N O; Gogishvili, Kh B

    2009-04-01

    The aim of the research was to study sensitivity of specific microorganisms from the periodontal pockets of patients with rapidly progressive periodontal disease to Taromentine. 95 patients aged 21 to 35 years (50 women (52,6+/-33,62) and 45 men (47,36+/-3,62)) with rapidly progressive form of periodontal desease were observed. Porphiromonas gingivalis was identifide in 83 out of 95 patients (87,36+/-2,06). Prevotella intermedia - in 31 patients (32,6+/-2,750); Actinobacillus actinomycetemcomitans - in 23 patients (24,2+/-2,050); Bacteroides forsythus - in 19 patients (20,0+/-2,360); Treponema denticola - in 16 patients (16,84+/-2,190); Candida - in 11 patients (11,57+/-1,80). The sensitivity of all cultures to Taromentine was investigated: 134 (77,9+/-1,89) out of 183 identified markers demonstrated sensitivity to Taromentine. Demostrated sensitivity to Taromentine: 64 (37,2+/-1,06) out of 83 identified cultures of Porphiromonas gingivalis, 24 (13,95+/-1,85) out of 31 identified cultures of Prevotela intermedia, 18 (10,47+/-1,05) out of 23 identified cultures of Actinobacillus actinomycetemcomitans, 15 (8,7+/-1,86) out of 19 identified cultures of Bacteroides forsythus, and 13 (7,84+/-1,09) out of 16 identified cultures of Treponema denticola. Totally 38 (22,1+/-1,59) out of 172 identified periodontal markers demonstrated resistence to Taromentine. The results of analysis showed that Taromentine could be recommended in complex treatment of periodontal diseases. PMID:19430039

  2. Uptake pathways of anionic and cationic photosensitizers into bacteria

    PubMed Central

    George, Saji; Hamblin, Michael R.; Kishen, Anil

    2009-01-01

    The effect of divalent cations (calcium and magnesium) and a permeabilizing agent (EDTA) on the uptake of a cationic photosensitizer (PS), methylene blue (MB), and two anionic PSs, rose bengal (RB) and indocyanine green (ICG), by Gram-positive Enterococcus faecalis and Gram-negative Actinobacillus actinomycetemcomitans was examined. The possible roles of multidrug efflux pumps and protein transporters in photosensitizer uptake were assessed in E. faecalis cells by studies using an efflux pump inhibitor (verapamil) and trypsin treatment respectively. Divalent cations enhanced the uptake and photodynamic inactivation potential of both RB and ICG in E. faecalis and A. actinomycetemcomitans, while they decreased the uptake and bacterial killing by MB. Verapamil increased the uptake of RB (possibly due to efflux pump inhibition), whereas trypsin treatment resulted in significant decrease in RB and ICG uptake. The results suggested that the uptake of anionic PSs by bacterial cells may be mediated through a combination of electrostatic charge interaction and by protein transporters, while the uptake of cationic PSs, as previously reported, is mediated by electrostatic interactions and self promoted uptake pathways. PMID:19492106

  3. A molecular survey of a captive wallaby population for periodontopathogens and the co-incidence of Fusobacterium necrophorum subspecies necrophorum with periodontal diseases.

    PubMed

    Antiabong, John F; Boardman, Wayne; Smith, Ian; Brown, Melissa H; Ball, Andrew S; Goodman, Amanda E

    2013-05-01

    Periodontal diseases (PD) are diseases of polymicrobial aetiology and constitute major health problems in captive macropods. Increasing knowledge of the causal pathogens is therefore crucial for effective management and prevention of these diseases. PCR survey and sequence analyses of potential periodontopathogens in captive wallaby populations revealed a co-incidence of the diseases with the detection of Fusobacterium necrophorum subsp. necrophorum (Fnn) and its encoded leukotoxin (lktA) gene. Sequence analyses showed that the outer membrane protein of Fnn in the GenBank database shared significant homology (99%) with the Fnn encoded haemagglutinin-related-protein gene fragment identified in this study. In addition, this report suggests the existence of a variant of Fnn with no detectable lktA gene and thus warrants further studies. In contrast to reports associating Porphyromonas gingivalis and F. nucleatum with PD, this study revealed that PD in macropods are associated with Porphyromonas gulae and Fnn and raises the question: is there a possible host pathogen co-evolution in the pathogenesis of PD in animals and humans? These findings contribute to the understanding of the aetiology of periodontal disease in macropods as well as opening up a new direction of research into the microbial interactions involved in the pathogenesis of PD in macropods.

  4. Molecular Characterization, Antibiotic Resistance, and Virulence Factors of Methicillin-Resistant Staphylococcus aureus Strains Isolated from Imported and Domestic Meat in Korea.

    PubMed

    Kim, Young Jo; Oh, Deog Hwan; Song, Bo Ra; Heo, Eun Jeong; Lim, Jong Su; Moon, Jin San; Park, Hyun Jung; Wee, Sung Hwan; Sung, Kidon

    2015-05-01

    During a nationwide surveillance in Korea, 13 methicillin-resistant Staphylococcus aureus (MRSA) strains were isolated from imported and domestic meat between 2009 and 2011. The predominant MRSA genotype was SCCmec type V, and only two agr types (I and II) were found. Unexpectedly, sequence type ST72 comprised more than 50% of the isolates; this is the first instance of type ST72 in food from Canada. Two Spanish pork isolates were ST398, which caused human disease in Europe, and they carried leukotoxin genes, lukS, lukF, and lukE-lukD. Furthermore, P71 and P6 harbored all of the known leukocidin genes, lukS-lukF-lukE-lukD-lukM. Our collected MRSA strains were multidrug resistant with various antimicrobial and heavy-metal resistance genes. Toxin genes that are commonly found in clinical MRSA also were detected in our meat strains. One MRSA strain exhibited an uncommon type of enterotoxin, sec-see-seg-sei-sel-sem-sen-seo-sep. Plasmids (1.5-15.0 kb) were found in 12 of the 13 MRSA isolates. Repetitive sequence-based polymerase chain reaction of the genomic DNA showed 3 clusters with 95% similarity. The presence of multidrug-resistant and toxigenic MRSA in meat products suggests that comprehensive surveillance should be continued for imported meats in Korea.

  5. RTX proteins: a highly diverse family secreted by a common mechanism

    PubMed Central

    Linhartová, Irena; Bumba, Ladislav; Mašín, Jiří; Basler, Marek; Osička, Radim; Kamanová, Jana; Procházková, Kateřina; Adkins, Irena; Hejnová-Holubová, Jana; Sadílková, Lenka; Morová, Jana; Šebo, Peter

    2010-01-01

    Repeats-in-toxin (RTX) exoproteins of Gram-negative bacteria form a steadily growing family of proteins with diverse biological functions. Their common feature is the unique mode of export across the bacterial envelope via the type I secretion system and the characteristic, typically nonapeptide, glycine- and aspartate-rich repeats binding Ca2+ ions. In this review, we summarize the current state of knowledge on the organization of rtx loci and on the biological and biochemical activities of therein encoded proteins. Applying several types of bioinformatic screens on the steadily growing set of sequenced bacterial genomes, over 1000 RTX family members were detected, with the biological functions of most of them remaining to be characterized. Activities of the so far characterized RTX family members are then discussed and classified according to functional categories, ranging from the historically first characterized pore-forming RTX leukotoxins, through the large multifunctional enzymatic toxins, bacteriocins, nodulation proteins, surface layer proteins, up to secreted hydrolytic enzymes exhibiting metalloprotease or lipase activities of industrial interest. PMID:20528947

  6. Insights on Evolution of Virulence and Resistance from the Complete Genome Analysis of an Early Methicillin-Resistant Staphylococcus aureus Strain and a Biofilm-Producing Methicillin-Resistant Staphylococcus epidermidis Strain†

    PubMed Central

    Gill, Steven R.; Fouts, Derrick E.; Archer, Gordon L.; Mongodin, Emmanuel F.; DeBoy, Robert T.; Ravel, Jacques; Paulsen, Ian T.; Kolonay, James F.; Brinkac, Lauren; Beanan, Mauren; Dodson, Robert J.; Daugherty, Sean C.; Madupu, Ramana; Angiuoli, Samuel V.; Durkin, A. Scott; Haft, Daniel H.; Vamathevan, Jessica; Khouri, Hoda; Utterback, Terry; Lee, Chris; Dimitrov, George; Jiang, Lingxia; Qin, Haiying; Weidman, Jan; Tran, Kevin; Kang, Kathy; Hance, Ioana R.; Nelson, Karen E.; Fraser, Claire M.

    2005-01-01

    Staphylococcus aureus is an opportunistic pathogen and the major causative agent of numerous hospital- and community-acquired infections. Staphylococcus epidermidis has emerged as a causative agent of infections often associated with implanted medical devices. We have sequenced the ∼2.8-Mb genome of S. aureus COL, an early methicillin-resistant isolate, and the ∼2.6-Mb genome of S. epidermidis RP62a, a methicillin-resistant biofilm isolate. Comparative analysis of these and other staphylococcal genomes was used to explore the evolution of virulence and resistance between these two species. The S. aureus and S. epidermidis genomes are syntenic throughout their lengths and share a core set of 1,681 open reading frames. Genome islands in nonsyntenic regions are the primary source of variations in pathogenicity and resistance. Gene transfer between staphylococci and low-GC-content gram-positive bacteria appears to have shaped their virulence and resistance profiles. Integrated plasmids in S. epidermidis carry genes encoding resistance to cadmium and species-specific LPXTG surface proteins. A novel genome island encodes multiple phenol-soluble modulins, a potential S. epidermidis virulence factor. S. epidermidis contains the cap operon, encoding the polyglutamate capsule, a major virulence factor in Bacillus anthracis. Additional phenotypic differences are likely the result of single nucleotide polymorphisms, which are most numerous in cell envelope proteins. Overall differences in pathogenicity can be attributed to genome islands in S. aureus which encode enterotoxins, exotoxins, leukocidins, and leukotoxins not found in S. epidermidis. PMID:15774886

  7. Cloning and comparison of bighorn sheep CD18 with that of domestic sheep, goats, cattle, humans and mice.

    PubMed

    Liu, Weiguo; Brayton, Kelly A; Lagerquist, John; Foreyt, William J; Srikumaran, Subramaniam

    2006-03-15

    Previously, we have shown that CD18, the beta-subunit of beta(2)-integrins, serves as a receptor for leukotoxin (Lkt) secreted by Mannheimia (Pasteurella) haemolytica on bovine leukocytes. Anti-CD18 monoclonal antibodies (mAbs) inhibit Lkt-induced cytolysis of bighorn sheep (Ovis canadensis) leukocytes suggesting that CD18 may serve as a receptor for Lkt on the leukocytes of this species as well. Confirmation of bighorn sheep CD18 as a receptor for Lkt, and elucidation of the enhanced Lkt-susceptibility of bighorn sheep polymorphonuclear leukocytes (PMNs), necessitates the cloning and sequencing of cDNA encoding bighorn sheep CD18. Hence, in this study we cloned and sequenced the cDNA encoding CD18 of bighorn sheep, and compared with that of other animal species. The cDNA of bighorn sheep CD18 has an open reading frame (ORF) of 2310bp. CD18 sequences obtained individually from peripheral blood mononuclear cells (PBMCs) and PMNs were identical to each other. Comparison of the deduced 770-amino acid sequence of CD18 of bighorn sheep with that of domestic sheep, goats, cattle, humans and mice revealed 99, 98, 95, 82 and 80% identity, respectively. Availability of cloned bighorn sheep CD18 cDNA should allow the molecular characterization of M. haemolytica Lkt-receptor interactions in bighorn sheep and other ruminants that are susceptible to this disease.

  8. Bacteriocin production by Fusobacterium isolates recovered from the oral cavity of human subjects with and without periodontal disease and of marmosets.

    PubMed

    Oliveira, A A; Farias, L M; Nicoli, J R; Costa, J E; Carvalho, M A

    1998-09-01

    Bacteriocin production has been studied in very few anaerobic bacteria, and no report is available for Fusobacterium species. In the present study a total of 167 Fusobacterium isolates were tested for bacteriocin production: 70 isolates were obtained from the oral cavity of patients with periodontal disease, 47 were recovered from healthy oral sites of human subjects and 50 from the oral cavity of Callithrix penicillata. Autoantagonism and isoantagonism were observed when the bacteriocin-producing isolates were tested against themselves. Heteroantagonism was detected by testing the Fusobacterium isolates against 14 reference strains and 2 strains of Actinobacillus actinomycetemcomitans from our laboratory collection. The auto-, iso- and heteroantagonism phenomena observed in this comparative study suggest a possible ecological role for this (these) antagonistic substance(s) in the oral environment.

  9. Combined orthodontic and periodontic treatment in a child with Papillon Lefèvre syndrome

    PubMed Central

    AlSarheed, Maha A.; Al-Sehaibany, Fares S.

    2015-01-01

    A 9-year-old girl with Papillon-Lefèvre syndrome (PLS) was treated orthodontically 24 months after the start of mechanical and antibiotic therapy in adjunct with periodontal treatment every 6 weeks. After achieving stable periodontal conditions, orthodontic treatment was commenced to correct the teeth position, facial profile, and maxillary protraction. Following the combination therapy and a failure to detect Actinobacillus actinomycetemcomitans from any site in the oral cavity, orthodontic treatment with a fixed appliance was performed aside from creating space for eruption of permanent teeth. We found that combined periodontal and orthodontic treatment of PLS may be successful with a complex interdisciplinary regimen and close follow up. This is a 2-year follow-up case report of a girl with PLS. Orthodontic and periodontic therapy were offered using combined treatments of orthodontic and periodontal with the benefit of prosthodontic consultation, resulting in a treatment plan. PMID:26219452

  10. Aspartame as a source of essential phenylalanine for the growth of oral anaerobes.

    PubMed

    Wyss, C

    1993-04-15

    Phenylalanine and aspartic acid requirements were determined for 13 species of oral bacteria using the chemically defined medium OMIZ-W1. None of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Eikenella corrodens, Selenomonas sputigena, Treponema pectinovorum, T. socranskii, or Wolinella recta required either of these amino acid constituents of aspartame (L-aspartyl-L-phenylalanine methylester). Phenylalanine was essential for the growth of Capnocytophaga gingivalis, Eubacterium timidum, Fusobacterium nucleatum, Porphyromonas gingivalis, T. denticola, and T. vincentii, while aspartic acid was not required. With the exception of E. timidum, all phenylalanine-dependent strains could grow when the free amino acid was replaced by aspartame at concentrations at least 10-fold lower than those used for aspartame as an artificial sweetener.

  11. Combined orthodontic and periodontic treatment in a child with Papillon Lefèvre syndrome.

    PubMed

    AlSarheed, Maha A; Al-Sehaibany, Fares S

    2015-08-01

    A 9-year-old girl with Papillon-Lefèvre syndrome (PLS) was treated orthodontically 24 months after the start of mechanical and antibiotic therapy in adjunct with periodontal treatment every 6 weeks. After achieving stable periodontal conditions, orthodontic treatment was commenced to correct the teeth position, facial profile, and maxillary protraction. Following the combination therapy and a failure to detect Actinobacillus actinomycetemcomitans from any site in the oral cavity, orthodontic treatment with a fixed appliance was performed aside from creating space for eruption of permanent teeth. We found that combined periodontal and orthodontic treatment of PLS may be successful with a complex interdisciplinary regimen and close follow up. This is a 2-year follow-up case report of a girl with PLS. Orthodontic and periodontic therapy were offered using combined treatments of orthodontic and periodontal with the benefit of prosthodontic consultation, resulting in a treatment plan. PMID:26219452

  12. Initial serum antibody titer to Porphyromonas gingivalis influences development of antibody avidity and success of therapy for chronic periodontitis.

    PubMed Central

    Mooney, J; Adonogianaki, E; Riggio, M P; Takahashi, K; Haerian, A; Kinane, D F

    1995-01-01

    This study assessed the effect of periodontal therapy on specific serum antibody concentration, expressed as titer, and antibody binding strength, expressed as relative avidity. The immune responses to Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans were investigated. Antibody titer was assayed by enzyme-linked immunosorbent assay (ELISA) and relative avidity was measured by thiocyanate elution in 17 adult periodontitis patients before and after therapy. Immunoglobulin G (IgG) avidities (expressed as thiocyanate molarity) to P. gingivalis increased from 1.01 to 1.38 M (P = 0.05) and IgA titers (expressed as ELISA units [EU]) increased from 89 to 237 EU (P = 0.012). There were no significant changes in avidity to A. actinomycetemcomitans, but the titer of all three immunoglobulin classes increased significantly (P < 0.03). More specifically, when patients were divided into subgroups which had originally been either IgG seropositive (i.e., having an IgG titer to this organism > 2 times the control median) or seronegative for P. gingivalis, only patients who were initially seropositive showed a significant increase in antibody avidity (P = 0.026; mean difference, 0.69 M). Patients who were originally seropositive in terms of IgG and IgA titer to P. gingivalis had demonstrably better treatment outcomes in terms of a reduced number of deep pockets and sites which bled on probing (P < 0.05). These findings suggest that periodontal therapy affects the magnitude and quality of the humoral immune response to suspected periodontopathogens, that this effect is dependent on initial serostatus, and that initial serostatus may have a bearing on treatment outcome. PMID:7642270

  13. Detection of putative periodontal pathogens in non-insulin-dependent diabetes mellitus and non-diabetes mellitus by polymerase chain reaction.

    PubMed

    Yuan, K; Chang, C J; Hsu, P C; Sun, H S; Tseng, C C; Wang, J R

    2001-02-01

    It has been assumed that there is a relationship between periodontal diseases and diabetes mellitus, however the putative periodontal microorganisms in non-diabetes mellitus (non-DM) individuals and non-insulin-dependent diabetes mellitus (NIDDM) patients have not been well studied. In this study, the detection rates of 5 putative periodontal pathogens: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Eikenella corrodens, Treponema denticola, and Candida albicans by polymerase chain reaction (PCR) between NIDDM and non-DM adults were compared. A total of 246 adults were randomly recruited and periodontal parameters including: plaque index (P1I), gingival index (GI), probing depth (PD) and attachment level (AL) were recorded. Subgingival plaque samples were collected by sterile curettes from the most diseased and healthy sites based on PD and AL. The differences in periodontal parameters and microbiological data in healthy and diseased sites between non-DM and NIDDM patients were compared by chi-square analysis. The results showed no significant differences in age, gender, GI, P1I, PD, and prevalence of the 5 microorganisms between the NIDDM and the non-diabetic groups. However, except for A. actinomycetemcomitans, the prevalence of the periodontal microorganisms tested was significantly higher (p <0.001) in diseased sites than in the healthy sites in both groups. The P1I, GI, PD and AL were significantly higher in T. denticola positive sites than in negative sites. The results suggested that P. gingivalis, T. denticola, E. corrodens and C. albicans may play important roles in the periodontitis of both NIDDM and non-DM individuals, however the etiology of periodontitis in both groups may not be different from each other.

  14. Antibacterial Activity of As-Annealed TiO2 Nanotubes Doped with Ag Nanoparticles against Periodontal Pathogens

    PubMed Central

    Yeniyol, Sinem; He, Zhiming; Yüksel, Behiye; Boylan, Robert Joseph; Ürgen, Mustafa; Özdemir, Tayfun; Ricci, John Lawrence

    2014-01-01

    It is important to develop functional transmucosal implant surfaces that reduce the number of initially adhering bacteria and they need to be modified to improve the anti-bacterial performance. Commercially pure Ti sheets were anodized in an electrolyte containing ethylene glycol, distilled water and ammonium fluoride at room temperature to produce TiO2 nanotubes. These structures were then annealed at 450°C to transform them to anatase. As-annealed TiO2 nanotubes were then treated in an electrolyte containing 80.7 g/L NiSO4·7H2O, 41 g/L MgSO4·7H2O, 45 g/L H3BO3, and 1.44 g/L Ag2SO4 at 20°C by the application of 9 V AC voltage for doping them with silver. As-annealed TiO2 nanotubes and as-annealed Ag doped TiO2 nanotubes were evaluated by SEM, FESEM, and XRD. Antibacterial activity was assessed by determining the adherence of A. actinomycetemcomitans, T. forsythia, and C. rectus to the surface of the nanotubes. Bacterial morphology was examined using an SEM. As-annealed Ag doped TiO2 nanotubes revealed intense peak of Ag. Bacterial death against the as-annealed Ag doped TiO2 nanotubes were detected against A. actinomycetemcomitans, T. forsythia, and C. rectus indicating antibacterial efficacy. PMID:25202230

  15. Physicochemical and structural investigation of the surfaces of some anaerobic subgingival bacteria.

    PubMed Central

    Cowan, M M; van der Mei, H C; Rouxhet, P G; Busscher, H J

    1992-01-01

    The surfaces of nine clinical isolates of Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, and Peptostreptococcus micros and that of laboratory strain P. gingivalis W83 were studied by using contact angle measurements, X-ray photoelectron spectroscopy, infrared spectroscopy, microelectrophoresis of whole cells, and transmission electron microscopy of whole and sectioned cells. P. intermedia strains were hydrophilic, as judged from their small water contact angles, and had highly negative zeta potentials, consistent with the presence of a prominent ruthenium red (RR)-staining layer and fibrillar appendages which are probably partly carbohydrate. The two clinical isolates of P. gingivalis were also hydrophilic and highly negatively charged despite the presence of prominent fibrils, which usually yield less negative zeta potentials. This finding suggests that the RR-staining layer dominates the suspension characteristics of P. gingivalis and P. intermedia strains. P. gingivalis W83 had no demonstrable fibrils and a morphologically distinct RR-staining layer, and it was more hydrophobic than the two clinical isolates of P. gingivalis. P. micros isolates were hydrophobic and much less negatively charged than the other species. The A. actinomycetemcomitans strains displayed long, prominent fibrils and a very thin RR-staining layer, which resulted in high hydrophobicity but distinctly different zeta potentials for the two. Physicochemical data on microbial cell surfaces usually have clear and predictable relationships with each other. For the strains in this study that did not follow these relationships, their aberrant behavior could be explained as due to a masking effect caused by specific surface architecture. We conclude that this combined analysis provides a detailed image of subgingival bacterial surface architecture. Images PMID:1599251

  16. Clonal Complexes and Diversity of Exotoxin Gene Profiles in Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Patients in a Spanish Hospital▿

    PubMed Central

    Argudín, M. A.; Mendoza, M. C.; Méndez, F. J.; Martín, M. C.; Guerra, B.; Rodicio, M. R.

