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Sample records for actinomycetemcomitans fusobacterium nucleatum

  1. 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.

  2. Carcinogenesis mechanisms of Fusobacterium nucleatum.

    PubMed

    Gholizadeh, Pourya; Eslami, Hosein; Kafil, Hossein Samadi

    2017-03-07

    Transformed cells of cancers may be related to stromal cells, immune cells, and some bacteria such as Fusobacterium nucleatum. This review aimed to evaluate carcinogenesis mechanisms of Fusobacterium spp. in the oral cavity, pancreatic and colorectal cancers. These cancers are the three of the ten most prevalence cancer in the worldwide. Recent findings demonstrated that F. nucleatum could be considered as the risk factor for these cancers. The most important carcinogenesis mechanisms of F. nucleatum are chronic infection, interaction of cell surface molecules of these bacteria with immune system and stromal cells, immune evasion and immune suppression. However, there are some uncertainty carcinogenesis mechanisms about these bacteria, but this review evaluates almost all the known mechanisms. Well-characterized virulence factors of F. nucleatum such as FadA, Fap2, LPS and cell wall extracts may act as effector molecules in the shift of normal epithelial cells to tumor cells. These molecules may provide new targets, drugs, and strategies for therapeutic intervention.

  3. 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

  4. Fusobacterium nucleatum: a commensal-turned pathogen

    PubMed Central

    Han, Yiping W.

    2015-01-01

    Fusobacterium nucleatum is an anaerobic oral commensal and a periodontal pathogen associated with a wide spectrum of human diseases. This article reviews its implication in adverse pregnancy outcomes (chorioamnionitis, preterm birth, stillbirth, neonatal sepsis, preeclampsia), GI disorders (colorectal cancer, inflammatory bowel disease, appendicitis), cardiovascular disease, rheumatoid arthritis, respiratory tract infections, Lemierre’s syndrom and Alzheimer’s disease. The virulence mechanisms involved in the diseases are discussed, with a particular emphasis on its colonization, systemic dissemination, and induction of host inflammatory and tumorigenic responses. The FadA adhesin/invasin conserved in F. nucleatum is a key virulence factor and a potential diagnostic marker for F. nucleatum-associated diseases. PMID:25576662

  5. Cytology of pulmonary Fusobacterium nucleatum infection. A case report.

    PubMed

    Hsu, C Y; Luh, K T

    1995-01-01

    A patient with pulmonary Fusobacterium nucleatum infection presenting as a solitary nodule is reported. The infectious nature of the lung nodule was disclosed by ultrasound-guided percutaneous transthoracic fine needle aspiration cytology (FNAC) with Liu's stain. The cytologic characteristics of F nucleatum infection are described. The usefulness of real-time ultrasound in fine needle aspiration diagnosis of a peripheral lung lesion is demonstrated, and the role of Liu's stain in FNAC is emphasized.

  6. Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma.

    PubMed

    Castellarin, Mauro; Warren, René L; Freeman, J Douglas; Dreolini, Lisa; Krzywinski, Martin; Strauss, Jaclyn; Barnes, Rebecca; Watson, Peter; Allen-Vercoe, Emma; Moore, Richard A; Holt, Robert A

    2012-02-01

    An estimated 15% or more of the cancer burden worldwide is attributable to known infectious agents. We screened colorectal carcinoma and matched normal tissue specimens using RNA-seq followed by host sequence subtraction and found marked over-representation of Fusobacterium nucleatum sequences in tumors relative to control specimens. F. nucleatum is an invasive anaerobe that has been linked previously to periodontitis and appendicitis, but not to cancer. Fusobacteria are rare constituents of the fecal microbiota, but have been cultured previously from biopsies of inflamed gut mucosa. We obtained a Fusobacterium isolate from a frozen tumor specimen; this showed highest sequence similarity to a known gut mucosa isolate and was confirmed to be invasive. We verified overabundance of Fusobacterium sequences in tumor versus matched normal control tissue by quantitative PCR analysis from a total of 99 subjects (p = 2.5 × 10(-6)), and we observed a positive association with lymph node metastasis.

  7. Fusobacterium nucleatum, inflammation, and immunity: the fire within human gut.

    PubMed

    Bashir, Arif; Miskeen, Abid Yousuf; Hazari, Younis Mohammad; Asrafuzzaman, Syed; Fazili, Khalid Majid

    2016-03-01

    Fusobacterium nucleatum is an identified proinflammatory autochthonous bacterium implicated in human colorectal cancer. It is also abundantly found in patients suffering from chronic gut inflammation (inflammatory bowel disease), consequently contributing to the pathogenesis of colorectal cancer. Majority of the studies have reported that colorectal tumors/colorectal adenocarcinomas are highly enriched with F. nucleatum compared to noninvolved adjacent colonic tissue. During the course of multistep development of colorectal cancer, tumors have evolved many mechanisms to resist the antitumor immune response. One of such favorite ploy is providing access to pathogenic bacteria, especially F. nucleatum in the colorectal tumor microenvironment, wherein both (colorectal tumors and F. nucleatum) exert profound effect on each other, consequently attracting tumor-permissive myeloid-derived suppressor cells, suppressing cytotoxic CD8+ T cells and inhibiting NK cell-mediated cancer cell killing. In this review, we have primarily focused on how this bug modulates the immune response, consequently rendering the antitumor immune cells inactive.