    2009-01-01

    Molecular epidemiology studies have allowed the identification of the methicillin (meticillin)-resistant (MRSA) and methicillin-susceptible (MSSA) clonal complexes (CCs) and clones of Staphylococcus aureus circulating in a Spanish hospital recently. Of 81 isolates tested, 32.1% were MRSA. Most of them carried staphylococcal cassette chromosome mec (SCCmec) IVc (88.5%) and belonged to CC5 (88.5%; multilocus sequence typing types ST125 [mainly associated with spa type t067], ST5, and ST228). A higher diversity was found among MSSA isolates (67.9%). Eighty percent shared the genetic background of major MRSA lineages (CC5 [38.2%; ST125 and ST5], CC30 [25.5%; ST30], CC45 [14.5%; ST45 and ST47], and CC8 [1.8%; ST8]), but CC12, CC15, CC51, and CC59 were also detected. Many exotoxin genes were present in each of the 81 isolates, independent of whether they were involved in sepsis (11 to 22) or other types of infections (13 to 21), and they appeared in 73 combinations. The relevant data are that (i) all isolates were positive for hemolysin and leukotoxin genes (98.8% for lukED and 25.9% for lukPV); (ii) all contained an enterotoxin gene cluster (egc with or without seu), frequently with one or more genes encoding classical enterotoxins; (iii) about half were positive for tst and 95% were positive for exfoliatin-encoding genes (eta, etb, and/or etd); and (iv) the four agr groups were detected, with agrII (55.6%) and agrIII (23.5%) being the most frequent. Taken together, results of the present study suggest a frequent acquisition and/or loss of exotoxin genes, which may be mediated by efficient intralineage transfer of mobile genetic elements and exotoxin genes therein and by eventual breakage of interlineage barriers. PMID:19458176

  17. A Multivalent Mannheimia-Bibersteinia Vaccine Protects Bighorn Sheep against Mannheimia haemolytica Challenge ▿

    PubMed Central

    Subramaniam, Renuka; Shanthalingam, Sudarvili; Bavananthasivam, Jegarubee; Kugadas, Abirami; Potter, Kathleen A.; Foreyt, William J.; Hodgins, Douglas C.; Shewen, Patricia E.; Barrington, George M.; Knowles, Donald P.; Srikumaran, Subramaniam

    2011-01-01

    Bighorn sheep (BHS) are more susceptible than domestic sheep (DS) to Mannheimia haemolytica pneumonia. Although both species carry M. haemolytica as a commensal bacterium in the nasopharynx, DS carry mostly leukotoxin (Lkt)-positive strains while BHS carry Lkt-negative strains. Consequently, antibodies to surface antigens and Lkt are present at much higher titers in DS than in BHS. The objective of this study was to determine whether repeated immunization of BHS with multivalent Mannheimia-Bibersteinia vaccine will protect them upon M. haemolytica challenge. Four BHS were vaccinated with a culture supernatant vaccine prepared from M. haemolytica serotypes A1 and A2 and Bibersteinia trehalosi serotype T10 on days 0, 21, 35, 49, and 77. Four other BHS were used as nonvaccinated controls. On the day of challenge, 12 days after the last immunization, the mean serum titers of Lkt-neutralizing antibodies and antibodies to surface antigens against M. haemolytica were 1:160 and 1:4,000, respectively. Following intranasal challenge with M. haemolytica A2 (1 × 105 CFU), all four control BHS died within 48 h. Necropsy revealed acute fibrinonecrotic pneumonia characteristic of M. haemolytica infection. None of the vaccinated BHS died during the 8 weeks postchallenge observation period. Radiography at 3 weeks postchallenge revealed no lung lesions in two vaccinated BHS and mild lesions in the other two, which resolved by 8 weeks postchallenge. These results indicate that if BHS can be induced to develop high titers of Lkt-neutralizing antibodies and antibodies to surface antigens, they are likely to survive M. haemolytica challenge which is likely to reduce the BHS population decline due to pneumonia. PMID:21832104

  18. Epizootic Pneumonia of Bighorn Sheep following Experimental Exposure to Mycoplasma ovipneumoniae

    PubMed Central

    Besser, Thomas E.; Cassirer, E. Frances; Potter, Kathleen A.; Lahmers, Kevin; Oaks, J. Lindsay; Shanthalingam, Sudarvili; Srikumaran, Subramaniam; Foreyt, William J.

    2014-01-01

    Background Bronchopneumonia is a population limiting disease of bighorn sheep (Ovis canadensis). The cause of this disease has been a subject of debate. Leukotoxin expressing Mannheimia haemolytica and Bibersteinia trehalosi produce acute pneumonia after experimental challenge but are infrequently isolated from animals in natural outbreaks. Mycoplasma ovipneumoniae, epidemiologically implicated in naturally occurring outbreaks, has received little experimental evaluation as a primary agent of bighorn sheep pneumonia. Methodology/Principal Findings In two experiments, bighorn sheep housed in multiple pens 7.6 to 12 m apart were exposed to M. ovipneumoniae by introduction of a single infected or challenged animal to a single pen. Respiratory disease was monitored by observation of clinical signs and confirmed by necropsy. Bacterial involvement in the pneumonic lungs was evaluated by conventional aerobic bacteriology and by culture-independent methods. In both experiments the challenge strain of M. ovipneumoniae was transmitted to all animals both within and between pens and all infected bighorn sheep developed bronchopneumonia. In six bighorn sheep in which the disease was allowed to run its course, three died with bronchopneumonia 34, 65, and 109 days after M. ovipneumoniae introduction. Diverse bacterial populations, predominantly including multiple obligate anaerobic species, were present in pneumonic lung tissues at necropsy. Conclusions/Significance Exposure to a single M. ovipneumoniae infected animal resulted in transmission of infection to all bighorn sheep both within the pen and in adjacent pens, and all infected sheep developed bronchopneumonia. The epidemiologic, pathologic and microbiologic findings in these experimental animals resembled those seen in naturally occurring pneumonia outbreaks in free ranging bighorn sheep. PMID:25302992

  19. Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia.

    PubMed

    Dassanayake, Rohana P; Shanthalingam, Sudarvili; Herndon, Caroline N; Subramaniam, Renuka; Lawrence, Paulraj K; Bavananthasivam, Jegarubee; Cassirer, E Frances; Haldorson, Gary J; Foreyt, William J; Rurangirwa, Fred R; Knowles, Donald P; Besser, Thomas E; Srikumaran, Subramaniam

    2010-10-26

    Mycoplasma ovipneumoniae has been isolated from the lungs of pneumonic bighorn sheep (BHS). However experimental reproduction of fatal pneumonia in BHS with M. ovipneumoniae was not successful. Therefore the specific role, if any, of M. ovipneumoniae in BHS pneumonia is unclear. The objective of this study was to determine whether M. ovipneumoniae alone causes fatal pneumonia in BHS, or predisposes them to infection by Mannheimia haemolytica. We chose M. haemolytica for this study because of its isolation from pneumonic BHS, and its consistent ability to cause fatal pneumonia under experimental conditions. Since in vitro culture could attenuate virulence of M. ovipneumoniae, we used ceftiofur-treated lung homogenates from pneumonic BHS lambs or nasopharyngeal washings from M. ovipneumoniae-positive domestic sheep (DS) as the source of M. ovipneumoniae. Two adult BHS were inoculated intranasally with lung homogenates while two others received nasopharyngeal washings from DS. All BHS developed clinical signs of respiratory infection, but only one BHS died. The dead BHS had carried leukotoxin-positive M. haemolytica in the nasopharynx before the onset of this study. It is likely that M. ovipneumoniae colonization predisposed this BHS to fatal infection with the M. haemolytica already present in this animal. The remaining three BHS developed pneumonia and died 1-5 days following intranasal inoculation with M. haemolytica. On necropsy, lungs of all four BHS showed lesions characteristic of bronchopneumonia. M. haemolytica and M. ovipneumoniae were isolated from the lungs. These results suggest that M. ovipneumoniae alone may not cause fatal pneumonia in BHS, but can predispose them to fatal pneumonia due to M. haemolytica infection.

  20. Cytoplasmic replication of Staphylococcus aureus upon phagosomal escape triggered by phenol-soluble modulin α

    PubMed Central

    Grosz, Magdalena; Kolter, Julia; Paprotka, Kerstin; Winkler, Ann-Cathrin; Schäfer, Daniel; Chatterjee, Som Subra; Geiger, Tobias; Wolz, Christiane; Ohlsen, Knut; Otto, Michael; Rudel, Thomas; Sinha, Bhanu; Fraunholz, Martin

    2013-01-01

    Staphylococcus aureus is a Gram-positive human pathogen that is readily internalized by professional phagocytes such as macrophages and neutrophils but also by non-professional phagocytes such as epithelial or endothelial cells. Intracellular bacteria have been proposed to play a role in evasion of the innate immune system and may also lead to dissemination within migrating phagocytes. Further, S. aureus efficiently lyses host cells with a battery of cytolytic toxins. Recently, phenol-soluble modulins (PSM) have been identified to comprise a genus-specific family of cytolytic peptides. Of these the PSMα peptides have been implicated in killing polymorphonuclear leukocytes after phagocytosis. We questioned if the peptides were active in destroying endosomal membranes to avoid lysosomal killing of the pathogen and monitored integrity of infected host cell endosomes by measuring the acidity of the intracellular bacterial microenvironment via flow cytometry and by a reporter recruitment technique. Isogenic mutants of the methicillin-resistant S. aureus (MRSA) strains USA300 LAC, USA400 MW2 as well as the strongly cytolytic methicillin-sensitive strain 6850 were compared to their respective wild type strains. In all three genetic backgrounds, PSMα mutants were unable to escape from phagosomes in non-professional (293, HeLa, EAhy.926) and professional phagocytes (THP-1), whereas mutants in PSMβ and δ-toxin as well as β-toxin, phosphatidyl inositol-dependent phospholipase C and Panton Valentine leukotoxin escaped with efficiencies of the parental strains. S. aureus replicated intracellularly only in presence of a functional PSMα operon thereby illustrating that bacteria grow in the host cell cytoplasm upon phagosomal escape. PMID:24164701

  1. Simvastatin and a Plant Galactolipid Protect Animals from Septic Shock by Regulating Oxylipin Mediator Dynamics through the MAPK-cPLA2 Signaling Pathway

    PubMed Central

    Apaya, Maria Karmella; Lin, Chih-Yu; Chiou, Ching-Yi; Yang, Chung-Chih; Ting, Chen-Yun; Shyur, Lie-Fen

    2015-01-01

    Sepsis remains a major medical issue despite decades of research. Identification of important inflammatory cascades and key molecular mediators are crucial for developing intervention and prevention strategies. In this study, we conducted a comparative oxylipin metabolomics study to gain a comprehensive picture of lipid mediator dynamics during the initial hyperinflammatory phase of sepsis, and demonstrated, in parallel, the efficacy of simvastatin and plant galactolipid, 1,2-di-O-α-linolenoyl-3-O-β-­galactopyranosyl-sn-glycerol (dLGG) in the homeostatic regulation of the oxylipin metabolome using a lipopolysaccharide (LPS)-induced sepsis C57BL/6J mouse model. LPS increased the systemic and organ levels of proinflammatory metabolites of linoleic acid including leukotoxin diols (9-,10-DHOME, 12-,13-DHOME) and octadecadienoic acids (9-HODE and 13-HODE) and arachidonic acid-derived prostanoid, PGE2, and hydroxyeicosatetraenoic acids (8-, 12- and 15-HETE). Treatment with either compound decreased the levels of proinflammatory metabolites and elevated proresolution lipoxin A4, 5(6)-EET, 11(12)-EET and 15-deoxy-PGJ2. dLGG and simvastatin ameliorated the effects of LPS-induced mitogen-activated protein kinase (MAPK)-dependent activation of cPLA2, cyclooxygenase-2, lipoxygenase, cytochrome P450 and/or epoxide hydrolase lowered systemic TNF-α and IL-6 levels and aminotransferase activities and decreased organ-specific infiltration of inflammatory leukocytes and macrophages, and septic shock-induced multiple organ damage. Furthermore, both dLGG and simvastatin increased the survival rates in the cecal ligation and puncture (CLP) sepsis model. This study provides new insights into the role of oxylipins in sepsis pathogenesis and highlights the potential of simvastatin and dLGG in sepsis therapy and prevention. PMID:26701313

  2. The Association of Panton-Valentine leukocidin and mecA Genes in Methicillin-Resistant Staphylococcus aureus Isolates From Patients Referred to Educational Hospitals in Ahvaz, Iran

    PubMed Central

    Motamedi, Hossein; Rahmat Abadi, Seyyed Soheil; Moosavian, Seyyed Mojtaba; Torabi, Maryam

    2015-01-01

    Background: Staphylococcus aureus, an important human pathogen is one of the main causative agents of nosocomial infection. Virulence genes play a major role in the pathogenicity of this agent and its infections. Methicillin-Resistant Staphylococcus aureus (MRSA) isolates are major challenge among infectious agents that can cause severe infections and mortality. Methicillin-resistant S. aureus produces a unique type of Penicillin Binding Protein 2a (PBP2a) that has low affinity for β-lactam antibiotics. Most of the MRSA bacterial strains can also produce a leukotoxin as Panton-Valentine Leukocidin (PVL) that increases the virulence of MRSA strains and can cause severe necrotic pneumonia. The presence of pvl gene is a genetic marker for the MRSA populations. Objectives: The aim of this study was to explore the association of pvl and mecA genes in clinical isolates of MRSA. Materials and Methods: Fifty MRSA isolates were collected from 200 clinical samples from three different educational hospitals in Ahvaz, Iran, and identified by biochemical tests including catalase, oxidase, tube coagulase, mannitol fermentation, and sensitivity to furazolidone, resistance to bacitracin, PYR test and Voges-Proskauer test. Their resistance to methicillin was evaluated using the disc diffusion method. DNA was extracted by boiling and then the presence of pvl and mecA genes was investigated by the polymerase chain reaction method using specific primers. Results: The results revealed that mecA and pvl genes were positive for 15 (30%) and 3 (6%) of the isolates, respectively. None of mecA positive isolates was positive for pvl gene. Conclusions: It can be concluded from these results that fortunately the prevalence of pvl gene is low in MRSA isolates in this region and there is no association between the presence of pvl and mecA genes in these isolates. PMID:26468365

  3. Subcutaneous Immunization with Inactivated Bacterial Components and Purified Protein of Escherichia coli, Fusobacterium necrophorum and Trueperella pyogenes Prevents Puerperal Metritis in Holstein Dairy Cows

    PubMed Central

    Machado, Vinícius Silva; Bicalho, Marcela Luccas de Souza; Meira Junior, Enoch Brandão de Souza; Rossi, Rodolfo; Ribeiro, Bruno Leonardo; Lima, Svetlana; Santos, Thiago; Kussler, Arieli; Foditsch, Carla; Ganda, Erika Korzune; Oikonomou, Georgios; Cheong, Soon Hon; Gilbert, Robert Owen; Bicalho, Rodrigo Carvalho

    2014-01-01

    In this study we evaluate the efficacy of five vaccine formulations containing different combinations of proteins (FimH; leukotoxin, LKT; and pyolysin, PLO) and/or inactivated whole cells (Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes) in preventing postpartum uterine diseases. Inactivated whole cells were produced using two genetically distinct strains of each bacterial species (E. coli, F. necrophorum, and T. pyogenes). FimH and PLO subunits were produced using recombinant protein expression, and LKT was recovered from culturing a wild F. necrophorum strain. Three subcutaneous vaccines were formulated: Vaccine 1 was composed of inactivated bacterial whole cells and proteins; Vaccine 2 was composed of proteins only; and Vaccine 3 was composed of inactivated bacterial whole cells only. Two intravaginal vaccines were formulated: Vaccine 4 was composed of inactivated bacterial whole cells and proteins; and Vaccine 5 was composed of PLO and LKT. To evaluate vaccine efficacy, a randomized clinical trial was conducted at a commercial dairy farm; 371 spring heifers were allocated randomly into one of six different treatments groups: control, Vaccine 1, Vaccine 2, Vaccine 3, Vaccine 4 and Vaccine 5. Late pregnant heifers assigned to one of the vaccine groups were each vaccinated twice: at 230 and 260 days of pregnancy. When vaccines were evaluated grouped as subcutaneous and intravaginal, the subcutaneous ones were found to significantly reduce the incidence of puerperal metritis. Additionally, subcutaneous vaccination significantly reduced rectal temperature at 6±1 days in milk. Reproduction was improved for cows that received subcutaneous vaccines. In general, vaccination induced a significant increase in serum IgG titers against all antigens, with subcutaneous vaccination again being more effective. In conclusion, subcutaneous vaccination with inactivated bacterial components and/or protein subunits of E. coli, F. necrophorum and T. pyogenes

  4. Moxifloxacin efficacy and vitreous penetration in a rabbit model of Staphylococcus aureus endophthalmitis and effect on gene expression of leucotoxins and virulence regulator factors.

    PubMed

    Bronner, Stéphane; Jehl, François; Peter, Jean-Daniel; Ploy, Marie-Cécile; Renault, Corinne; Arvis, Pierre; Monteil, Henri; Prevost, Gilles

    2003-05-01

    Bacterial endophthalmitis is a serious complication of ocular surgery and of eye trauma; the leading causative organisms are Staphylococcus aureus strains. Tissue damage is due both to the host inflammatory response and to toxin synthesis by bacteria. Systemic treatment remains difficult because most antibiotics show poor ocular penetration. Moxifloxacin (MXF), a novel fluoroquinolone, was evaluated for its penetration into the vitreous of normal rabbit eyes and of eyes of rabbits infected for 24 h with methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA) following a single intravenous administration of 5 or 20 mg/kg. MXF penetration was rapid and efficient regardless of the dose, ranging from 28 to 52%. An inflammatory state of the vitreous significantly increased penetration after the 20-mg/kg dose, with penetration reaching 52%. Concentrations determined in the vitreous cavity following a 20-mg/kg administration showed a 3.5-fold decrease of the bacterial density within 5 h for MSSA (MIC, 0.125 micro g/ml) and a 1.6-fold decrease for MRSA (MIC, 4 micro g/ml) strains, respectively. By using a semiquantitative reverse transcription-PCR method, the expression of luk-PV and hlgCB, but not hlgA, encoding staphylococcal leukotoxins, was detected in the vitreous without MXF treatment. A slight decrease in the expression of leucotoxins and sarA, agr, and sigB virulence regulatory factors was observed 1 h following the administration of 5 mg of MXF per kg.

  5. The effects of iron limitation and cell density on prokaryotic metabolism and gene expression: Excerpts from Fusobacterium necrophorum strain 774 (sheep isolate).

    PubMed

    Antiabong, John F; Ball, Andrew S; Brown, Melissa H

    2015-05-25

    Fusobacterium necrophorum is a Gram-negative obligate anaerobe associated with several diseases in humans and animals. Despite its increasing clinical significance, there is little or no data on the relationship between its metabolism and virulence. Previous studies have shown that bacteria grown under iron-limitation express immunogenic antigens similar to those generated in vivo. Thus, this paper describes the relationship between F. necrophorum subsp. necrophorum (Fnn) metabolism and the expression of the encoded putative virulence factors under iron-restricted conditions. At the midlog phase, iron limitation reduced Fnn growth but the cell density was dependent on the size of the inoculum. Preferential utilization of glucose-1-phosphate, d-mannitol and l-phenylalanine; production of 2-hydroxycaproic acid and termination of dimethyl sulphide production were major Fnn response-factors to iron limitation. Ultimately, iron restriction resulted in an increased ability of Fnn to metabolize diverse carbon sources and in the expression of stress-specific virulence factors. Iron starvation in low Fnn cell density was associated with the up-regulation of haemagglutinin (HA) and leukotoxin (lktA) genes (2.49 and 3.72 fold change respectively). However, Fnn encoded Haemolysin (Hly), yebN homologue (febN) and tonB homologue, were down-regulated (0.15, 0.79 and 0.33, fold changes respectively). Interestingly, cell density appeared to play a regulatory role in the final bacteria cell biomass, induction of a metabolic gene expression and the expression pattern virulence factors in Fnn suggesting the role of a cell density-associated regulatory factor. This report suggest that future studies on differential expression of bacterial genes under altered environmental condition(s) should consider testing the effect of cell concentrations as this is often neglected in such studies. In conclusion, iron restriction induces preferential utilization of carbon sources and altered

  6. Expression and characterization of an epoxide hydrolase from Anopheles gambiae with high activity on epoxy fatty acids

    PubMed Central

    Xu, Jiawen; Morisseau, Christophe; Hammock, Bruce D.

    2014-01-01

    In insects, epoxide hydrolases (EHs) play critical roles in the metabolism of xenobiotic epoxides from the food resources and in the regulation of endogenous chemical mediators, such as juvenile hormones. Using the baculovirus expression system, we expressed and characterized an epoxide hydrolase from Anopheles gambiae (AgEH) that is distinct in evolutionary history from insect juvenile hormone epoxide hydrolases (JHEHs). We partially purified the enzyme by ion exchange chromatography and isoelectric focusing. The experimentally determined molecular weight and pI were estimated to be 35kD and 6.3 respectively, different than the theoretical ones. The AgEH had the greatest activity on long chain epoxy fatty acids such as 14,15-epoxyeicosatrienoic acids (14,15-EET) and 9,10-epoxy-12Z-octadecenoic acids (9,10-EpOME or leukotoxin) among the substrates evaluated. Juvenile hormone III, a terpenoid insect growth regulator, was the next best substrate tested. The AgEH showed kinetics comparable to the mammalian soluble epoxide hydrolases, and the activity could be inhibited by AUDA [12-(3-adamantan-1-yl-ureido) dodecanoic acid], a urea-based inhibitor designed to inhibit the mammalian soluble epoxide hydrolases. The rabbit serum generated against the soluble epoxide hydrolase of Mus musculus can both cross-react with natural and denatured forms of the AgEH, suggesting immunologically they are similar. The study suggests there are mammalian sEH homologs in insects, and epoxy fatty acids may be important chemical mediators in insects. PMID:25173592

  7. Staphylococcus aureus Leukocidin A/B (LukAB) Kills Human Monocytes via Host NLRP3 and ASC when Extracellular, but Not Intracellular

    PubMed Central

    DuMont, Ashley L.; Torres, Victor J.; Duncan, Joseph A.

    2015-01-01

    Staphylococcus aureus infections are a growing health burden worldwide, and paramount to this bacterium’s pathogenesis is the production of virulence factors, including pore-forming leukotoxins. Leukocidin A/B (LukAB) is a recently discovered toxin that kills primary human phagocytes, though the underlying mechanism of cell death is not understood. We demonstrate here that LukAB is a major contributor to the death of human monocytes. Using a variety of in vitro and ex vivo intoxication and infection models, we found that LukAB activates Caspase 1, promotes IL-1β secretion and induces necrosis in human monocytes. Using THP1 cells as a model for human monocytes, we found that the inflammasome components NLRP3 and ASC are required for LukAB-mediated IL-1β secretion and necrotic cell death. S. aureus was shown to kill human monocytes in a LukAB dependent manner under both extracellular and intracellular ex vivo infection models. Although LukAB-mediated killing of THP1 monocytes from extracellular S. aureus requires ASC, NLRP3 and the LukAB-receptor CD11b, LukAB-mediated killing from phagocytosed S. aureus is independent of ASC or NLRP3, but dependent on CD11b. Altogether, this study provides insight into the nature of LukAB-mediated killing of human monocytes. The discovery that S. aureus LukAB provokes differential host responses in a manner dependent on the cellular contact site is critical for the development of anti-infective/anti-inflammatory therapies that target the NLRP3 inflammasome. PMID:26069969

  8. Immunogenicity of Mannheimia haemolytica Recombinant Outer Membrane Proteins Serotype 1-Specific Antigen, OmpA, OmpP2, and OmpD15▿

    PubMed Central

    Ayalew, Sahlu; Shrestha, Binu; Montelongo, Marie; Wilson, Amanda E.; Confer, Anthony W.

    2011-01-01

    We previously identified Mannheimia haemolytica outer membrane proteins (OMPs) that may be important immunogens by using immunoproteomic analyses. Genes for serotype 1-specific antigen (SSA-1), OmpA, OmpP2, and OmpD15 were cloned and expressed, and recombinant proteins were purified. Objective 1 of this study was to demonstrate immunogenicity of the four recombinant OMPs in mice and cattle. Objective 2 was to determine if the addition of individual recombinant OMPs or combinations of them would modify immune responsiveness of mice to the recombinant chimeric protein SAC89, containing the main epitope from M. haemolytica outer membrane lipoprotein PlpE and the neutralizing epitope of M. haemolytica leukotoxin. Mice vaccinated with recombinant OmpA (rOmpA), rSSA-1, rOmpD15, and rOmpP2 developed significant antibody responses to M. haemolytica outer membranes and to the homologous recombinant OMP. Cattle vaccinated with rOmpA and rSSA-1 developed significant antibodies to M. haemolytica outer membranes by day 28, whereas cattle vaccinated with rOmpD15 and rOmpP2 developed only minimal responses. Sera from cattle vaccinated with each of the recombinant proteins stimulated complement-mediated killing of the bacterium. Concurrent vaccination with SAC89 plus any of the four rOMPs singly resulted in increased endpoint anti-SAC89 titers, and for the SAC89/rSSA-1 vaccinees, the response was increased significantly. In contrast, the SAC89/P2/SSA-1 and SAC89/OmpA/P2/D15/SSA-1 combination vaccines resulted in significant decreases in anti-SAC89 antibodies compared to SAC89 vaccination alone. In conclusion, under the conditions of these experiments, vaccination of mice and cattle with rOmpA and rSSA-1 stimulated high antibody responses and may have protective vaccine potential. PMID:21976226

  9. Brief heat treatment causes a structural change and enhances cytotoxicity of the Escherichia coli α-hemolysin.

    PubMed

    Aulik, Nicole A; Atapattu, Dhammika N; Czuprynski, Charles J; McCaslin, Darrel R

    2013-02-01

    α-Hemolysin (HLY) is an important virulence factor for uropathogenic Escherichia coli. HLY is a member of the RTX family of exotoxins secreted by a number of Gram-negative bacteria. Recently, it was reported that a related RTX toxin, the Mannheimia haemolytica leukotoxin, exhibits increased cytotoxicity following brief heat treatment. In this article, we show that brief heat treatment (1 min at 100°C) increases cytotoxicity of HLY for human bladder cells, kidney epithelial cells (A498) and neutrophils. Heat treatment also increased hemolysis of human red blood cells (RBCs). Furthermore, heat treatment of previously inactived HLY restored its cytotoxicity. Heat-activated and native HLY both required glycophorin A to lyse RBCs. Native and heat-activated HLY appeared to bind equally well to the surface of A498 cells; although, Western blot analyses demonstrated binding to different proteins on the surface. Confocal microscopy revealed that heat-activated HLY bound more extensively to internal structures of permeabilized A498 cells than did native HLY. Several lines of spectroscopic evidence demonstrate irreversible changes in the structure of heat activated compared to native HLY. We show changes in secondary structure, increased exposure of tryptophan residues to the aqueous environment, an increase in molecular dimension and an increase in hydrophobic surface area. These properties are among the most common characteristics described for the molten globule state, first identified as an intermediate in protein folding. We hypothesize that brief heat treatment of HLY causes a conformational change leading to significant differences in protein-protein interactions that result in increased cytotoxicity for target cells. PMID:22994841

  10. IL12, IL10, IFNγ and TNFα Expression in Human Primary Monocytes Stimulated with Bacterial Heat Shock GroEL (Hsp64) Protein

    PubMed Central

    Nalbant, Ayten; Saygılı, Tahsin

    2016-01-01

    Actinobacillus (Aggregatibacter) actinomycetemicomitans (Aa) is a bacterium that lives in the oral cavity and plays an important role in periodontal diseases. The effect of A.actinomycetemcomitans’s heat shock family protein GroEL on host or immune cells including monocytes is quite limited. In this study, the recombinant A.actinomycetemcomitans’s GroEL protein (rAaGroEL) was used as an antigen and its effects on monocytes of peripheral blood mononuclear cells (PBMCs) was investigated. To do that, PBMCs were stimulated with rAaGroEL protein at different time points (16h to 96h) and the cytokines of CD14+ monocytes were detected with intracellular cytokine staining by Flow cytometry. Data showed that AaGroEL protein has an antigenic effect on human primary monocytes. AaGroEL protein responsive CD14 monocytes stimulates the expression of IL12, IL10, IFNγ and TNFα cytokines with different kinetics and expression profile. Therefore, A. actinomycetemcomitans’s heat shock GroEL protein can modulate innate and adaptive immune responses and contribute to an inflammatory diseases pathology. PMID:27119521

  11. 16S rRNA based microarray analysis of ten periodontal bacteria in patients with different forms of periodontitis.

    PubMed

    Topcuoglu, Nursen; Kulekci, Guven

    2015-10-01

    DNA microarray analysis is a computer based technology, that a reverse capture, which targets 10 periodontal bacteria (ParoCheck) is available for rapid semi-quantitative determination. The aim of this three-year retrospective study was to display the microarray analysis results for the subgingival biofilm samples taken from patient cases diagnosed with different forms of periodontitis. A total of 84 patients with generalized aggressive periodontitis (GAP,n:29), generalized chronic periodontitis (GCP, n:25), peri-implantitis (PI,n:14), localized aggressive periodontitis (LAP,n:8) and refractory chronic periodontitis (RP,n:8) were consecutively selected from the archives of the Oral Microbiological Diagnostic Laboratory. The subgingival biofilm samples were analyzed by the microarray-based identification of 10 selected species. All the tested species were detected in the samples. The red complex bacteria were the most prevalent with very high levels in all groups. Fusobacterium nucleatum was detected in all samples at high levels. The green and blue complex bacteria were less prevalent compared with red and orange complex, except Aggregatibacter actinomycetemcomitas was detected in all LAP group. Positive correlations were found within all the red complex bacteria and between red and orange complex bacteria especially in GCP and GAP groups. Parocheck enables to monitoring of periodontal pathogens in all forms of periodontal disease and can be alternative to other guiding and reliable microbiologic tests.

  12. The use of genomic DNA fingerprinting in studies of the epidemiology of bacteria in periodontitis.

    PubMed

    Genco, R J; Loos, B G

    1991-07-01

    Recent studies of microbial epidemiology emphasizing the genetic organization and distribution of organisms associated with orofacial infections have led to new insights into the possible origins of pathogenicity. Studies into genetic heterogeneity, acquisition and transmission of these organisms have been markedly advanced by the utilization of the powerful technique of genomic DNA fingerprinting. Characteristic fingerprints for each bacterial isolate can be produced by cleavage of high molecular weight genomic DNA by restriction endonucleases. It is assumed that each DNA fingerprint represents a clonal type. In this report, we review and analyze studies of the epidemiology of bacteria associated with orofacial infections with an emphasis on periodontal disease. Studies of nontypable (NT) Haemophilus influenzae associated with recurrent otitis media illustrate the utility of this technique. DNA fingerprinting clearly demonstrates genetic heterogeneity of NT H. influenzae isolates, and clonality of infection of any individual. Furthermore, DNA fingerprinting has shown that the same clonal type is seen in siblings concurrently suffering from otitis media, suggesting horizontal transmission within the family. Studies of mutans Streptococci also show extensive genetic heterogeneity and show vertical transmission of a predominant clonal type only from mother to infant, but not from father to infant. Studies of Actinobacillus actinomycetemcomitans show considerable genetic heterogeneity among monkey isolates. Thus far, three clonal types have been reported with DNA fingerprinting among isolates from periodontal patients, but additional genetic heterogeneity can be found using specific DNA fragments as probes in hybridization experiments. Intrafamilial transmission of A. actinomycetemcomitans has been demonstrated. Porphyromonas (Bacteroides) gingivalis shows extensive genetic heterogeneity and case reports suggest clonal infection of any one individual. In contrast

  13. Phylogeny of 54 representative strains of species in the family Pasteurellaceae as determined by comparison of 16S rRNA sequences.

    PubMed Central

    Dewhirst, F E; Paster, B J; Olsen, I; Fraser, G J

    1992-01-01

    Virtually complete 16S rRNA sequences were determined for 54 representative strains of species in the family Pasteurellaceae. Of these strains, 15 were Pasteurella, 16 were Actinobacillus, and 23 were Haemophilus. A phylogenetic tree was constructed based on sequence similarity, using the Neighbor-Joining method. Fifty-three of the strains fell within four large clusters. The first cluster included the type strains of Haemophilus influenzae, H. aegyptius, H. aphrophilus, H. haemolyticus, H. paraphrophilus, H. segnis, and Actinobacillus actinomycetemcomitans. This cluster also contained A. actinomycetemcomitans FDC Y4, ATCC 29522, ATCC 29523, and ATCC 29524 and H. aphrophilus NCTC 7901. The second cluster included the type strains of A. seminis and Pasteurella aerogenes and H. somnus OVCG 43826. The third cluster was composed of the type strains of Pasteurella multocida, P. anatis, P. avium, P. canis, P. dagmatis, P. gallinarum, P. langaa, P. stomatis, P. volantium, H. haemoglobinophilus, H. parasuis, H. paracuniculus, H. paragallinarum, and A. capsulatus. This cluster also contained Pasteurella species A CCUG 18782, Pasteurella species B CCUG 19974, Haemophilus taxon C CAPM 5111, H. parasuis type 5 Nagasaki, P. volantium (H. parainfluenzae) NCTC 4101, and P. trehalosi NCTC 10624. The fourth cluster included the type strains of Actinobacillus lignieresii, A. equuli, A. pleuropneumoniae, A. suis, A. ureae, H. parahaemolyticus, H. parainfluenzae, H. paraphrohaemolyticus, H. ducreyi, and P. haemolytica. This cluster also contained Actinobacillus species strain CCUG 19799 (Bisgaard taxon 11), A. suis ATCC 15557, H. ducreyi ATCC 27722 and HD 35000, Haemophilus minor group strain 202, and H. parainfluenzae ATCC 29242. The type strain of P. pneumotropica branched alone to form a fifth group. The branching of the Pasteurellaceae family tree was quite complex. The four major clusters contained multiple subclusters. The clusters contained both rapidly and slowly evolving

  14. The relationship of serum IgG antibody titers to periodontal pathogens to indicators of the host response in crevicular fluid.

    PubMed

    Lamster, I B; Celenti, R; Ebersole, J L

    1990-08-01

    In this study; the relationship of indicators of the local host response in gingival crevicular fluid (GCF) to the serum antibody titer to periodontal pathogens was examined. 15 patients with chronic adult periodontitis were studied. GCF was collected and analyzed for the total amount of IgG, IgM, the lysosomal enzyme B-glucuronidase (BG) and alpha-2-macroglobulin (alpha 2M). At the same examination, serum from these patients was collected, and enzyme-linked immunosorbent assays used to determine the serum IgG antibody titer to a panel of 17 periodontal pathogens (Actinobacillus actinomycetemcomitans (3 strains), Bacteroides gingivalis (4), Eikenella corrodens (2), Wolinella recta, Bacteroides intermedius (3), Fusobacterium nucleatum, and 3 Capnocytophaga species). Using Spearman rank order correlation analysis, correlation coefficients were calculated to relate the 4 indicators of host response in GCF to the serum IgG antibody titer to each of the 17 micro-organisms. The mean correlation between total IgG in GCF and the serum IgG antibody titer was positive (r = +0.30), and statistically significant correlations between total IgG in GCF and serum IgG antibody titer were observed for one strain of B. intermedius and C. ochracea. A weaker positive correlation was observed for IgM (r = 0.18). In contrast, the mean correlation between total BG in GCF and the serum antibody titer was negative (r = -0.34). Statistically significant negative correlations were observed for all 3 strains of A. actinomycetemcomitans, one strain of E. corrodens and W. recta. The mean correlation for alpha 2M was r = -0.06. These data suggest that elevated BG activity in GCF, believed to be a marker for lysosomal enzyme released from polymorphonuclear leukocytes in the crevicular environment, may be associated with a reduced serum IgG antibody response to suspected periodontal pathogens. Furthermore, these findings imply that the development of a serum IgG antibody response to suspected

  15. Modification of cystatin C activity by bacterial proteinases and neutrophil elastase in periodontitis.

    PubMed Central

    Abrahamson, M; Wikström, M; Potempa, J; Renvert, S; Hall, A

    1997-01-01

    AIM: To study the interaction between the human cysteine proteinase inhibitor, cystatin C, and proteinases of periodontitis associated bacteria. METHODS: Gingival crevicular fluid samples were collected from discrete periodontitis sites and their cystatin C content was estimated by enzyme linked immunosorbent assay (ELISA). The interaction between cystatin C and proteolytic enzymes from cultured strains of the gingival bacteria Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans was studied by measuring inhibition of enzyme activity against peptidyl substrates, by detection of break down patterns of solid phase coupled and soluble cystatin C, and by N-terminal sequence analysis of cystatin C products resulting from the interactions. RESULTS: Gingival crevicular fluid contained cystatin C at a concentration of approximately 15 nM. Cystatin C did not inhibit the principal thiol stimulated proteinase activity of P gingivalis. Instead, strains of P gingivalis and P intermedia, but not A actinomycetemcomitans, released cystatin C modifying proteinases. Extracts of five P gingivalis and five P intermedia strains all hydrolysed bonds in the N-terminal region of cystatin C at physiological pH values. The modified cystatin C resulting from incubation with one P gingivalis strain was isolated and found to lack the eight most N-terminal residues. The affinity of the modified inhibitor for cathepsin B was 20-fold lower (Ki 5 nM) than that of full length cystatin C. A 50 kDa thiol stimulated proteinase, gingipain R, was isolated from P gingivalis and shown to be responsible for the Arg8-bond hydrolysis in cystatin C. The cathepsin B inhibitory activity of cystatin C incubated with gingival crevicular fluid was rapidly abolished after Val10-bond cleavage by elastase from exudate neutrophils, but cleavage at the gingipain specific Arg8-bond was also demonstrated. CONCLUSIONS: The physiological control of cathepsin B activity is impeded in

  16. Chronic gingivitis: the prevalence of periodontopathogens and therapy efficiency.

    PubMed

    Igic, M; Kesic, L; Lekovic, V; Apostolovic, M; Mihailovic, D; Kostadinovic, L; Milasin, J

    2012-08-01

    The purpose of this study was to determine the level of gingival inflammation and the prevalence of periodontopathogenic microorganisms in adolescents with chronic gingivitis, as well as to compare the effectiveness of two approaches in gingivitis treatment-basic therapy alone and basic therapy + adjunctive low-level laser therapy (LLLT). After periodontal evaluation, the content of gingival pockets of 140 adolescents with gingivitis was analyzed by multiplex PCR for the presence of P. gingivalis, A. actinomycetemcomitans, T. forsythensis and P. intermedia. Subsequent to bacteria detection, the examinees were divided into two groups with homogenous clinical and microbiological characteristics. Group A was subjected to basic gingivitis therapy, and group B underwent basic therapy along with adjunctive LLLT. A statistically significant difference between the values of plaque-index (PI) and sulcus bleeding index (SBI) before and after therapy was confirmed in both groups (p<0.001), though more pronounced in group B. Following therapy, the incidence of periodontopathogenic microorganisms decreased considerably. The best result was obtained in P. gingivalis eradication by combined therapy (p=0.003). The presence of periodontopathogens in adolescents with gingivitis should be regarded as a sign for dentists to foster more effective oral health programs. LLLT appears to be beneficial as adjuvant to basic therapy.

  17. Comparative cytotoxicity of periodontal bacteria

    SciTech Connect

    Stevens, R.H.; Hammond, B.F.

    1988-11-01

    The direct cytotoxicity of sonic extracts (SE) from nine periodontal bacteria for human gingival fibroblasts (HGF) was compared. Equivalent dosages (in terms of protein concentration) of SE were used to challenge HGF cultures. The cytotoxic potential of each SE was assessed by its ability to (1) inhibit HGF proliferation, as measured by direct cell counts; (2) inhibit 3H-thymidine incorporation in HGF cultures; or (3) cause morphological alterations of the cells in challenged cultures. The highest concentration (500 micrograms SE protein/ml) of any of the SEs used to challenge the cells was found to be markedly inhibitory to the HGFs by all three of the criteria of cytotoxicity. At the lowest dosage tested (50 micrograms SE protein/ml); only SE from Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Fusobacterium nucleatum caused a significant effect (greater than 90% inhibition or overt morphological abnormalities) in the HGFs as determined by any of the criteria employed. SE from Capnocytophaga sputigena, Eikenella corrodens, or Wolinella recta also inhibited cell proliferation and thymidine incorporation at this dosage; however, the degree of inhibition (5-50%) was consistently, clearly less than that of the first group of three organisms named above. The SE of the three other organisms tested (Actinomyces odontolyticus, Bacteroides intermedius, and Streptococcus sanguis) had little or no effect (0-10% inhibition) at this concentration. The data suggest that the outcome of the interaction between bacterial components and normal resident cells of the periodontium is, at least in part, a function of the bacterial species.

  18. In vitro Evaluation of Copaifera oblongifolia Oleoresin Against Bacteria Causing Oral Infections and Assessment of Its Cytotoxic Potential.

    PubMed

    da S Moraes, Thaís; Leandro, Luis F; de O Silva, Larissa; Santiago, Mariana B; Souza, Ariana B; Furtado, Ricardo A; Tavares, Denise C; Veneziani, Rodrigo C S; Ambrósio, Sérgio R; Bastos, Jairo K; Martins, Carlos H G

    2016-01-01

    The oral cavity, which harbors more than 750 bacterial species, is one of the most diverse sites of the human body. Some of these bacteria have been associated with oral diseases, such as dental caries and endodontic infections. We report on the antimicrobial and cytotoxic activities of Copaifera oblongifolia oleoresin against bacteria that cause caries and endodontic infections. The aim of this study is to determine the minimum (MIC) and the bactericidal (MBC) inhibitory concentrations as well as the biofilm inhibition ability (through determination of MBIC50) of the C. oblongifolia oleoresin. This study also investigated the bactericidal kinetics (time-kill curves) and the synergistic effect of the C. oblongifolia oleoresin. Additionally, this study evaluated the cytotoxic activity of the oleoresin toward V79 cells by means of the colony-forming assay. The C. oblongifolia oleoresin gave promising MIC and MBC values, which ranged from 25 to 200 μg/mL. Analysis of the MBIC50values of the oleoresin revealed it displayed biofilm inhibitory activity against all the assayed bacteria. Analysis of the bactericidal kinetics showed different behaviors of the oleoresin against the tested bacteria at the different time intervals and concentrations assayed in this study. An additive effect of the oleoresin with chlorhexidine dihydrochloride occurred only for S. mitis and A. actinomycetemcomitans. The C. oblongifolia oleoresin showed cytotoxic activity at concentrations ≥ 625 μg/mL.

  19. Inhibitory effect of quercetin on periodontal pathogens in vitro.

    PubMed

    Geoghegan, F; Wong, R W K; Rabie, A B M

    2010-06-01

    Actinobacillus actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) are bacteria strongly associated with early onset, progressive and refractory periodontal disease and associated alveolar bone loss. Quercetin is a flavonoid found in many foods including apples, onions and tea. The aim of this study was to evaluate the effect of quercetin on in vitro growth of periodontal pathogens Aa and Pg. For comparison, quercetin's effect on several oral microbes was also evaluated. Different concentrations of quercetin solution were added to calibrated suspensions of Aa and Pg. All suspensions were incubated for 1, 3, 6, and 24 h in an anaerobic chamber at 37 degrees C. At each time point, selected dilutions from each culture broth were plated on blood agar plates. Colonies appearing on blood agar plates were visually counted on 3 days for Aa and 5 days for Pg. Minimum inhibitory concentrations of both periodontal pathogens were also determined. Both periodontal bacteria showed a significant decrease (p < 0.05) in viable counts after 1 h. No colony forming units of Pg could be observed after 24 h. The results suggest that quercetin possesses significant antimicrobial properties on periodontal pathogens in vitro. PMID:19957242

  20. [Clinical and microbiological study of adult periodontal disease].

    PubMed

    Nogueira Moreira, A; Fernández Canigia, L; Furman, C; Chiappe, V; Marcantoni, M; Bianchini, H

    2001-01-01

    The aim of this study was to carry out a microbiological evaluation of sites with and without clinical evidence of moderate and severe periodontitis and their correlation with clinical parameters. A total of 52 disease sites and 10 healthy sites were selected according to clinical criteria. The following clinical indexes were measured for all the sites: plaque index, gingival index, blood on probing, depth on probing and insertion level. Samples of subgingival plaque were collected for culture and for differential counts of microbial morphotypes. In disease sites the most frequently isolated were: Prevotella intermedia/nigrescens (65%), Porphyromonas gingivalis (23%), Actinobacillus actinomycetemcomitans (23%), Fusobacterium nucleatum (10%) and Peptostreptococcus sp. (31%). The aerobic gram-positive microflora was predominant in healthy sites. Significant differences were observed in microbial morphotypes between healthy and disease sites: cocci 18.71% and 78.90%, motile rods 46.12% and 16.70%, total spirochetes 26.48% and 2.80%, respectively. The presence of motile rods, spirochetes and P. intermedia/nigrescens were the parameters with most sensitivity to suspect periodontal disease. There were significant differences in the subgingival microflora between healthy and disease sites in patients with moderate and severe periodontitis. PMID:11594003

  1. Anaerobic infections in the head and neck region.

    PubMed

    Tabaqchali, S

    1988-01-01

    Anaerobic bacteria form the predominant flora of the oral cavity, outnumbering facultative organisms by 10-1,000: 1. The type of anaerobic bacteria and their concentration depend on the anatomical site and the degree of anaerobiosis in the different sites in the mouth. Three groups of anaerobic bacteria inhabit the oral cavity; the strict anaerobes, the moderate anaerobes, and the microaerophilic group of organisms. The majority of anaerobic bacterial infections occurring in the region of the mouth, head and neck are caused by the commensal flora. These infections include dental and periodontal disease where the predominant organisms are Bacteroides species, Veillonella, Bifidobacteria, Peptococcus, Peptostreptococcus and Propionibacterium species. More recently, Bacteroides endontalis has been isolated from a periapical abscess of endodontal origin and B. gingivalis, B. intermedius, Haemophilus actinomycetemcomitans and Wollinella species in chronic periodontal disease. Treponema species and other strict anaerobes are seen in smears of severe periodontal disease and acute necrotising gingivitis, but have not yet been isolated in pure culture. Until such time, their role in disease remains uncertain. Fusobacterium nucleatum is specially associated with severe orofacial infections which may extend into the mediastinum. Other anaerobic infections include chronic otitis media, chronic sinusitis and mastoiditis, and brain abscess. Treatment of these conditions should include the use of beta-lactamase resistant antimicrobials, such as clindamycin or one of the nitroimidazoles with penicillin.

  2. Interleukin-1 haplotype and periodontal disease progression following therapy.

    PubMed

    Ehmke, B; Kress, W; Karch, H; Grimm, T; Klaiber, B; Flemmig, T F

    1999-12-01

    The purpose of this study was to assess the prognostic value of the IL-1 haplotype on the progression of periodontal disease following therapy. 48 adult patients with untreated periodontitis harboring Actinobacillus actinomycetemcomitans and/or Porphyromonas gingivalis were randomly assigned to receive full-mouth scaling alone (control) or in combination with systemic metronidazole plus amoxicillin and supragingival irrigation with chlorhexidine digluconate (test). All patients received supportive periodontal therapy at 3 to 6 months intervals. In 33 patients, lymphocyte DNA was analyzed for polymorphism in the IL-1A gene at position -889 and IL-1B gene at position +3953. Overall, 16 of 33 patients (7 of 17 test and 9 of 16 control) carried the IL-1 haplotype. 2 years following initial periodontal therapy, no differences in the survival rates of sites or teeth not exhibiting probing attachment loss of 2 mm or more compared to baseline, were found between patients who tested positive (85% sites, 53% teeth) and patients who tested negative (89% sites, 56% teeth) for the IL-1 haplotype. The results indicated that the IL-1 haplotype may be of limited value for the prognosis of periodontal disease progression following non-surgical periodontal therapy.

  3. Antimicrobial activity of Substance P and Neuropeptide Y against laboratory strains of bacteria and oral microorganisms.

    PubMed

    Hansen, Christopher J; Burnell, Kindra K; Brogden, Kim A

    2006-08-01

    Infection and inflammation of mucosal tissue may induce the production of neuropeptides, specifically Substance P and Neuropeptide Y. Since these neuropeptides are similar to antimicrobial peptides in their amino acid composition, amphipathic design, cationic charge, and size, we wanted to determine if they had antimicrobial activity against a panel of common bacteria and oral microorganisms using the radial diffusion assay. Neuropeptide Y and Substance P had antimicrobial activity against E. coli (MIC 20.6+/-5.5 microg/ml SEM and 71.5+/-15 SEM microg/ml, respectively), but did not have activity against laboratory strains of Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Serratia marcescens (MIC>500 microg/ml) nor oral strains of Streptococcus mutans, Candida albicans, and Actinobacillus actinomycetemcomitans (MIC>500 microg/ml). While Substance P and Neuropeptide Y did not have direct antimicrobial activity against the microorganisms tested, they still may stimulate local epithelial cells to produce other innate immune factors like defensins and cathelicidins. However, this remains to be determined.

  4. [Clinical and microbiological study of adult periodontal disease].

    PubMed

    Nogueira Moreira, A; Fernández Canigia, L; Furman, C; Chiappe, V; Marcantoni, M; Bianchini, H

    2001-01-01

    The aim of this study was to carry out a microbiological evaluation of sites with and without clinical evidence of moderate and severe periodontitis and their correlation with clinical parameters. A total of 52 disease sites and 10 healthy sites were selected according to clinical criteria. The following clinical indexes were measured for all the sites: plaque index, gingival index, blood on probing, depth on probing and insertion level. Samples of subgingival plaque were collected for culture and for differential counts of microbial morphotypes. In disease sites the most frequently isolated were: Prevotella intermedia/nigrescens (65%), Porphyromonas gingivalis (23%), Actinobacillus actinomycetemcomitans (23%), Fusobacterium nucleatum (10%) and Peptostreptococcus sp. (31%). The aerobic gram-positive microflora was predominant in healthy sites. Significant differences were observed in microbial morphotypes between healthy and disease sites: cocci 18.71% and 78.90%, motile rods 46.12% and 16.70%, total spirochetes 26.48% and 2.80%, respectively. The presence of motile rods, spirochetes and P. intermedia/nigrescens were the parameters with most sensitivity to suspect periodontal disease. There were significant differences in the subgingival microflora between healthy and disease sites in patients with moderate and severe periodontitis.

  5. Rapid quantification of periodontitis-related bacteria using a novel modification of Invader PLUS technologies.

    PubMed

    Tadokoro, Kenichi; Yamaguchi, Toshikazu; Kawamura, Katsumi; Shimizu, Hajime; Egashira, Toru; Minabe, Masato; Yoshino, Toshiaki; Oguchi, Hirokazu

    2010-01-01

    The Invader PLUS technology is a sensitive, rapid method for the detection and quantification of nucleic acid. While the original technology is based on the amplification by polymerase chain reaction (PCR) of the target sequence followed by its detection using the Invader technology, the current modification allows simultaneous PCR amplification and Invader reaction. The PCR primers and the Invader probes are designed to operate at the same temperature. This allows simpler design and faster results. This technology has been applied for the quantification of six periodontitis-related bacteria (Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Toreponema denticola, Tannerella forsythensis and Fusobacterium nucleatum). Direct comparison of this modified Invader PLUS with real-time PCR demonstrated similar linear range. Furthermore, testing of 64 volunteers showed a good correlation between both technologies with correlation factors r2 spanning between 0.827 and 0.987. We demonstrated here that the proposed improvement of the Invader PLUS allows the detection and quantification of DNA sequences using a simple design and protocol that can be implemented in clinical testing. PMID:18718748

  6. Brazilian propolis: physicochemical properties, plant origin and antibacterial activity on periodontopathogens.

    PubMed

    Santos, F A; Bastos, E M A F; Maia, A B R A; Uzeda, M; Carvalho, M A R; Farias, L M; Moreira, E S A

    2003-03-01

    Propolis samples collected in the dry and rainy seasons, from an experimental apiary located in a cerrado vegetation region in Brazil were used in this study. Microscopic analysis showed the presence of 31 pollen types, secretory hairs (genus Baccharis) and fragments of plant epidermis. The oxidation rates and the wax content of the samples after physicochemical analyses were in agreement with the Cuban Guideline NRAG 870-88. A high performance liquid chromatography analysis showed a similar pattern of chromatograms, characterized by the presence of ten phenolic compounds. There was no significant difference in the pro fi le of phenolic compounds and also in the total flavonoid concentration in propolis samples collected in different seasons. Antibacterial assays were performed by the method of dilution of an ethanol extract of propolis (EEP) in agar (v/v%) and showed that all 16 A. actinomycetemcomitans strains tested were inhibited by propolis concentrations of 0.1% to 0.25%, and did not grow at all at 0.5%. The growth inhibition of six Fusobacterium spp. and 16 black-pigmented anaerobes was observed at concentrations of 0.05% to 0.1%, and no growth was observed at 0.25%. There was no effect of seasonality on the inhibitory activity of propolis. The antibiotics tetracycline and meropenem were used as positive controls.

  7. Oral Chlamydia trachomatis in Patients with Established Periodontitis

    PubMed Central

    Reed, Susan G.; Lopatin, Dennis E.; Foxman, Betsy; Burt, Brian A.

    2009-01-01

    Periodontitis is considered a consequence of a pathogenic microbial infection at the periodontal site and host susceptibility factors. Periodontal research supports the association of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, and Bacteroides forsythus, and periodontitis; however causality has not been demonstrated. In pursuit of the etiology of periodontitis, we hypothesized that the intracellular bacteria, Chlamydia trachomatis, may play a role. As a first step, a cross-sectional study of dental school clinic patients with established periodontitis were assessed for the presence of C. trachomatis in the oral cavity, and in particular from the lining epithelium of periodontal sites. C. trachomatis was detected using a direct fluorescent monoclonal antibody (DFA) in oral specimens from 7% (6/87) of the patients. Four patients tested positive in specimens from the lining epithelium of diseased periodontal sites, one patient tested positive in healthy periodontal sites, and one patient tested positive in the general mucosal specimen. In conclusion, this study provides preliminary evidence of C. trachomatis in the periodontal sites. Planned studies include the use of a more precise periodontal epithelial cell collection device, the newer nucleic acid amplification techniques to detect C. trachomatis, and additional populations to determine the association of C. trachomatis and periodontitis. PMID:11218493

  8. Transcriptional activation of MMP-13 by periodontal pathogenic LPS requires p38 MAP kinase.

    PubMed

    Rossa, Carlos; Liu, Min; Bronson, Paul; Kirkwood, Keith L

    2007-01-01

    Matrix metalloprotease-13 (MMP-13) is induced by pro-inflammatory cytokines and increased expression is associated with a number of pathological conditions such as tumor metastasis, osteoarthritis, rheumatoid arthritis and periodontal diseases. MMP-13 gene regulation and the signal transduction pathways activated in response to bacterial LPS are largely unknown. In these studies, the role of the mitogen-activated protein kinase (MAPK) pathways in the regulation of MMP-13 induced by lipopolysaccharide was investigated. Lipopolysaccharide from Escherichia coli and Actinobacillus actinomycetemcomitans significantly (P < 0.05) increased MMP-13 steady-state mRNA (average of 27% and 46% increase, respectively) in murine periodontal ligament fibroblasts. MMP-13 mRNA induction was significantly reduced by inhibition of p38 MAP kinase. Immunoblot analysis indicated that p38 signaling was required for LPS-induced MMP-13 expression. Lipopolysaccharide induced proximal promoter reporter (-660/+32 mMMP-13) gene activity required p38 signaling. Collectively, these results indicate that lipopolysaccharide-induced murine MMP-13 is regulated by p38 signaling through a transcriptional mechanism.

  9. Dissemination of Periodontal Pathogens in the Bloodstream after Periodontal Procedures: A Systematic Review

    PubMed Central

    Horliana, Anna Carolina Ratto Tempestini; Chambrone, Leandro; Foz, Adriana Moura; Artese, Hilana Paula Carillo; Rabelo, Mariana de Sousa; Pannuti, Cláudio Mendes; Romito, Giuseppe Alexandre

    2014-01-01

    Background To date, there is no compilation of evidence-based information associating bacteremia and periodontal procedures. This systematic review aims to assess magnitude, duration, prevalence and nature of bacteremia caused by periodontal procedures. Study Design Systematic Review Types of Studies Reviewed MEDLINE, EMBASE and LILACS databases were searched in duplicate through August, 2013 without language restriction. Observational studies were included if blood samples were collected before, during or after periodontal procedures of patients with periodontitis. The methodological quality was assessed in duplicate using the modified Newcastle-Ottawa scale (NOS). Results Search strategy identified 509 potentially eligible articles and nine were included. Only four studies demonstrated high methodological quality, whereas five were of medium or low methodological quality. The study characteristics were considered too heterogeneous to conduct a meta-analysis. Among 219 analyzed patients, 106 (49.4%) had positive bacteremia. More frequent bacteria were S. viridans, A. actinomycetemcomitans P. gingivalis, M. micros and species Streptococcus and Actinomyces, although identification methods of microbiologic assays were different among studies. Clinical Implications Although half of the patients presented positive bacteremia after periodontal procedures, accurate results regarding the magnitude, duration and nature of bacteremia could not be confidentially assessed. PMID:24870125

  10. Acute Haemophilus parainfluenzae endocarditis: a case report

    PubMed Central

    2009-01-01

    Introduction Numerous pathogens can cause infective endocarditis, including Haemophilus parainfluenzae. H. parainfluenzae is part of the H. aphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae group that may cause about 3% of the total endocarditis cases, and is characterized by a subacute course and large vegetations. Case presentation Acute H. parainfluenzae endocarditis developed in a 54-year-old woman, with no underlying predisposing factors. The patient presented with fever of 3 days duration and a severe headache. Magnetic resonance imaging of the brain revealed multiple cerebral emboli with hemorrhagic foci. Upon suspicion of endocarditis, cardiac transesophageal ultrasonography was performed and revealed massive vegetations. The patient underwent emergency mitral valve replacement, and was further treated with ceftriaxone. Blood cultures grew H. parainfluenzae only after valve replacement, and a 6-week course of ceftriaxone was prescribed. Conclusion We underline the typical presentation of large vegetations in H. parainfluenzae endocarditis, which are associated with embolic phenomena and resulting severity. Although the majority of the few cases reported in the literature are subacute in progress, our case further underlines the possibility that H. parainfluenzae endocarditis may develop rapidly. Thus, awareness of the imaging characteristics of the pathogen may enhance early appropriate diagnosis and therapeutic response. PMID:19830211

  11. Gingival crevicular fluid levels of clindamycin compared with its minimal inhibitory concentrations for periodontal bacteria.

    PubMed Central

    Walker, C B; Gordon, J M; Cornwall, H A; Murphy, J C; Socransky, S S

    1981-01-01

    Clindamycin concentrations in gingival crevicular fluid and in blood were determined over a 7-h period and were related to the minimal inhibitory concentrations of this agent for 340 bacterial strains isolated from diseased periodontal sites. The clindamycin levels after administration of single 300-mg oral doses were measured in gingival crevicular fluids by using an agar diffusion bioassay. Minimal inhibitory concentrations were determined by agar dilution techniques for 30 species of periodontal bacteria. With the exception of Eikenella corrodens and Actinobacillus actinomycetemcomitans, most of the bacteria were inhibited by a concentration of 1.0 microgram of clindamycin per ml or less. The peak concentrations in crevicular fluid (2.0 +/- 0.3 microgram/ml) and in blood (1.9 +/- 0.3 micrograms/ml) were approximately the same. However, crevicular fluid levels of 1.0 micrograms/ml and above were present for up to 6 h, whereas blood concentrations dropped below 1.0 micrograms/ml within 2 h after administration. Based on its minimal inhibitory concentrations, clindamycin at crevicular fluid levels of 1.0 micrograms/ml or above should inhibit most bacteria associated with diseased periodontal sites. PMID:6794446

  12. Putative periodontopathic bacteria and herpesviruses in pregnant women: a case-control study

    PubMed Central

    Lu, Haixia; Zhu, Ce; Li, Fei; Xu, Wei; Tao, Danying; Feng, Xiping

    2016-01-01

    Little is known about herpesvirus and putative periodontopathic bacteria in maternal chronic periodontitis. The present case-control study aimed to explore the potential relationship between putative periodontopathic bacteria and herpesviruses in maternal chronic periodontitis.Saliva samples were collected from 36 pregnant women with chronic periodontitis (cases) and 36 pregnant women with healthy periodontal status (controls). Six putative periodontopathic bacteria (Porphyromonas gingivalis [Pg], Aggregatibacer actinomycetemcomitans [Aa], Fusobacterium nucleatum [Fn], Prevotella intermedia [Pi], Tannerella forsythia [Tf], and Treponema denticola [Td]) and three herpesviruses (Epstein-Barr virus [EBV], human cytomegalovirus [HCMV], and herpes simplex virus [HSV]) were detected. Socio-demographic data and oral health related behaviors, and salivary estradiol and progesterone levels were also collected. The results showed no significant differences in socio-demographic background, oral health related behaviors, and salivary estradiol and progesterone levels between the two groups (all P > 0.05). The detection rates of included periodontopathic microorganisms were not significantly different between the two groups (all P > 0.05), but the coinfection rate of EBV and Pg was significantly higher in the case group than in the control group (P = 0.028). EBV and Pg coinfection may promote the development of chronic periodontitis among pregnant women. PMID:27301874

  13. Genetic and Molecular Characterization of a Dental Pathogen Using Genome-Wide Approaches

    PubMed Central

    Actis, Luis A.; Rhodes, Eric; Tomaras, Andrew P.

    2005-01-01

    Actinobacillus actinomycetemcomitans causes periodontitis, a costly chronic infection that affects a large number of patients. The pathogenesis of this dental infection is a multifactorial process that results in a serious degenerative disease of the periodontium. Although significant progress has been achieved after the identification of this gram-negative bacterium as the etiological agent of this infection, much remains to be done to understand in detail the bacterial factors and host-pathogen interactions involved in the pathogenesis of this disease. Classical research approaches have resulted in the identification of important virulence factors and cellular processes, although they have provided a rather narrow picture of some of steps of this complex process. In contrast, a much wider picture could be obtained with the application of tools such as bioinformatics and genomics. These tools will provide global information regarding the differential expression of genes encoding factors and processes that lead to the pathogenesis of this disease. Furthermore, comparative genomics has the potential of helping to understand the emergence and evolution of this human pathogen. This genome-wide approach should provide a more complete picture of the pathogenesis process of this disease, and will facilitate the development of efficient diagnostic, preventive, and therapeutic measures for this disease. PMID:15126217

  14. Liver abscesses in cattle: A review of incidence in Holsteins and of bacteriology and vaccine approaches to control in feedlot cattle.

    PubMed

    Amachawadi, R G; Nagaraja, T G

    2016-04-01

    oils and vaccines, to control liver abscesses. Because liver abscess is a bacterial infection and the pathogenicity and virulence factors of have been studied widely, there have been considerable interest and efforts to develop an efficacious vaccine. The 2 antigens that have been targeted for vaccine production are leukotoxin and outer membrane proteins of . PMID:27136021

  15. Characterization of Mannheimia haemolytica isolated from feedlot cattle that were healthy or treated for bovine respiratory disease

    PubMed Central

    Klima, Cassidy L.; Alexander, Trevor W.; Hendrick, Steve; McAllister, Tim A.

    2014-01-01

    Mannheimia haemolytica is the principal bacterial pathogen associated with bovine respiratory disease (BRD). As an opportunistic pathogen, M. haemolytica is also frequently isolated from the respiratory tract of healthy cattle. This study examined the characteristics of M. haemolytica collected using deep nasal swabs from healthy cattle (n = 49) and cattle diagnosed with BRD (n = 41). Isolates were analyzed by pulsed-field gel electrophoresis (PFGE), serotyped, and tested for antimicrobial susceptibility. Polymerase chain reaction (PCR) was used to screen isolates for virulence [leukotoxin C (lktC), putative adhesin (ahs), outer-membrane lipoprotein (gs60), O-sialoglycoprotease (gcp), transferring-binding protein B (tbpB) and UDP-N-acetyl-D-glucosamine-2-epimerase (nmaA)] and antimicrobial resistance [tet(H), blaROB-1, erm(X), erm(42), msr(E)-mph(E) and aphA-1] genes. Isolates were genetically diverse but in three instances, M. haemolytica with the same pulsotype, resistance phenotype, and genotype were collected from cattle with BRD. This occurred once between cattle located in two different feedlots, once between cattle in the same feedlot, but in different pens, and once among cattle from the same feedlot in the same pen. Isolates from healthy cattle were primarily serotype 2 (75.5%) while those from individuals with BRD were serotype 1 (70.7%) or 6 (19.5%). Resistance to at least one antibiotic occurred more frequently (P < 0.001) in M. haemolytica collected from cattle with BRD (37%) compared with those that were healthy (2%). Overall, tetracycline resistance (18%) was the most prevalent resistant phenotype. All tetracycline-resistant M. haemolytica encoded tet(H). Ampicillin resistance (6%) and neomycin resistance (15%) were detected and corresponded to the presence of the blaROB-1 and aphA-1 genes, respectively. Tilmicosin resistance (6%) was also detected, but the resistance genes responsible were not identified. The virulence genes lktC, ahs, gs60, and gcp

  16. Free-Living Species of Carnivorous Mammals in Poland: Red Fox, Beech Marten, and Raccoon as a Potential Reservoir of Salmonella, Yersinia, Listeria spp. and Coagulase-Positive Staphylococcus.

    PubMed

    Nowakiewicz, Aneta; Zięba, Przemysław; Ziółkowska, Grażyna; Gnat, Sebastian; Muszyńska, Marta; Tomczuk, Krzysztof; Majer Dziedzic, Barbara; Ulbrych, Łukasz; Trościańczyk, Aleksandra

    2016-01-01

    The objective of the study was to examine a population of free-living carnivorous mammals most commonly found in Poland (red fox, beech marten, and raccoon) for the occurrence of bacteria that are potentially pathogenic for humans and other animal species and to determine their virulence potential (the presence of selected virulence genes). From the total pool of isolates obtained (n = 328), we selected 90 belonging to species that pose the greatest potential threat to human health: Salmonella spp. (n = 19; 4.51%), Yersinia enterocolitica (n = 10; 2.37%), Listeria monocytogenes and L. ivanovii (n = 21), and Staphylococcus aureus (n = 40; 9.5%). The Salmonella spp. isolates represented three different subspecies; S. enterica subsp. enterica accounted for a significant proportion (15/19), and most of the serotypes isolated (S. Typhimurium, S. Infantis, S. Newport and S. Enteritidis) were among the 10 non-typhoidal Salmonella serotypes that are most often responsible for infections in Europe, including Poland. Y. enterococlitica was detected in the smallest percentage of animals, but 60% of strains among the isolates tested possessed the ail gene, which is responsible for attachment and invasion. Potentially pathogenic Listeria species were isolated from approx. 5% of the animals. The presence of all tested virulence genes was shown in 35% of L. monocytogenes strains, while in the case of the other strains, the genes occurred in varying numbers and configurations. The presence of the inlA, inlC, hlyA, and iap genes was noted in all strains, whereas the genes encoding PI-PLC, actin, and internalin Imo2821 were present in varying percentages (from 80% to 55%). S. aureus was obtained from 40 individuals. Most isolates possessed the hla, hld (95% for each), and hlb (32.5%) genes encoding hemolysins as well as the gene encoding leukotoxin lukED (70%). In a similar percentage of strains (77.5%), the presence of at least one gene encoding enterotoxin was found, with 12

  17. Free-Living Species of Carnivorous Mammals in Poland: Red Fox, Beech Marten, and Raccoon as a Potential Reservoir of Salmonella, Yersinia, Listeria spp. and Coagulase-Positive Staphylococcus

    PubMed Central

    Nowakiewicz, Aneta; Zięba, Przemysław; Ziółkowska, Grażyna; Gnat, Sebastian; Muszyńska, Marta; Tomczuk, Krzysztof; Majer Dziedzic, Barbara; Ulbrych, Łukasz; Trościańczyk, Aleksandra

    2016-01-01

    The objective of the study was to examine a population of free-living carnivorous mammals most commonly found in Poland (red fox, beech marten, and raccoon) for the occurrence of bacteria that are potentially pathogenic for humans and other animal species and to determine their virulence potential (the presence of selected virulence genes). From the total pool of isolates obtained (n = 328), we selected 90 belonging to species that pose the greatest potential threat to human health: Salmonella spp. (n = 19; 4.51%), Yersinia enterocolitica (n = 10; 2.37%), Listeria monocytogenes and L. ivanovii (n = 21), and Staphylococcus aureus (n = 40; 9.5%). The Salmonella spp. isolates represented three different subspecies; S. enterica subsp. enterica accounted for a significant proportion (15/19), and most of the serotypes isolated (S. Typhimurium, S. Infantis, S. Newport and S. Enteritidis) were among the 10 non-typhoidal Salmonella serotypes that are most often responsible for infections in Europe, including Poland. Y. enterococlitica was detected in the smallest percentage of animals, but 60% of strains among the isolates tested possessed the ail gene, which is responsible for attachment and invasion. Potentially pathogenic Listeria species were isolated from approx. 5% of the animals. The presence of all tested virulence genes was shown in 35% of L. monocytogenes strains, while in the case of the other strains, the genes occurred in varying numbers and configurations. The presence of the inlA, inlC, hlyA, and iap genes was noted in all strains, whereas the genes encoding PI-PLC, actin, and internalin Imo2821 were present in varying percentages (from 80% to 55%). S. aureus was obtained from 40 individuals. Most isolates possessed the hla, hld (95% for each), and hlb (32.5%) genes encoding hemolysins as well as the gene encoding leukotoxin lukED (70%). In a similar percentage of strains (77.5%), the presence of at least one gene encoding enterotoxin was found, with 12

  18. Characterization of Mannheimia haemolytica isolated from feedlot cattle that were healthy or treated for bovine respiratory disease.

    PubMed

    Klima, Cassidy L; Alexander, Trevor W; Hendrick, Steve; McAllister, Tim A

    2014-01-01

    Mannheimia haemolytica is the principal bacterial pathogen associated with bovine respiratory disease (BRD). As an opportunistic pathogen, M. haemolytica is also frequently isolated from the respiratory tract of healthy cattle. This study examined the characteristics of M. haemolytica collected using deep nasal swabs from healthy cattle (n = 49) and cattle diagnosed with BRD (n = 41). Isolates were analyzed by pulsed-field gel electrophoresis (PFGE), serotyped, and tested for antimicrobial susceptibility. Polymerase chain reaction (PCR) was used to screen isolates for virulence [leukotoxin C (lktC), putative adhesin (ahs), outer-membrane lipoprotein (gs60), O-sialoglycoprotease (gcp), transferring-binding protein B (tbpB) and UDP-N-acetyl-D-glucosamine-2-epimerase (nmaA)] and antimicrobial resistance [tet(H), bla ROB-1, erm(X), erm(42), msr(E)-mph(E) and aphA-1] genes. Isolates were genetically diverse but in three instances, M. haemolytica with the same pulsotype, resistance phenotype, and genotype were collected from cattle with BRD. This occurred once between cattle located in two different feedlots, once between cattle in the same feedlot, but in different pens, and once among cattle from the same feedlot in the same pen. Isolates from healthy cattle were primarily serotype 2 (75.5%) while those from individuals with BRD were serotype 1 (70.7%) or 6 (19.5%). Resistance to at least one antibiotic occurred more frequently (P < 0.001) in M. haemolytica collected from cattle with BRD (37%) compared with those that were healthy (2%). Overall, tetracycline resistance (18%) was the most prevalent resistant phenotype. All tetracycline-resistant M. haemolytica encoded tet(H). Ampicillin resistance (6%) and neomycin resistance (15%) were detected and corresponded to the presence of the bla ROB-1 and aphA-1 genes, respectively. Tilmicosin resistance (6%) was also detected, but the resistance genes responsible were not identified. The virulence genes lktC, ahs, gs60, and

  19. Identification of livestock-associated methicillin-resistant Staphylococcus aureus isolates in Korea and molecular comparison between isolates from animal carcasses and slaughterhouse workers.

    PubMed

    Moon, Dong Chan; Tamang, Migma Dorji; Nam, Hyang-Mi; Jeong, Jin-Ha; Jang, Geum-Chan; Jung, Suk-Chan; Park, Yong-Ho; Lim, Suk-Kyung

    2015-04-01

    This study was undertaken to screen methicillin-resistant Staphylococcus aureus (MRSA) in animal carcasses and slaughterhouse workers and characterize MRSA isolates identified during 2010-2012 in Korea. A total of 830 (16.4%) S. aureus and 65 (1.3%) MRSA were isolated from 9669 carcass samples. MRSA was more frequently detected in chicken carcasses (1.2%) than in cattle (0.3%) and pig carcasses (0.6%). The prevalence of MRSA in workers was 6.9% (4/58) in chicken slaughterhouse workers, but no MRSA was detected in pig and cattle slaughterhouse workers (0/41). Two different lineages of MRSA were identified (i.e., human-associated type [ST5, ST59, and ST72] and livestock-associated [LA] type [ST398, ST541, and ST692]); only LA MRSA was observed in chicken carcasses, whereas both types were found in cattle and pig carcasses and workers. All human-associated MRSA isolates carried enterotoxin and/or leukotoxin genes, whereas LA MRSA types did not carry these genes, except ST692 type. However, all LA MRSA isolates were multiresistant, whereas human-associated types were susceptible or resistant to fewer than two antimicrobials except ST5. Furthermore, one or more resistance genes were attributed for resistance to aminoglycosides (aac(6')-Ie-aph(2″), ant(4')-Ia, and aph(3')-IIIa), tetracycline [tet(K), tet(L), tet(M), and tet(S)], macrolide-lincosamide-streptogramin B (ermA, ermB, ermC, and ermT), lincosamide [lnu(B)], phenicol-lincosamide-oxazolidinone-pleuromutilin-streptogramin A (cfr), chloramphenicol (fexA), and fusidic acid [fus(C)]. To our knowledge, this is the first report of tet(S) gene in MRSA isolates and first detection of a unique (ST692) type of MRSA in occupational workers. Detection of new types of human-associated and LA MRSA with multiple resistance and virulence genes in food animal products constitutes a potential threat to public health.

  20. Free-Living Species of Carnivorous Mammals in Poland: Red Fox, Beech Marten, and Raccoon as a Potential Reservoir of Salmonella, Yersinia, Listeria spp. and Coagulase-Positive Staphylococcus.

    PubMed

    Nowakiewicz, Aneta; Zięba, Przemysław; Ziółkowska, Grażyna; Gnat, Sebastian; Muszyńska, Marta; Tomczuk, Krzysztof; Majer Dziedzic, Barbara; Ulbrych, Łukasz; Trościańczyk, Aleksandra

    2016-01-01

    The objective of the study was to examine a population of free-living carnivorous mammals most commonly found in Poland (red fox, beech marten, and raccoon) for the occurrence of bacteria that are potentially pathogenic for humans and other animal species and to determine their virulence potential (the presence of selected virulence genes). From the total pool of isolates obtained (n = 328), we selected 90 belonging to species that pose the greatest potential threat to human health: Salmonella spp. (n = 19; 4.51%), Yersinia enterocolitica (n = 10; 2.37%), Listeria monocytogenes and L. ivanovii (n = 21), and Staphylococcus aureus (n = 40; 9.5%). The Salmonella spp. isolates represented three different subspecies; S. enterica subsp. enterica accounted for a significant proportion (15/19), and most of the serotypes isolated (S. Typhimurium, S. Infantis, S. Newport and S. Enteritidis) were among the 10 non-typhoidal Salmonella serotypes that are most often responsible for infections in Europe, including Poland. Y. enterococlitica was detected in the smallest percentage of animals, but 60% of strains among the isolates tested possessed the ail gene, which is responsible for attachment and invasion. Potentially pathogenic Listeria species were isolated from approx. 5% of the animals. The presence of all tested virulence genes was shown in 35% of L. monocytogenes strains, while in the case of the other strains, the genes occurred in varying numbers and configurations. The presence of the inlA, inlC, hlyA, and iap genes was noted in all strains, whereas the genes encoding PI-PLC, actin, and internalin Imo2821 were present in varying percentages (from 80% to 55%). S. aureus was obtained from 40 individuals. Most isolates possessed the hla, hld (95% for each), and hlb (32.5%) genes encoding hemolysins as well as the gene encoding leukotoxin lukED (70%). In a similar percentage of strains (77.5%), the presence of at least one gene encoding enterotoxin was found, with 12

  1. Effect of copper, manganese, and zinc supplementation on the performance, clinical signs, and mineral status of calves following exposure to bovine viral diarrhea virus type 1b and subsequent infection.

    PubMed

    Wilson, B K; Vazquez-Anon, M; Step, D L; Moyer, K D; Haviland, C L; Maxwell, C L; O'Neill, C F; Gifford, C A; Krehbiel, C R; Richards, C J

    2016-03-01

    Research has indicated that trace mineral (TM) supplementation may alter immune function and reduce morbidity associated with bovine respiratory disease. The objective of this experiment was to determine the influence of dietary Cu, Mn, and Zn supplementation on the performance, clinical signs, and TM balance of calves following a bovine viral diarrhea virus (BVDV) and (MH) combination respiratory pathogen challenge. Steers ( = 16; 225 ± 20 kg BW) from a single ranch were processed, weaned, and randomly pairwise assigned to either the TM-supplemented (MIN) or the control (CON) experimental treatments. The MIN calves received an additional 150 mg of Cu, 130 mg of Mn, and 320 mg of Zn daily and the CON calves received the basal diet with no additional Cu, Mn, or Zn supplementation. The basal diet contained sufficient Mn and Zn but inadequate Cu based on published nutrient requirements. After 46 d on the experimental treatments, all calves were naturally exposed to a heifer persistently infected with BVDV type 1b for 4 d and then subsequently intratracheally challenged with MH. Data were analyzed using the GLIMMIX procedure of SAS with sampling time serving as a repeated measure and calf serving as the experimental unit. The respiratory challenge was validated via increased BVDV type 1b antibody concentrations, MH whole cell and leukotoxin antibody concentrations, rectal temperatures (TEMP), and subjective clinical severity scores (CS). Calf performance ( ≥ 0.48) was not affected by TM supplementation. Mineral supplementation also did not impact the CS or TEMP of calves ( ≥ 0.53). There was a treatment × time ( < 0.001) interaction observed for liver Cu concentrations. The concentrations of Cu, Mn, Zn, and Fe within the liver; Cu, Mn, and Zn within the muscle; and Cu, Zn, and Fe within the serum were all impacted by time ( ≤ 0.03). Calves receiving the MIN treatment had greater ( < 0.01) liver Cu and Mn concentrations compared with CON calves. In contrast

  2. Genotyping of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) in a tertiary care centre in Mysore, South India: ST2371-SCCmec IV emerges as the major clone.

    PubMed

    Rajan, Vineeth; Schoenfelder, Sonja M K; Ziebuhr, Wilma; Gopal, Shubha

    2015-08-01

    The burden of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) is on the rise in population and clinical settings on account of the adaptability and virulence traits of this pathogen. We characterized 45 non-duplicate CA-MRSA strains implicated mainly in skin and soft tissue infections (SSTIs) in a tertiary care hospital in Mysore, South India. All the isolates were genotyped by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) typing, accessory gene regulator (agr) typing, and multi-locus sequence typing (MLST). Four sequence types (STs) belonging to three major clonal complexes (CCs) were identified among the isolates: CC22 (ST2371 and ST22), CC1 (ST772) and CC8 (ST8). The majority (53.3%) of the isolates was of the genotype ST2371-t852-SCCmec IV [sequence type-spa type-SCCmec type], followed by ST22-t852-SCCmec IV (22.2%), ST772-t657-SCCmec V (13.3%) and ST8-t008-SCCmec IV (11.1%). ST237I, a single locus variant of ST22 (EMRSA-15 clone), has not been reported previously from any of the Asian countries. Our study also documents for the first time, the appearance of ST8-SCCmec IV (USA300) strains in India. Representative strains of the STs were further analyzed by pulsed field gel electrophoresis (PFGE). agr typing detected type I or II alleles in the majority of the isolates. All the isolates were positive for the leukotoxin gene, pvl (Panton-Valentine leukocidin) and the staphylococcal enterotoxin gene cluster, egc. Interestingly, multidrug resistance (resistance to ⩾3 classes of non-beta-lactam antibiotics) was observed in 77.8% (n=35) of the isolates. The highest (75.5%) resistance was recorded for ciprofloxacin, followed by erythromycin (53.3%), and quinupristin-dalfopristin (51.1%). Inducible clindamycin-resistance was identified in 37.7% of the isolates and it was attributed to the presence of erm(A), erm(C) and a combination of erm(A) and erm(C) genes. Isolates which showed a phenotypic

  3. Microbiota of deciduous endodontic infections analyzed by MDA and Checkerboard DNA-DNA hybridization

    PubMed Central

    Tavares, WLF; de Brito, LC Neves; Teles, RP; Massara, MLA; Sobrinho, AP Ribeiro; Haffajee, AD; Socransky, SS; Teles, FR

    2011-01-01

    Aims To evaluate the microbiota of endodontic infections in deciduous teeth by checkerboard DNA-DNA hybridization after uniform amplification of DNA in samples by multiple displacement amplification (MDA). Methodology Forty samples from the root canal system of deciduous teeth exhibiting pulp necrosis with or without radiographically detectable periradicular/interadicular bone resorption were collected and 32 were analyzed, with 3 individuals contributing 2 samples; these were MDA- amplified and analyzed by Checkerboard DNA-DNA hybridization for levels of 83 bacterial taxa. Two outcome measures were used: the percentage of teeth colonized by each species; and the mean proportion of each bacterial taxon present across all samples were computed. Results The mean amount of DNA in the samples prior to amplification was 5.2 (± 4.7) ng and 6.1 (± 2.3) μg after MDA. The mean number of species detected per sample was 19 (± 4) (range: 3–66) to the nearest whole number. The most prevalent taxa were Prevotella intermedia (96.9%), Neisseria mucosa (65.6%), Prevotella nigrescens (56.2%) and Tannerella forsythia (56.2%). Aggregatibacter (Haemophilus) aphrophilus and Helicobacter pylori were not detected. P. intermedia (10%), Prevotella tannerae (7%) and Prevotella nigrescens (4.3%) presented the highest mean proportions of the target species averaged across the positive samples. Conclusion Root canals of infected deciduous teeth had a diverse bacterial population. Prevotella sp were commonly found with P. intermedia, Prevotella tannerae and Prevotella nigrescens among the most prominent species detected. PMID:21083570

  4. Massive Parallel Sequencing Provides New Perspectives on Bacterial Brain Abscesses

    PubMed Central

    Wilhelmsen, Marianne Thulin; Skrede, Steinar; Meisal, Roger; Jakovljev, Aleksandra; Gaustad, Peter; Hermansen, Nils Olav; Vik-Mo, Einar; Solheim, Ole; Ambur, Ole Herman; Sæbø, Øystein; Høstmælingen, Christina Teisner; Helland, Christian

    2014-01-01

    Rapid development within the field of massive parallel sequencing (MPS) is about to bring this technology within reach for diagnostic microbiology laboratories. We wanted to explore its potential for improving diagnosis and understanding of polymicrobial infections, using bacterial brain abscesses as an example. We conducted a prospective nationwide study on bacterial brain abscesses. Fifty-two surgical samples were included over a 2-year period. The samples were categorized as either spontaneous intracerebral, spontaneous subdural, or postoperative. Bacterial 16S rRNA genes were amplified directly from the specimens and sequenced using Ion Torrent technology, with an average of 500,000 reads per sample. The results were compared to those from culture- and Sanger sequencing-based diagnostics. Compared to culture, MPS allowed for triple the number of bacterial identifications. Aggregatibacter aphrophilus, Fusobacterium nucleatum, and Streptococcus intermedius or combinations of them were found in all spontaneous polymicrobial abscesses. F. nucleatum was systematically detected in samples with anaerobic flora. The increased detection rate for Actinomyces spp. and facultative Gram-negative rods further revealed several species associations. We suggest that A. aphrophilus, F. nucleatum, and S. intermedius are key pathogens for the establishment of spontaneous polymicrobial brain abscesses. In addition, F. nucleatum seems to be important for the development of anaerobic flora. MPS can accurately describe polymicrobial specimens when a sufficient number of reads is used to compensate for unequal species concentrations and principles are defined to discard contaminant bacterial DNA in the subsequent data analysis. This will contribute to our understanding of how different types of polymicrobial infections develop. PMID:24671797

  5. The ecological proportion of indigenous bacterial populations in saliva is correlated with oral health status.

    PubMed

    Takeshita, Toru; Nakano, Yoshio; Kumagai, Takashi; Yasui, Masaki; Kamio, Noriaki; Shibata, Yukie; Shiota, Susumu; Yamashita, Yoshihisa

    2009-01-01

    To obtain deeper insights into the etiology of oral disease, an understanding of the composition of the surrounding bacterial environments that lead to health or disease is required, which is attracting increasing attention. In this study, the bacterial compositions in the saliva of 200 subjects aged 15-40 years were depicted as peak patterns by terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes. The subjects were classified into three clusters by partitioning around medoids clustering based on their T-RFLP profiles, and the clinical oral health parameters of the clusters were compared. The clustering of the T-RFLP profiles in this study was mainly based on differences in the abundance distribution of the dominant terminal restriction fragments (TRFs) detected in most of the subjects. Predicted from the sizes of the TRFs, the characteristically more predominant members of each were Prevotella and Veillonella species in cluster I; Streptococcus species in cluster II and Neisseria, Haemophilus or Aggregatibacter species and Porphyromonas species in cluster III. The parameters associated with periodontal disease were significantly different among the clusters. Clusters I and II had a higher percentage of sites of periodontal pockets greater than 4 mm than cluster III, and cluster I contained sites exhibiting bleeding on probing more often than cluster II or III; no significant differences were observed in other parameters. These results suggest that the abundance distribution of commensal bacteria in saliva is correlated with periodontal health, and might be involved in the susceptibility of an individual to periodontal disease. PMID:18830275

  6. Subgingival microbial profile and production of proinflammatory cytokines in chronic periodontitis.

    PubMed

    Dosseva-Panova, Velichka T; Popova, Christina L; Panov, Vladimir E

    2014-01-01

    This review examines literature data concerning the bacterial findings in chronic periodontitis depending on pocket depth, and presents the latest published information on the presence of proinflammatory factors in periodontal environment. It has been found that chronic periodontitis affects as much as 80% of the middle-aged population; by comparison, the prevalence of aggressive periodontitis reaches up to 1-1.5%. It is accepted that this social disease is multifactorial in etiology, but the evidence in the literature suggests that the levels of specific Gram-negative organisms in subgingival plaque biofilm play a major role in the initiation and progression of the disease. Of the many bacterial species inhabiting the periodontal environment, three types--Porphyromonas gingivalis (PG), Treponema denticola (TD), Tannerella forsythia (TF)--are strongly associated with the initiation and progression of periodontitis. Microbiological studies suggest that Porphyromonas gingivalis should be considered a major etiologic agent. Currently, Porphyromonas gingivalis is strongly associated with the pathogenesis of chronic periodontitis. On the other hand, the presence of Aggregatibacter actinomycetemeomitans in patients with chronic periodontitis may be related to the severity of the disease and thus modify the therapeutic plan. The increased amount of periodontal pathogens in the subgingival area can activate a cascade of defense mechanisms of the body associated with the production of factors causing inflammation and destruction, which suggests a correlation between the bacterial findings and the body response implemented by enhancing the local cytokine expression. Studies in the literature show that the presence of certain micro-organisms in the periodontal environment is associated to increased levels of proinflammatory cytokines in the gingival fluid and gingival tissue. These levels have been associated with destructive tissues response. There is little evidence in the

  7. HIV Infection and Microbial Diversity in Saliva

    PubMed Central

    Saxena, Deepak; Chen, Zhou; Liu, Gaoxia; Abrams, Willam R.; Phelan, Joan A.; Norman, Robert G.; Fisch, Gene S.; Corby, Patricia M.; Dewhirst, Floyd; Paster, Bruce J.; Kokaras, Alexis S.; Malamud, Daniel

    2014-01-01

    Limited information is available about the effects of HIV and subsequent antiretroviral treatment on host-microbe interactions. This study aimed to determine the salivary microbial composition for 10 HIV-seropositive subjects, before and 6 months after highly active antiretroviral therapy (HAART), compared with that for 10 HIV-seronegative subjects. A conventional culture and two culture-independent analyses were used and consistently demonstrated differences in microbial composition among the three sets of samples. HIV-positive subjects had higher levels of total cultivable microbes, including oral streptococci, lactobacilli, Streptococcus mutans, and Candida, in saliva than did HIV-negative subjects. The total cultivable microbial levels were significantly correlated with CD4+ T cell counts. Denaturing gradient gel electrophoresis (DGGE), which compared the overall microbial profiles, showed distinct fingerprinting profiles for each group. The human oral microbe identification microarray (HOMIM) assay, which compared the 16S rRNA genes, showed clear separation among the three sample groups. Veillonella, Synergistetes, and Streptococcus were present in all 30 saliva samples. Only minor changes or no changes in the prevalence of Neisseria, Haemophilus, Gemella, Leptotrichia, Solobacterium, Parvimonas, and Rothia were observed. Seven genera, Capnocytophaga, Slackia, Porphyromonas, Kingella, Peptostreptococcaceae, Lactobacillus, and Atopobium, were detected only in HIV-negative samples. The prevalences of Fusobacterium, Campylobacter, Prevotella, Capnocytophaga, Selenomonas, Actinomyces, Granulicatella, and Atopobium were increased after HAART. In contrast, the prevalence of Aggregatibacter was significantly decreased after HAART. The findings of this study suggest that HIV infection and HAART can have significant effects on salivary microbial colonization and composition. PMID:24523469

  8. A high-throughput microfluidic dental plaque biofilm system to visualize and quantify the effect of antimicrobials

    PubMed Central

    Nance, William C.; Dowd, Scot E.; Samarian, Derek; Chludzinski, Jeffrey; Delli, Joseph; Battista, John; Rickard, Alexander H.

    2013-01-01

    Objectives Few model systems are amenable to developing multi-species biofilms in parallel under environmentally germane conditions. This is a problem when evaluating the potential real-world effectiveness of antimicrobials in the laboratory. One such antimicrobial is cetylpyridinium chloride (CPC), which is used in numerous over-the-counter oral healthcare products. The aim of this work was to develop a high-throughput microfluidic system that is combined with a confocal laser scanning microscope (CLSM) to quantitatively evaluate the effectiveness of CPC against oral multi-species biofilms grown in human saliva. Methods Twenty-four-channel BioFlux microfluidic plates were inoculated with pooled human saliva and fed filter-sterilized saliva for 20 h at 37°C. The bacterial diversity of the biofilms was evaluated by bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). The antimicrobial/anti-biofilm effect of CPC (0.5%–0.001% w/v) was examined using Live/Dead stain, CLSM and 3D imaging software. Results The analysis of biofilms by bTEFAP demonstrated that they contained genera typically found in human dental plaque. These included Aggregatibacter, Fusobacterium, Neisseria, Porphyromonas, Streptococcus and Veillonella. Using Live/Dead stain, clear gradations in killing were observed when the biofilms were treated with CPC between 0.5% and 0.001% w/v. At 0.5% (w/v) CPC, 90% of the total signal was from dead/damaged cells. Below this concentration range, less killing was observed. In the 0.5%–0.05% (w/v) range CPC penetration/killing was greatest and biofilm thickness was significantly reduced. Conclusions This work demonstrates the utility of a high-throughput microfluidic–CLSM system to grow multi-species oral biofilms, which are compositionally similar to naturally occurring biofilms, to assess the effectiveness of antimicrobials. PMID:23800904

  9. HACEK infective endocarditis: characteristics and outcomes from a large, multi-national cohort.

    PubMed

    Chambers, Stephen T; Murdoch, David; Morris, Arthur; Holland, David; Pappas, Paul; Almela, Manel; Fernández-Hidalgo, Nuria; Almirante, Benito; Bouza, Emilio; Forno, Davide; del Rio, Ana; Hannan, Margaret M; Harkness, John; Kanafani, Zeina A; Lalani, Tahaniyat; Lang, Selwyn; Raymond, Nigel; Read, Kerry; Vinogradova, Tatiana; Woods, Christopher W; Wray, Dannah; Corey, G Ralph; Chu, Vivian H

    2013-01-01

    The HACEK organisms (Haemophilus species, Aggregatibacter species, Cardiobacterium hominis, Eikenella corrodens, and Kingella species) are rare causes of infective endocarditis (IE). The objective of this study is to describe the clinical characteristics and outcomes of patients with HACEK endocarditis (HE) in a large multi-national cohort. Patients hospitalized with definite or possible infective endocarditis by the International Collaboration on Endocarditis Prospective Cohort Study in 64 hospitals from 28 countries were included and characteristics of HE patients compared with IE due to other pathogens. Of 5591 patients enrolled, 77 (1.4%) had HE. HE was associated with a younger age (47 vs. 61 years; p<0.001), a higher prevalence of immunologic/vascular manifestations (32% vs. 20%; p<0.008) and stroke (25% vs. 17% p = 0.05) but a lower prevalence of congestive heart failure (15% vs. 30%; p = 0.004), death in-hospital (4% vs. 18%; p = 0.001) or after 1 year follow-up (6% vs. 20%; p = 0.01) than IE due to other pathogens (n = 5514). On multivariable analysis, stroke was associated with mitral valve vegetations (OR 3.60; CI 1.34-9.65; p<0.01) and younger age (OR 0.62; CI 0.49-0.90; p<0.01). The overall outcome of HE was excellent with the in-hospital mortality (4%) significantly better than for non-HE (18%; p<0.001). Prosthetic valve endocarditis was more common in HE (35%) than non-HE (24%). The outcome of prosthetic valve and native valve HE was excellent whether treated medically or with surgery. Current treatment is very successful for the management of both native valve prosthetic valve HE but further studies are needed to determine why HE has a predilection for younger people and to cause stroke. The small number of patients and observational design limit inferences on treatment strategies. Self selection of study sites limits epidemiological inferences.

  10. Detection of periodontopathogenic bacteria in pregnant women by traditional anaerobic culture method and by a commercial molecular genetic method.

    PubMed

    Urbán, Edit; Terhes, Gabriella; Radnai, Márta; Gorzó, István; Nagy, Elisabeth

    2010-06-01

    To culture facultative and strict anaerobic bacteria is a well-established method for analyzing subgingival plaque samples. Micro-IDent and micro-IDent Plus (HAIN Lifescience GmbH, Nehren, Germany) tests are two commercially available rapid PCR-based methods for the identification and quantification of putative periodontopathogen bacteria. In this study, we compared these commercial PCR-based hybridization methods with conventional anaerobic culture technique. A total of 36 subgingival plaque samples were collected from periodontal pockets of pregnant women with chronic localized periodontitis. Aliquots of these samples were evaluated with species-specific probes provided by micro-IDent and micro-IDent Plus tests simultaneously, and from the same samples anaerobic and capnophylic bacteria were cultured on selective media. The overall agreement between both methods was excellent for Eubacterium nodatum, Tannerella forsythia and Porphyromonas gingivalis (97-92%), fair for Capnocytophaga sp, Eikenella corrodens, Actinobacillus actinomycetemcomitans, and Prevotella intermedia (91-89%) and poor for Fusobacterium nucleatum, Parvimonas micra (Micromonas micros), and Campylobacter rectus (86-78%). Discrepancies in the results may be explained by inability of culture method to distinguish between closely related taxa (e.i P. intermedia/Prevotella. nigrescens), and problems of keeping periodontopathogen bacteria viable, which is required for successful detection by standard culture method. Nucleic acid-based methods may replace cultivation method as frequently used methods in microbiological diagnosis of progressive periodontitis, thus micro-IDent and micro-IDent Plus tests can be recommended where culture of periodontopathogenic bacteria is not performed in routine microbiology laboratories to analyze subgingival plaque samples.

  11. [Purine regulon of gamma-proteobacteria: a detailed description].

    PubMed

    Ravcheev, D A; Gel'fand, M S; Mironov, A A; Rakhmaninova, A B

    2002-09-01

    The structure of the purine regulon was studied by a comparative genomic approach in seven genomes of gamma-proteobacteria: Escherichia coli, Salmonella typhi, Yersinia pestis, Haemophilus influenzae, Pasteurella multocida, Actinobacillus actinomycetemcomitans, and Vibrio cholerae. The palindromic binding site of the purine repressor (consensus ACGCAAACGTTTGCGT) is fairly well retained of genes encoding enzymes that participate in the synthesis of inosinemonophosphate from phosphoribozylpyrophosphate and in transfer of unicarbon groups, and also upstream of some transport protein genes. These genes may be regarded as the main part of the purine regulon. In terms of physiology, the regulation of the purC and gcvTHP/folD genes seems to be especially important, because the PurR site was found upstream of nonorthologous but functionally replaceable genes. However, the PurR site is poorly retained in front of orthologs of some genes belonging to the E. coli purine regulon, such as genes involved in general nitrogen metabolism, biosynthesis of pyrimidines, and synthesis of AMP and GMP from IMP, and also upstream of the purine repressor gene. It is predicted that purine regulons of the examined bacteria include the following genes: upp participating in synthesis of pyrimidines; uraA encoding an uracil transporter gene; serA involved in serine biosynthesis; folD responsible for the conversion of N5,N10-methenyl tetrahydrofolate into N10-formyltetrahydrofolate; rpiA involved in ribose metabolism; and protein genes with an unknown function (yhhQ and ydiK). The PurR site was shown to have different structure in different genomes. Thus, the tendency for a decline of the conservatism of site positions 2 and 15 was observed in genomes of bacteria belonging to the Pasteurellaceae and Vibrionaceae groups.

  12. An N-terminal segment of the active component of the bacterial genotoxin cytolethal distending toxin B (CDTB) directs CDTB into the nucleus.

    PubMed

    Nishikubo, Shuichi; Ohara, Masaru; Ueno, Yoko; Ikura, Masae; Kurihara, Hidemi; Komatsuzawa, Hitoshi; Oswald, Eric; Sugai, Motoyuki

    2003-12-12

    Cytolethal distending toxin (CDT), produced by Actinobacillus actinomycetemcomitans, is a putative virulence factor in the pathogenesis of periodontal diseases. It is a cell cycle specific inhibitor at the G2/M transition. CDTB, one of the subunits of the CDT holotoxin, is implicated in a genotoxic role after entering the target cells, whereby chromosomal damage induces checkpoint phosphorylation cascades. CDTB microinjected into the cytoplasm was shown to localize in the nucleus and induce chromatin collapse. To investigate the molecular mechanism involved in nuclear transport of CDTB, we used transient expression and microinjection of a CDTB-green fluorescent protein (GFP) fusion protein. After microinjection, His-tagged CDTB-GFP entered the nucleus in 3-4 h. Leptomycin B did not increase the speed of entry of the fusion protein, suggesting that the relatively slow entry of the fusion protein is not due to the CRM1-dependent nuclear export of the protein. Nuclear localization of the CDTBGFP was temperature-dependent. An in vitro transport assay demonstrated that the nuclear localization of CDTB is mediated by active transport. An assay using transient expression of a series of truncated CDTB-GFP fusion proteins revealed that residues 48-124 constitute the minimum region involved in nuclear transport of CDTB. A domain swapping experiment of the region involved in nuclear transport of CDTB with an SV40 T nuclear localization signal indicated that CDTB is composed of two domains, an N-terminal domain for nuclear transport and a C-terminal active domain. Our results strongly suggest that nuclear localization of CDTB is required for the holotoxin to induce cytodistension and cell cycle block. This is the first demonstration that a bacterial toxin possessing a unique domain for nuclear transport is transferred to the animal cell nucleus by active transport. PMID:12947116

  13. Targeted profiling of oral bacteria in human saliva and in vitro biofilms with quantitative real-time PCR.

    PubMed

    Price, R R; Viscount, H B; Stanley, M C; Leung, K-P

    2007-01-01

    An in vitro plaque model based on the use of human salivary bacteria and tooth-like surfaces was previously developed for studying the formation of oral biofilm and its use for pre-clinical testing of candidate antimicrobial or antiplaque agents. In this study, a quantitative Taqman PCR assay (QPCR) was developed to compare the bacterial compositions of in vitro biofilms to parent saliva samples, and to determine the relative contributions of different species in the formation of the oral biofilm. In addition, the growth inhibition of saliva-derived plaque was evaluated by chlorhexidine. With this assay, which consisted of primer/probe sets targeting either 16S rDNA sequences present in public databases or cloned ribosomal intergenic spacer region (ISR) sequences, 15 oral bacteria derived from saliva as well as those that were responsible for biofilm formation in an in vitro plaque model were rapidly identified and quantified. Among the target organisms were Actinobacillus actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Lactobacillus acidophilus, Micromonas micros, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus mutans, Streptococcus sobrinus, Tannerella forsythensis, and Veillonella parvula. Primer and probe sets developed were both sensitive and specific. The relative profiles of a number of bacteria in 45-h-old biofilms were determined and, when compared to saliva samples, it was found that most of the bacteria identified in saliva also populated the in vitro plaque, including some anaerobes. Brief exposure of biofilms to chlorhexidine resulted in significant losses in viability. This new broad spectrum QPCR assay in combination with the in vitro plaque model will be of significant value in the quantitative study of the microbial composition of human saliva, saliva-derived plaque, and pre-clinical evaluation of potential antimicrobial and antiplaque molecules.

  14. Non-inflammatory destructive periodontal disease: a clinical, microbiological, immunological and genetic investigation

    PubMed Central

    REPEKE, Carlos Eduardo; CARDOSO, Cristina Ribeiro; CLAUDINO, Marcela; SILVEIRA, Elcia Maria; TROMBONE, Ana Paula Favaro; CAMPANELLI, Ana Paula; SILVA, João Santana; MARTINS JÚNIOR, Walter; GARLET, Gustavo Pompermaier

    2012-01-01

    Periodontitis comprises a group of multifactorial diseases in which periodontopathogens accumulate in dental plaque and trigger host chronic inflammatory and immune responses against periodontal structures, which are determinant to the disease outcome. Although unusual cases of non-inflammatory destructive periodontal disease (NIDPD) are described, their pathogenesis remains unknown. A unique NIDPD case was investigated by clinical, microbiological, immunological and genetic tools. The patient, a non-smoking dental surgeon with excessive oral hygiene practice, presented a generalized bone resorption and tooth mobility, but not gingival inflammation or occlusion problems. No hematological, immunological or endocrine alterations were found. No periodontopathogens (A. actinomycetemcomitans, P. gingivalis, F. nucleatum and T. denticola) or viruses (HCMV, EBV-1 and HSV-1) were detected, along with levels of IL-1β and TNF-α in GCF compatible with healthy tissues. Conversely ALP, ACP and RANKL GCF levels were similar to diseased periodontal sites. Genetic investigation demonstrated that the patient carried some SNPs, as well HLA-DR4 (*0404) and HLA-B27 alleles, considered risk factors for bone loss. Then, a less vigorous and diminished frequency of toothbrushing was recommended to the patient, resulting in the arrest of alveolar bone loss, associated with the return of ALP, ACP and RANKL in GCF to normality levels. In conclusion, the unusual case presented here is compatible with the previous description of NIDPD, and the results that a possible combination of excessive force and frequency of mechanical stimulation with a potentially bone loss prone genotype could result in the alveolar bone loss seen in NIDPD. PMID:22437688

  15. The Active Partition Gene incC of IncP Plasmids Is Required for Stable Maintenance in a Broad Range of Hosts

    PubMed Central

    Siddique, Azeem; Figurski, David H.

    2002-01-01

    Plasmids of incompatibility group P (IncP) are capable of replication and stable inheritance in a wide variety of gram-negative bacteria. Three determinants of IncP plasmids are components of an active partition locus that is predicted to function in the segregation of plasmid copies to daughter cells. These determinants are incC, which codes for a member of the ParA family of partition ATPases; korB, which specifies a DNA-binding protein that also functions as a global transcriptional repressor; and OB, the DNA target for KorB, which occurs at multiple locations on IncP plasmids. To determine the importance and host range of the IncC/KorB partition system in the maintenance of IncP plasmids, we constructed an in-frame deletion of incC in the otherwise intact 60-kb IncPα plasmid R995. R995ΔincC was found to be highly unstable in Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Agrobacterium tumefaciens, and Acinetobacter calcoaceticus, whereas wild-type R995 is stable in all these hosts. In addition, R995ΔincC could not be established in Actinobacillus actinomycetemcomitans. trans-Complementation analysis showed that the coding region for IncC2 polypeptide, which is expressed from an internal translational start within the incC gene, was sufficient to restore stable maintenance to wild-type levels. The results show that the IncC/KorB active partition system of IncP plasmids is remarkably proficient for stable maintenance in diverse bacteria. PMID:11872733

  16. Relationship between invasion of the periodontium by periodontal pathogens and periodontal disease: a systematic review.

    PubMed

    Mendes, Luzia; Azevedo, Nuno Filipe; Felino, António; Pinto, Miguel Gonçalves

    2015-01-01

    Bacterial invasion of the periodontal tissues has been suggested as a relevant step in the etiopathogenesis of periodontal disease. However, its exact importance remains to be defined. The present systematic review assessed the scientific evidence concerning the relationship between the quality or quantity of periodontal microbiota in periodontal tissues and development of periodontal disease. The databases Medline-PubMed, Cochrane-CENTRAL, ISI Web of Knowledge and SCOPUS were searched, up to January 2014. Studies that reported evaluation of periodontal pathogens invasion on human tissues were selected. The screening of 440 title/abstracts elected 26 papers for full-text reading. Twenty three papers were subsequently excluded because of insufficient data or a study protocol not related to the objectives of this systematic review. All included studies were case-control studies that evaluated intracellular or adherent bacteria to epithelial cells from periodontal pockets versus healthy sulci. Study protocols presented heterogeneity regarding case and control definitions and methodological approaches for microbial identification. No consistent significant differences were found related to the presence/absence or proportion of specific periopathogens across the studies, as only one study found statistically significant differences regarding the presence of A. actinomycetemcomitans (p = 0.043), T. forsythia (P < 0.001), P. intermedia (P < 0.001), C. ochracea (P < 0.001) and C. rectus (P = 0.003) in epithelial cells from periodontal pockets vs. healthy sulci. All studies reported a larger unspecific bacterial load in or on the epithelial cells taken from a diseased site compared to a healthy sulcus. The current available data is of low to moderate quality and inconsistent mainly due to study design, poor reporting and methodological diversity. As so, there is insufficient evidence to support or exclude the invasion by periodontal pathogens as a key step in the

  17. Relationship between invasion of the periodontium by periodontal pathogens and periodontal disease: a systematic review

    PubMed Central

    Mendes, Luzia; Azevedo, Nuno Filipe; Felino, António; Pinto, Miguel Gonçalves

    2015-01-01

    Bacterial invasion of the periodontal tissues has been suggested as a relevant step in the etiopathogenesis of periodontal disease. However, its exact importance remains to be defined. The present systematic review assessed the scientific evidence concerning the relationship between the quality or quantity of periodontal microbiota in periodontal tissues and development of periodontal disease. The databases Medline-PubMed, Cochrane-CENTRAL, ISI Web of Knowledge and SCOPUS were searched, up to January 2014. Studies that reported evaluation of periodontal pathogens invasion on human tissues were selected. The screening of 440 title/abstracts elected 26 papers for full-text reading. Twenty three papers were subsequently excluded because of insufficient data or a study protocol not related to the objectives of this systematic review. All included studies were case-control studies that evaluated intracellular or adherent bacteria to epithelial cells from periodontal pockets versus healthy sulci. Study protocols presented heterogeneity regarding case and control definitions and methodological approaches for microbial identification. No consistent significant differences were found related to the presence/absence or proportion of specific periopathogens across the studies, as only one study found statistically significant differences regarding the presence of A. actinomycetemcomitans (p = 0.043), T. forsythia (P < 0.001), P. intermedia (P < 0.001), C. ochracea (P < 0.001) and C. rectus (P = 0.003) in epithelial cells from periodontal pockets vs. healthy sulci. All studies reported a larger unspecific bacterial load in or on the epithelial cells taken from a diseased site compared to a healthy sulcus. The current available data is of low to moderate quality and inconsistent mainly due to study design, poor reporting and methodological diversity. As so, there is insufficient evidence to support or exclude the invasion by periodontal pathogens as a key step in the

  18. Subgingival temperature: relation to gingival crevicular fluid enzymes, cytokines, and subgingival plaque micro-organisms.

    PubMed

    Wolff, L F; Koller, N J; Smith, Q T; Mathur, A; Aeppli, D

    1997-12-01

    There have been no reports on the relationship of subgingival temperature to specific gingival crevicular fluid (GCF) components. Therefore, the purpose of this cross-sectional study was to determine whether there was any relationship between subgingival temperature and GCF levels of neutrophil elastase (NE), myeloperoxidase (MPO), beta-glucuronidase (BG), interleukin-1 alpha (IL-1), and interferon alpha (IFN). Furthermore, another objective was to confirm an association of subgingival temperature with clinical parameters and specific subgingival plaque micro-organisms as has been reported earlier. 27 human subjects each having healthy (n = 50), gingivitis (n = 59) and periodontitis (n = 53) sites were evaluated. The plaque index (PI), subgingival temperature, probing depth, attachment loss, bleeding index and gingival index were measured. GCF was sampled following the measurement of the PI and removal of the supragingival plaque. GCF samples were assayed for the enzymes NE, BG, MPO and the cytokines IFN-alpha and IL-1 alpha. A sterile Gracey curette was utilized at each sampled site to collect subgingival plaque. The plaque samples were evaluated using an immunoassay. Subgingival temperature was found to directly correlate with all clinical parameters (p < 0.001). Significant, albeit not large, correlations were found between subgingival temperature and NE (r = 0.35, p < 0.001), MPO (r = 0.26, p < 0.001) and BG (r = 0.23, p < 0.01). Temperature was found to correlate positively with E. corrodens (r = 0.33, p < 0.02) and F. nucleatum (r = 0.25, p < 0.05) but not with P. intermedia (r = 0.02, p = 0.9), P. gingivalis (r = 0.20, p = 0.1) and A. actinomycetemcomitans (r = 0.01, p > 0.9). In conclusion, subgingival temperature is correlated with the GCF enzymes, NE, MPO and BG as well as the clinical parameters and specific plaque micro-organisms associated with periodontal disease.

  19. Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes.

    PubMed Central

    Kuhnert, P; Heyberger-Meyer, B; Burnens, A P; Nicolet, J; Frey, J

    1997-01-01

    The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance. PMID:9172345

  20. Microbial profile on metallic and ceramic bracket materials.

    PubMed

    Anhoury, Patrick; Nathanson, Dan; Hughes, Christopher V; Socransky, Sigmund; Feres, Magda; Chou, Laisheng Lee

    2002-08-01

    The placement of orthodontic appliances creates a favorable environment for the accumulation of a microbiota and food residues, which, in time, may cause caries or exacerbate any pre-existing periodontal disease. The purpose of the present study was to compare the total bacterial counts present on metallic and ceramic orthodontic brackets in order to clarify which bracket type has a higher plaque retaining capacity and to determine the levels of Streptococcus mutans and Lactobacillus spp on both types of brackets. Thirty-two metallic brackets and 24 ceramic brackets were collected from orthodontic patients at the day of debonding. Two brackets were collected from each patient; one from a maxillary central incisor and another from a maxillary second premolar. Sixteen patients who used metallic brackets and 12 patients who used ceramic brackets were sampled. Bacterial populations were studied using "checkerboard" DNA-DNA hybridization, which uses DNA probes to identify species in complex microbial samples. The significance of differences between groups was determined using the Mann-Whitney U-test. Results showed no significant differences between metallic and ceramic brackets with respect to the caries-inducing S mutans and L acidophilus spp counts. Mean counts of 8 of 35 additional species differed significantly between metallic and ceramic brackets with no obvious pattern favoring one bracket type over the other. This study showed higher mean counts of Treponema denticola, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum ss vincentii, Streptococcus anginosus, and Eubacterium nodatum on metallic brackets while higher counts of Eikenella corrodens, Campylobacter showae, and Selenomonas noxia were found on ceramic brackets.

  1. Putative periodontopathogens in "diseased" and "non-diseased" persons exhibiting poor oral hygiene.

    PubMed

    Dahlén, G; Manji, F; Baelum, V; Fejerskov, O

    1992-01-01

    The aim of the study was to assess the occurrence of some putative periodonto-pathogens in "test" and "control" sites in "diseased" and "non-diseased" persons, respectively, from an adult rural Kenyan population exhibiting poor oral hygiene and widespread loss of attachment (LA). 14 persons (less than 35 years) were assigned to a "diseased" category on the basis of at least 4 sites with LA greater than or equal to 4 mm; at least 5 mm LA and a pocket greater than or equal to 4 mm interproximally in a lower incisor ("test" site): and less than 2 mm LA and no pocket greater than or equal to 4 mm distal to a lower canine or mesial to a lower first premolar ("control" site). Age-matched "non-diseased" persons were identified on the basis of no sites with LA greater than 2 mm and no pockets greater than or equal to 4 mm associated with LA. Paperpoint samples from test and control sites as well as a scraping sample from the dorsum of tongue were examined for presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Bacteroides intermedius, B. melaninogenicus group, Capnocytophaga, Selenomonas spp., and Wolinella recta. P. gingivalis was found in 79% of test sites and 36% of control sites in "diseased" persons, and in 18% and 35% of test and control sites, respectively, in "non-diseased" persons. "No other bacterial group discriminated significantly between test and control sites or between diseased and non-diseased subjects. The surprisingly high occurrence of P. gingivalis in non-diseased subjects, both subgingivally and on tongue, indicates that deep periodontal pockets are not prerequisite ecological environments for P. gingivalis establishment.

  2. Cell adhesion molecules in the pathogenesis of and host defence against microbial infection.

    PubMed Central

    Kerr, J R

    1999-01-01

    Eukaryotic cell adhesion molecules (CAMs) are used by various cells and extracellular molecules in host defence against infection. They are involved in many processes including recognition by circulating phagocytes of a site of inflammation, transmigration through the endothelial barrier, diapedesis through basement membrane and extracellular matrix, and release of effector mechanisms at the infected site. CAMs involved in leucocyte-endothelial cell interaction include the selectins, integrins, and members of the immunoglobulin superfamily. However, CAMs are also used by various microorganisms (protozoa, fungi, bacteria, and viruses) during their pathogenesis. For example, bacteria that utilise CAMs include Mycobacterium tuberculosis, Listeria monocytogenes, Yersinia spp, enteropathogenic Escherichia coli, Shigella spp, Neisseria spp, Bordetella spp, and Borrelia burgdorferi. In addition, CAMs are involved in the pathogenetic effects of the RTX toxins of Pasteurella haemolytica, Actinobacillus actinomycetemcomitans, and the superantigen exotoxins of Staphylococcus aureus and Streptococcus pyogenes. A recurrent and topical theme of potential importance within the bacterial group is the intimate relation between CAMs, bacterial protein receptors, and type III secretion systems. For example, the IpaBCD protein complex is secreted by the type III system of Shigella flexneri and interacts with alpha 5 beta 1 integrin on the eukaryotic cell surface, followed by Rho mediated internalisation; this illustrates the relevance of cellular microbiology. CAMs might prove to be novel therapeutic targets. Comparative genomics has provided the knowledge of shared virulence determinants among diverse bacterial genera, and will continue to deepen our understanding of microbial pathogenesis, particularly in the context of the interaction of prokaryotic and eukaryotic molecules. PMID:10694943

  3. Quantitative molecular detection of putative periodontal pathogens in clinically healthy and periodontally diseased subjects.

    PubMed

    Göhler, André; Hetzer, Adrian; Holtfreter, Birte; Geisel, Marie Henrike; Schmidt, Carsten Oliver; Steinmetz, Ivo; Kocher, Thomas

    2014-01-01

    Periodontitis is a multi-microbial oral infection with high prevalence among adults. Putative oral pathogens are commonly found in periodontally diseased individuals. However, these organisms can be also detected in the oral cavity of healthy subjects. This leads to the hypothesis, that alterations in the proportion of these organisms relative to the total amount of oral microorganisms, namely their abundance, rather than their simple presence might be important in the transition from health to disease. Therefore, we developed a quantitative molecular method to determine the abundance of various oral microorganisms and the portion of bacterial and archaeal nucleic acid relative to the total nucleic acid extracted from individual samples. We applied quantitative real-time PCRs targeting single-copy genes of periodontal bacteria and 16S-rRNA genes of Bacteria and Archaea. Testing tongue scrapings of 88 matched pairs of periodontally diseased and healthy subjects revealed a significantly higher abundance of P. gingivalis and a higher total bacterial abundance in diseased subjects. In fully adjusted models the risk of being periodontally diseased was significantly higher in subjects with high P. gingivalis and total bacterial abundance. Interestingly, we found that moderate abundances of A. actinomycetemcomitans were associated with reduced risk for periodontal disease compared to subjects with low abundances, whereas for high abundances, this protective effect leveled off. Moderate archaeal abundances were health associated compared to subjects with low abundances. In conclusion, our methodological approach unraveled associations of the oral flora with periodontal disease, which would have gone undetected if only qualitative data had been determined.

  4. Evaluation of antimicrobial-antibiofilm activity of a hydrogen peroxide decontaminating system used in dental unit water lines.

    PubMed

    Orrù, Germano; Del Nero, Susanna; Tuveri, Enrica; Laura Ciusa, Maria; Pilia, Francesca; Erriu, Matteo; Orrù, Ginevra; Liciardi, Manuele; Piras, Vincenzo; Denotti, Gloria

    2010-01-01

    A dental unit water line (DUWL) equipped with a device designed to automatically and continually flush a bacteriostatic solution of hydrogen peroxide (WHE) and a discontinuous disinfecting system (BIOSTER) was evaluated. In the first instance a preliminary sensitivity test on a large number of microorganisms (bacteria and fungi) was tried with a H(2)O(2) range from 100 to 800 ppm. The bacteria frequently reported in DUWL (including Pseudomonas spp, Streptococcus spp., Staphylococcus spp., E. coli) and some periodontal pathogens showed a minimum inhibitory concentration from 100 to 300 H(2)O(2 )ppm (also including M. marinum and C. albicans). However, H(2)O(2) did not show any inhibitory effects against: A. actinomycetemcomitans, C. glabrata C. parapsilos, F. nucleatum, M. micros. In a second step, the DUWL was experimentally infected with S. faecalis, E. coli, P. aeruginosa, S. aureus. After disinfection steps with 3% H(2)O(2), the inhibitory effect on planktonic forms and on sessile biofilm was measured. In a third step, the count of 16S rRNA gene copies by real time PCR at different points of the DUWL described an accrue of bacterial slime in "hot spot" regions characterized by irregular/slow water flux (valves, elbows). However these results suggest that hydrogen peroxide is not only able to inhibit bursts of planktonic bacteria inside the DUWL, but that it could also be effective against sessile biofilm containing heterotrophic microorganisms derived from domestic water line contamination. In addition some oral pathogens could be contaminating and surviving in DUWL.

  5. L-arginine destabilizes oral multi-species biofilm communities developed in human saliva.

    PubMed

    Kolderman, Ethan; Bettampadi, Deepti; Samarian, Derek; Dowd, Scot E; Foxman, Betsy; Jakubovics, Nicholas S; Rickard, Alexander H

    2015-01-01

    The amino acid L-arginine inhibits bacterial coaggregation, is involved in cell-cell signaling, and alters bacterial metabolism in a broad range of species present in the human oral cavity. Given the range of effects of L-arginine on bacteria, we hypothesized that L-arginine might alter multi-species oral biofilm development and cause developed multi-species biofilms to disassemble. Because of these potential biofilm-destabilizing effects, we also hypothesized that L-arginine might enhance the efficacy of antimicrobials that normally cannot rapidly penetrate biofilms. A static microplate biofilm system and a controlled-flow microfluidic system were used to develop multi-species oral biofilms derived from pooled unfiltered cell-containing saliva (CCS) in pooled filter-sterilized cell-free saliva (CFS) at 37° C. The addition of pH neutral L-arginine monohydrochloride (LAHCl) to CFS was found to exert negligible antimicrobial effects but significantly altered biofilm architecture in a concentration-dependent manner. Under controlled flow, the biovolume of biofilms (μm(3)/μm(2)) developed in saliva containing 100-500 mM LAHCl were up to two orders of magnitude less than when developed without LAHCI. Culture-independent community analysis demonstrated that 500 mM LAHCl substantially altered biofilm species composition: the proportion of Streptococcus and Veillonella species increased and the proportion of Gram-negative bacteria such as Neisseria and Aggregatibacter species was reduced. Adding LAHCl to pre-formed biofilms also reduced biovolume, presumably by altering cell-cell interactions and causing cell detachment. Furthermore, supplementing 0.01% cetylpyridinium chloride (CPC), an antimicrobial commonly used for the treatment of dental plaque, with 500 mM LAHCl resulted in greater penetration of CPC into the biofilms and significantly greater killing compared to a non-supplemented 0.01% CPC solution. Collectively, this work demonstrates that LAHCl moderates multi

  6. IL-4/CCL22/CCR4 Axis Controls Regulatory T-Cell Migration That Suppresses Inflammatory Bone Loss in Murine Experimental Periodontitis

    PubMed Central

    Araujo-Pires, Ana Claudia; Vieira, Andreia Espindola; Francisconi, Carolina Favaro; Biguetti, Claudia Cristina; Glowacki, Andrew; Yoshizawa, Sayuri; Campanelli, Ana Paula; Trombone, Ana Paula Favaro; Sfeir, Charles S.; Little, Steven R.; Garlet, Gustavo Pompermaier

    2015-01-01

    Inflammatory bone resorption is a hallmark of periodontitis, and Tregs and Th2 cells are independently associated with disease progression attenuation. In this study, we employed an infection-triggered inflammatory osteolysis model to investigate the mechanisms underlying Treg and Th2 cell migration and the impact on disease outcome. Aggregatibacter actinomycetemcomitans–infected C57Bl/6 (wild-type [WT]) mice develop an intense inflammatory reaction and alveolar bone resorption, and Treg and Th2 cell migration is temporally associated with disease progression attenuation. Tregs extracted from the lesions preferentially express CCR4 and CCR8, whereas Th2 cells express CCR3, CCR4, and CCR8. The absence of CCR5 and CCR8 did not significantly impact the migration of Tregs and Th2 cells or affect the disease outcome. CCR4KO mice presented a minor reduction in Th2 cells in parallel with major impairment of Treg migration, which was associated with increased inflammatory bone loss and higher proinflammatory and osteoclastogenic cytokine levels. The blockade of the CCR4 ligand CCL22 in WT mice resulted in an increased inflammatory bone loss phenotype similar to that in the CCR4KO strain. Adoptive transfer of CCR4+ Tregs to the CCR4KO strain revert the increased disease phenotype to WT mice–like levels; also, the in situ production of CCL22 in the lesions is mandatory for Tregs migration and the consequent bone loss arrest. The local release of exogenous CCL22 provided by poly(lactic-co-glycolic acid) (PLGA) microparticles promotes migration of Tregs and disease arrest in the absence of endogenous CCL22 in the IL-4KO strain, characterized by the lack of endogenous CCL22 production, defective migration of Tregs, and exacerbated bone loss. In summary, our results show that the IL-4/CCL22/CCR4 axis is involved in the migration of Tregs to osteolytic lesion sites, and attenuates development of lesions by inhibiting inflammatory migration and the production of

  7. L-arginine destabilizes oral multi-species biofilm communities developed in human saliva.

    PubMed

    Kolderman, Ethan; Bettampadi, Deepti; Samarian, Derek; Dowd, Scot E; Foxman, Betsy; Jakubovics, Nicholas S; Rickard, Alexander H

    2015-01-01

    The amino acid L-arginine inhibits bacterial coaggregation, is involved in cell-cell signaling, and alters bacterial metabolism in a broad range of species present in the human oral cavity. Given the range of effects of L-arginine on bacteria, we hypothesized that L-arginine might alter multi-species oral biofilm development and cause developed multi-species biofilms to disassemble. Because of these potential biofilm-destabilizing effects, we also hypothesized that L-arginine might enhance the efficacy of antimicrobials that normally cannot rapidly penetrate biofilms. A static microplate biofilm system and a controlled-flow microfluidic system were used to develop multi-species oral biofilms derived from pooled unfiltered cell-containing saliva (CCS) in pooled filter-sterilized cell-free saliva (CFS) at 37° C. The addition of pH neutral L-arginine monohydrochloride (LAHCl) to CFS was found to exert negligible antimicrobial effects but significantly altered biofilm architecture in a concentration-dependent manner. Under controlled flow, the biovolume of biofilms (μm(3)/μm(2)) developed in saliva containing 100-500 mM LAHCl were up to two orders of magnitude less than when developed without LAHCI. Culture-independent community analysis demonstrated that 500 mM LAHCl substantially altered biofilm species composition: the proportion of Streptococcus and Veillonella species increased and the proportion of Gram-negative bacteria such as Neisseria and Aggregatibacter species was reduced. Adding LAHCl to pre-formed biofilms also reduced biovolume, presumably by altering cell-cell interactions and causing cell detachment. Furthermore, supplementing 0.01% cetylpyridinium chloride (CPC), an antimicrobial commonly used for the treatment of dental plaque, with 500 mM LAHCl resulted in greater penetration of CPC into the biofilms and significantly greater killing compared to a non-supplemented 0.01% CPC solution. Collectively, this work demonstrates that LAHCl moderates multi

  8. HACEK Infective Endocarditis: Characteristics and Outcomes from a Large, Multi-National Cohort

    PubMed Central

    Chambers, Stephen T.; Murdoch, David; Morris, Arthur; Holland, David; Pappas, Paul; Almela, Manel; Fernández-Hidalgo, Nuria; Almirante, Benito; Bouza, Emilio; Forno, Davide; del Rio, Ana; Hannan, Margaret M.; Harkness, John; Kanafani, Zeina A.; Lalani, Tahaniyat; Lang, Selwyn; Raymond, Nigel; Read, Kerry; Vinogradova, Tatiana; Woods, Christopher W.; Wray, Dannah; Corey, G. Ralph; Chu, Vivian H.

    2013-01-01

    The HACEK organisms (Haemophilus species, Aggregatibacter species, Cardiobacterium hominis, Eikenella corrodens, and Kingella species) are rare causes of infective endocarditis (IE). The objective of this study is to describe the clinical characteristics and outcomes of patients with HACEK endocarditis (HE) in a large multi-national cohort. Patients hospitalized with definite or possible infective endocarditis by the International Collaboration on Endocarditis Prospective Cohort Study in 64 hospitals from 28 countries were included and characteristics of HE patients compared with IE due to other pathogens. Of 5591 patients enrolled, 77 (1.4%) had HE. HE was associated with a younger age (47 vs. 61 years; p<0.001), a higher prevalence of immunologic/vascular manifestations (32% vs. 20%; p<0.008) and stroke (25% vs. 17% p = 0.05) but a lower prevalence of congestive heart failure (15% vs. 30%; p = 0.004), death in-hospital (4% vs. 18%; p = 0.001) or after 1 year follow-up (6% vs. 20%; p = 0.01) than IE due to other pathogens (n = 5514). On multivariable analysis, stroke was associated with mitral valve vegetations (OR 3.60; CI 1.34–9.65; p<0.01) and younger age (OR 0.62; CI 0.49–0.90; p<0.01). The overall outcome of HE was excellent with the in-hospital mortality (4%) significantly better than for non-HE (18%; p<0.001). Prosthetic valve endocarditis was more common in HE (35%) than non-HE (24%). The outcome of prosthetic valve and native valve HE was excellent whether treated medically or with surgery. Current treatment is very successful for the management of both native valve prosthetic valve HE but further studies are needed to determine why HE has a predilection for younger people and to cause stroke. The small number of patients and observational design limit inferences on treatment strategies. Self selection of study sites limits epidemiological inferences. PMID:23690995

  9. Robust species taxonomy assignment algorithm for 16S rRNA NGS reads: application to oral carcinoma samples

    PubMed Central

    Al-Hebshi, Nezar Noor; Nasher, Akram Thabet; Idris, Ali Mohamed; Chen, Tsute

    2015-01-01

    Background Usefulness of next-generation sequencing (NGS) in assessing bacteria associated with oral squamous cell carcinoma (OSCC) has been undermined by inability to classify reads to the species level. Objective The purpose of this study was to develop a robust algorithm for species-level classification of NGS reads from oral samples and to pilot test it for profiling bacteria within OSCC tissues. Methods Bacterial 16S V1-V3 libraries were prepared from three OSCC DNA samples and sequenced using 454's FLX chemistry. High-quality, well-aligned, and non-chimeric reads ≥350 bp were classified using a novel, multi-stage algorithm that involves matching reads to reference sequences in revised versions of the Human Oral Microbiome Database (HOMD), HOMD extended (HOMDEXT), and Greengene Gold (GGG) at alignment coverage and percentage identity ≥98%, followed by assignment to species level based on top hit reference sequences. Priority was given to hits in HOMD, then HOMDEXT and finally GGG. Unmatched reads were subject to operational taxonomic unit analysis. Results Nearly, 92.8% of the reads were matched to updated-HOMD 13.2, 1.83% to trusted-HOMDEXT, and 1.36% to modified-GGG. Of all matched reads, 99.6% were classified to species level. A total of 228 species-level taxa were identified, representing 11 phyla; the most abundant were Proteobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Actinobacteria. Thirty-five species-level taxa were detected in all samples. On average, Prevotella oris, Neisseria flava, Neisseria flavescens/subflava, Fusobacterium nucleatum ss polymorphum, Aggregatibacter segnis, Streptococcus mitis, and Fusobacterium periodontium were the most abundant. Bacteroides fragilis, a species rarely isolated from the oral cavity, was detected in two samples. Conclusion This multi-stage algorithm maximizes the fraction of reads classified to the species level while ensuring reliable classification by giving priority to the human, oral reference

  10. L-Arginine Destabilizes Oral Multi-Species Biofilm Communities Developed in Human Saliva

    PubMed Central

    Kolderman, Ethan; Bettampadi, Deepti; Samarian, Derek; Dowd, Scot E.; Foxman, Betsy; Jakubovics, Nicholas S.; Rickard, Alexander H.

    2015-01-01

    The amino acid L-arginine inhibits bacterial coaggregation, is involved in cell-cell signaling, and alters bacterial metabolism in a broad range of species present in the human oral cavity. Given the range of effects of L-arginine on bacteria, we hypothesized that L-arginine might alter multi-species oral biofilm development and cause developed multi-species biofilms to disassemble. Because of these potential biofilm-destabilizing effects, we also hypothesized that L-arginine might enhance the efficacy of antimicrobials that normally cannot rapidly penetrate biofilms. A static microplate biofilm system and a controlled-flow microfluidic system were used to develop multi-species oral biofilms derived from pooled unfiltered cell-containing saliva (CCS) in pooled filter-sterilized cell-free saliva (CFS) at 37oC. The addition of pH neutral L-arginine monohydrochloride (LAHCl) to CFS was found to exert negligible antimicrobial effects but significantly altered biofilm architecture in a concentration-dependent manner. Under controlled flow, the biovolume of biofilms (μm3/μm2) developed in saliva containing 100-500 mM LAHCl were up to two orders of magnitude less than when developed without LAHCI. Culture-independent community analysis demonstrated that 500 mM LAHCl substantially altered biofilm species composition: the proportion of Streptococcus and Veillonella species increased and the proportion of Gram-negative bacteria such as Neisseria and Aggregatibacter species was reduced. Adding LAHCl to pre-formed biofilms also reduced biovolume, presumably by altering cell-cell interactions and causing cell detachment. Furthermore, supplementing 0.01% cetylpyridinium chloride (CPC), an antimicrobial commonly used for the treatment of dental plaque, with 500 mM LAHCl resulted in greater penetration of CPC into the biofilms and significantly greater killing compared to a non-supplemented 0.01% CPC solution. Collectively, this work demonstrates that LAHCl moderates multi

  11. Increasing the metabolizable protein supply enhanced growth performance and led to variable results on innate and humoral immune response of preconditioning beef steers.

    PubMed

    Moriel, P; Artioli, L F A; Poore, M H; Confer, A W; Marques, R S; Cooke, R F

    2015-09-01

    We evaluated the effects of MP supply on growth performance before and after preconditioning and measurements of innate and humoral immune response of beef steers following vaccination. Angus steers ( = 36; BW = 231 ± 21 kg; age = 184 ± 18 d) were weaned on d -6, stratified by BW and age on d 0, and randomly assigned to 1 of 18 drylot pens (2 steers/pen). Treatments were assigned to pens (6 pens/treatment) and consisted of corn silage-based diets formulated to provide 85%, 100%, or 115% of the daily MP requirements of a beef steer gaining 1.1 kg/d from d 0 to 42. Steers were vaccinated against infectious bovine rhinotracheitis virus, bovine viral diarrhea (BVDV) types 1 and 2 viruses, and clostridium on d 14 and 28. Blood samples were collected on d 0, 14, 15, 17, 21, 28, 29, 30, 35, and 42. Body weight did not differ ( ≥ 0.17) among treatments from d 0 to 28. On d 42, 115% MP steers were heaviest, 100% MP steers were intermediate, and 85% MP steers were lightest ( = 0.05; 297, 290, and 278 ± 7 kg, respectively). Overall, ADG and G:F did not differ ( ≥ 0.13) between 100% and 115% MP steers and were least ( < 0.01) for 85% MP steers (1.2, 1.4, and 0.8 ± 0.07 kg/d and 0.23, 0.24, and 0.19 ± 0.008, respectively). Plasma haptoglobin (Hp) concentrations did not differ among treatments ( ≥ 0.46), whereas plasma ceruloplasmin (Cp) concentrations were greatest ( ≤ 0.04) for 85% MP steers, intermediate for 100% MP steers, and least for 115% MP steers on d 30, 35, and 42. Plasma cortisol concentrations were greater ( ≤ 0.03) for 85% vs. 100% and 115% MP steers on d 14 and 28. Liver mRNA expression of Cp and Hp and muscle mRNA expression of m-calpain, mammalian target of rapamycin, and ubiquitin did not differ among treatments ( ≥ 0.17). Serum neutralization titers to BVDV-1b titers were greater ( ≤ 0.02) for 115% vs. 85% and 100% MP steers on d 42 (5.8, 3.0, and 3.7 ± 0.60 log, respectively), whereas mean serum leukotoxin titers were greater for 85% vs

  12. CrossLaps and beta-glucuronidase in peri-implant and gingival crevicular fluid.

    PubMed

    Schubert, U; Kleber, B M; Strietzel, F P; Dörfling, P

    2001-01-01

    Collagen degradation products of the carboxyterminal region possibly reflect bone and attachment loss. In the present study, the Serum CrossLaps One-Step enzyme-linked immunosorbent assay was used to determine a specific part of the carboxyterminal region of type I collagen, the CrossLaps. Samples of peri-implant and gingival crevicular fluid of 111 implants and 53 teeth from 47 partially or completely edentulous patients were examined in reference to levels of CrossLaps and beta-glucuronidase (beta G), an established marker of periodontal disease. Clinical probing pocket depth (PPD), bleeding on probing (BOP), plaque accumulation, mobility, radiographic bone loss, and the occurrence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia were assessed. The mean values were: for PPD at implants 3.76 +/- 1.41 mm, at teeth 3.44 +/- 0.88 mm; for beta G at implants 0.364 +/- 0.392 pU/min, at teeth 0.314 +/- 0.209 pU/min; for CrossLaps at implants 0.069 +/- 0.059 pmol/min, at teeth 0.082 +/- 0.053 pmol/min. Bleeding on probing was significantly higher on implants than on teeth (McNemar test, P = .004). No significant difference of beta G levels was found between teeth and implants (Wilcoxon test). A negative correlation was found between beta G levels and CrossLaps levels at teeth (Pearson-rank correlation, P = .002). On implants, no significant correlation of these 2 parameters was seen, but significant correlations were found between sulcus fluid flow rate and PPD (P = .012), beta G levels and bone loss (P < 0.0005), and CrossLaps levels and PPD (P = .011). CrossLaps can be detected in both gingival and peri-implant crevicular fluid. While rising levels of beta G may indicate acute peri-implantitis, CrossLaps may not, but could play a role as a marker of ongoing attachment loss.

  13. Interleukin-8 and Intercellular Adhesion Molecule 1 Regulation in Oral Epithelial Cells by Selected Periodontal Bacteria: Multiple Effects of Porphyromonas gingivalis via Antagonistic Mechanisms

    PubMed Central

    Huang, George T.-J.; Kim, Daniel; Lee, Jonathan K.-H.; Kuramitsu, Howard K.; Haake, Susan Kinder

    2001-01-01

    Interaction of bacteria with mucosal surfaces can modulate the production of proinflammatory cytokines and adhesion molecules produced by epithelial cells. Previously, we showed that expression of interleukin-8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) by gingival epithelial cells increases following interaction with several putative periodontal pathogens. In contrast, expression of IL-8 and ICAM-1 is reduced after Porphyromonas gingivalis ATCC 33277 challenge. In the present study, we investigated the mechanisms that govern the regulation of these two molecules in bacterially infected gingival epithelial cells. Experimental approaches included bacterial stimulation of gingival epithelial cells by either a brief challenge (1.5 to 2 h) or a continuous coculture throughout the incubation period. The kinetics of IL-8 and ICAM-1 expression following brief challenge were such that (i) secretion of IL-8 by gingival epithelial cells reached its peak 2 h following Fusobacterium nucleatum infection whereas it rapidly decreased within 2 h after P. gingivalis infection and remained decreased up to 30 h and (ii) IL-8 and ICAM-1 mRNA levels were up-regulated rapidly 2 to 4 h postinfection and then decreased to basal levels 8 to 20 h after infection with either Actinobacillus actinomycetemcomitans, F. nucleatum, or P. gingivalis. Attenuation of IL-8 secretion was facilitated by adherent P. gingivalis strains. The IL-8 secreted from epithelial cells after F. nucleatum stimulation could be down-regulated by subsequent infection with P. gingivalis or its culture supernatant. Although these results suggested that IL-8 attenuation at the protein level might be associated with P. gingivalis proteases, the Arg- and Lys-gingipain proteases did not appear to be solely responsible for IL-8 attenuation. In addition, while P. gingivalis up-regulated IL-8 mRNA expression, this effect was overridden when the bacteria were continuously cocultured with the epithelial cells. The IL-8

  14. Subgingival microbiota in squirrel monkeys with naturally occurring periodontal diseases.

    PubMed Central

    Beem, J E; Hurley, C G; Magnusson, I; McArthur, W P; Clark, W B

    1991-01-01

    The squirrel monkey (Saimiri sciureus) has been proposed as an in vivo model for the study of subgingival colonization by suspected periodontopathogens, such as black-pigmented porphyromonads and prevotellas (BP/P). However, the indigenous microbiota of the squirrel monkey has not been well described. Therefore, in order to more fully characterize the oral microbiota of these animals, we studied two groups of squirrel monkeys from widely different sources. Group I consisted of 50 breeding colony monkeys ranging in age from 9 months to over 6 years which had been raised in captivity; group II consisted of 16 young sexually mature monkeys recently captured in the wild in Guyana. Group I animals in captivity had developed moderate to severe gingivitis, with a mean gingival index (GI) of 2.6; 52% of the sites bled, 26% had detectable calculus, and 83% had detectable BP/P. A group I subset (six animals), for which predominant cultivable microbiota was described, had a mean GI of 2.4. Colony morphology enumeration revealed that five of the six subset animals were detectably colonized with BP/P (range, 0 to 16.9%) and Actinobacillus actinomycetemcomitans (range, 0 to 3.9%); all subset animals were colonized with Fusobacterium species (range, 0.8 to 3.6%), Actinomyces species (range, 2.3 to 11%), and gram-positive cocci (range, 1.4 to 21.4%). Predominant cultivable microbiota results revealed the presence of many bacterial species commonly found in the human gingival sulcus. At baseline, group II animals were clinically healthy and had a mean GI of 1.4; 67% of the sites bled and 2.1% had calculus, and none of the animals had detectable BP/P. Neisseriae were very common in noninflamed sites. Subsequently, when inflamed sites were compared with noninflamed sites in group II animals after they had been maintained in captivity for 6 months, inflamed sites exhibited a more complex microbiota and increased proportions of gram-negative rods and asaccharolytic bacteria. PMID

  15. Indicators of periodontal disease activity: an evaluation.

    PubMed

    Fine, D H; Mandel, I D

    1986-05-01

    It is becoming increasingly apparent that the traditional clinical criteria are inadequate for: determining active disease sites in periodontitis, monitoring quantitatively the response to therapy or measuring the degree of susceptibility to future breakdown. In an attempt to develop objective measures, a wide variety of studies have been undertaken using saliva, blood, plaque and gingival crevicular fluid (GCF) as the specimen source. Examination has included: specific bacteria and their products; host cells and their products (enzymatic and antibacterial, both immunologic and non-immunologic); products of tissue injury derived from local epithelial and connective tissues and bone. Although most of the work to date has failed to provide reliable aids to the clinician, refinements in techniques for sampling and the availability of more sophisticated analytic techniques give cause for optimism. Methods proposed for detection of disease-associated bacteria in subgingival plaque vary in their sensitivity and specificity. Dark field microscopy shows some correlation with existing disease; however, the limited specificity of this method imposes severe restrictions on its usefulness. Highly specific polyclonal and monoclonal antisera to suspected pathogens Bacteroides gingivalis and Actinobacillus actinomycetemcomitans have been developed and improved methods of identification of these microbes in plaque by ELISA immunofluorescence and flow cytometry are under development. With respect to the host response, a strong correlation between antibody patterns to specific bacteria and periodontal disease categories appears to be emerging. Although most studies have focused on serum antibody derived from peripheral blood, a shift to detection of local antibody response appears to be likely. Techniques of measurement that are exquisitely sensitive have been developed for detection of major immune recognition proteins such as antibody and complement in crevicular fluid. Research

  16. Clinical evaluation of the effect of a hydrogen peroxide mouth rinse, sodium bicarbonate dentifrice, and mouth moisturizer on oral health.

    PubMed

    Shibly, O; Ciancio, S G; Kazmierczak, M; Cohen, R E; Mather, M L; Ho, A; Bessinger, M

    1997-01-01

    The objective of this 60-day single-blind, parallel trial, using 150 subjects, was to evaluate the effect of a 20% sodium bicarbonate dentifrice, a 1.5% hydrogen peroxide solution and a mouth moisturizer on oral tissues and microflora. Subjects were randomly assigned to one of five groups. The treatments were: 1) Sage dentifrice (sodium bicarbonate). Toothette Plus containing baking soda saturated with the hydrogen peroxide solution and use of a mouth moisturizer, 2) Crest dentifrice, Toothette Plus containing baking soda saturated with the hydrogen peroxide solution and use of a mouth moisturizer, 3) Crest dentifrice, Toothette Plus containing baking soda with a control solution and no mouth moisturizer, 4) Crest dentifrice, Toothette (without baking soda), saturated with a control solution and no mouth moisturizer, and 5) Crest dentifrice, Toothette saturated with 1.5% flavored H2O2 and no mouth moisturizer. From a subgroup of 35 patients (seven from each group) buccal smears for exfoliative cytology were taken as were supragingival microbiological samples from the mesial aspect of first molars (pooled). Buccal smears were evaluated for signs of histopathological changes. Microbiological samples from supra- and subgingival plaque for P. gingivalis, P. intermedia, A. actinomycetemcomitans. A viscosus, F. nucleatum, F. sanguis and C. albicans were evaluated. Clinical parameters measured were a stain index (SI), the modified gingival index (MGI), and a plaque index (PI). There were no adverse changes in the oral microflora and no anaplastic or other pathological changes in any subjects. Clinical parameters showed a statistically significant reduction in the MGI ranging from 26.7-29.9% with no significant differences among the groups (p > 0.05). The PI showed small reductions in all groups except group 2, but the differences were not statistically significant from each other or baseline (p > 0.05). The SI revealed slight increases in all groups and no differences

  17. Clinical evaluation of the effect of a hydrogen peroxide mouth rinse, sodium bicarbonate dentifrice, and mouth moisturizer on oral health.

    PubMed

    Shibly, O; Ciancio, S G; Kazmierczak, M; Cohen, R E; Mather, M L; Ho, A; Bessinger, M

    1997-01-01

    The objective of this 60-day single-blind, parallel trial, using 150 subjects, was to evaluate the effect of a 20% sodium bicarbonate dentifrice, a 1.5% hydrogen peroxide solution and a mouth moisturizer on oral tissues and microflora. Subjects were randomly assigned to one of five groups. The treatments were: 1) Sage dentifrice (sodium bicarbonate). Toothette Plus containing baking soda saturated with the hydrogen peroxide solution and use of a mouth moisturizer, 2) Crest dentifrice, Toothette Plus containing baking soda saturated with the hydrogen peroxide solution and use of a mouth moisturizer, 3) Crest dentifrice, Toothette Plus containing baking soda with a control solution and no mouth moisturizer, 4) Crest dentifrice, Toothette (without baking soda), saturated with a control solution and no mouth moisturizer, and 5) Crest dentifrice, Toothette saturated with 1.5% flavored H2O2 and no mouth moisturizer. From a subgroup of 35 patients (seven from each group) buccal smears for exfoliative cytology were taken as were supragingival microbiological samples from the mesial aspect of first molars (pooled). Buccal smears were evaluated for signs of histopathological changes. Microbiological samples from supra- and subgingival plaque for P. gingivalis, P. intermedia, A. actinomycetemcomitans. A viscosus, F. nucleatum, F. sanguis and C. albicans were evaluated. Clinical parameters measured were a stain index (SI), the modified gingival index (MGI), and a plaque index (PI). There were no adverse changes in the oral microflora and no anaplastic or other pathological changes in any subjects. Clinical parameters showed a statistically significant reduction in the MGI ranging from 26.7-29.9% with no significant differences among the groups (p > 0.05). The PI showed small reductions in all groups except group 2, but the differences were not statistically significant from each other or baseline (p > 0.05). The SI revealed slight increases in all groups and no differences

  18. Aggressive and acute periodontal diseases.

    PubMed

    Albandar, Jasim M

    2014-06-01

    Inflammatory periodontal diseases are highly prevalent, although most of these diseases develop and progress slowly, often unnoticed by the affected individual. However, a subgroup of these diseases include aggressive and acute forms that have a relatively low prevalence but show a rapid-course, high rate of progression leading to severe destruction of the periodontal tissues, or cause systemic symptoms that often require urgent attention from healthcare providers. Aggressive periodontitis is an early-onset, destructive disease that shows a high rate of periodontal progression and distinctive clinical features. A contemporary case definition of this disease is presented. Population studies show that the disease is more prevalent in certain geographic regions and ethnic groups. Aggressive periodontitis is an infectious disease, and recent data show that in affected subjects the subgingival microbiota is composed of a mixed microbial infection, with a wide heterogeneity in the types and proportions of microorganisms recovered. Furthermore, there are significant differences in the microbiota of the disease among different geographic regions and ethnicities. There is also evidence that the Aggregatibacter actinomycetemycomitans-JP2 clone may play an important role in the development of the disease in certain populations. The host response plays an important role in the susceptibility to aggressive periodontitis, where the immune response may be complex and involve multiple mechanisms. Also, genetic factors seem to play an important role in the pathogenesis of this disease, but the mechanisms of increased susceptibility are complex and not yet fully understood. The available data suggest that aggressive periodontitis is caused by mutations either in a few major genes or in multiple small-effect genes, and there is also evidence of gene-gene and gene-environment interaction effects. Diagnostic methods for this disease, based on a specific microbiologic, immunologic or