  8. Lectinlike interactions of Fusobacterium nucleatum with human neutrophils.

    PubMed Central

    Mangan, D F; Novak, M J; Vora, S A; Mourad, J; Kriger, P S

    1989-01-01

    Fusobacterium nucleatum expresses lectinlike adherence factors which mediate binding to a variety of human tissue cells. Adherence is selectively inhibited by galactose, lactose, and N-acetyl-D-galactosamine. In this study, adherence of F. nucleatum to human peripheral blood polymorphonuclear neutrophils (PMNs) was investigated. The results indicated that the fusobacteria adhered to live and metabolically inactivated or fixed PMNs. Adherence of F. nucleatum resulted in activation of PMNs as determined by PMN aggregation, membrane depolarization, increased intracellular free Ca2+, superoxide anion production, and lysozyme release. Transmission electron micrographs showed that F. nucleatum was phagocytized by the PMNs. Microbicidal assays indicated that greater than 98% of F. nucleatum organisms were killed by PMNs within 60 min. Adherence to and activation of PMNs by F. nucleatum were inhibited by N-acetyl-D-galactosamine or lactose greater than galactose, whereas equal concentrations of glucose, N-acetyl-D-glucosamine, mannose, and fucose had little or no effect on F. nucleatum-PMN interactions. Pretreatment of the fusobacteria with heat (80 degrees C, 20 min) or proteases inhibited adherence to and activation of PMNs, but superoxide production was also stimulated by heated bacteria. The results indicate that interaction of F. nucleatum with PMNs is lectinlike and is probably mediated by fusobacterial proteins which bind to other human tissue cells. Adherence of F. nucleatum to PMNs in the absence of serum opsonins, such as antibodies and complement, may play an important role in PMN recognition and killing of F. nucleatum in the gingival sulcus and in the subsequent release of PMN factors associated with tissue destruction. Images PMID:2553609

  9. Synergy in biofilm formation between Fusobacterium nucleatum and Prevotella species.

    PubMed

    Okuda, Tamaki; Kokubu, Eitoyo; Kawana, Tomoko; Saito, Atsushi; Okuda, Katsuji; Ishihara, Kazuyuki

    2012-02-01

    The formation of biofilm by anaerobic, Gram-negative bacteria in the subgingival crevice plays an important role in the development of chronic periodontitis. The aim of this study was to characterize the role of coaggregation between Fusobacterium nucleatum and Prevotella species in biofilm formation. Coaggregation between F. nucleatum and Prevotella species was determined by visual assay. Effect of co-culture of the species on biofilm formation was assessed by crystal violet staining. Effect of soluble factor on biofilm formation was also examined using culture supernatant and two-compartment co-culture separated by a porous membrane. Production of autoinducer-2 (AI-2) by the organisms was evaluated using Vibrio harveyi BB170. Cells of all F. nucleatum strains coaggregated with Prevotella intermedia or Prevotella nigrescens with a score of 1-4. Addition of ethylenediamine tetraacetic acid or l-lysine inhibited coaggregation. Coaggregation disappeared after heating of P. intermedia or P. nigrescens cells, or Proteinase K treatment of P. nigrescens cells. Co-culture of F. nucleatum ATCC 25586 with P. intermedia or P. nigrescens strains increased biofilm formation compared with single culture (p < 0.01); co-culture with culture supernatant of these strains, however, did not enhance biofilm formation by F. nucleatum. Production of AI-2 in Prevotella species was not related to enhancement of biofilm formation by F. nucleatum. These findings indicate that physical contact by coaggregation of F. nucleatum strains with P. intermedia or P. nigrescens plays a key role in the formation of biofilm by these strains.

  10. Cellular Components Mediating Coadherence of Candida albicans and Fusobacterium nucleatum

    PubMed Central

    Wu, T.; Cen, L.; Kaplan, C.; Zhou, X.; Lux, R.; Shi, W.; He, X.

    2015-01-01

    Candida albicans is an opportunistic fungal pathogen found as part of the normal oral flora. It can be coisolated with Fusobacterium nucleatum, an opportunistic bacterial pathogen, from oral disease sites, such as those involved in refractory periodontitis and pulp necrosis. The physical coadherence between these 2 clinically important microbes has been well documented and suggested to play a role in facilitating their oral colonization and colocalization and contributing to polymicrobial pathogenesis. Previous studies indicated that the physical interaction between C. albicans and F. nucleatum was mediated by the carbohydrate components on the surface of C. albicans and the protein components on the Fusobaterium cell surface. However, the identities of the components involved still remain elusive. This study was aimed at identifying the genetic determinants involved in coaggregation between the 2 species. By screening a C. albicans SN152 mutant library and a panel of F. nucleatum 23726 outer membrane protein mutants, we identified FLO9, which encodes a putative adhesin-like cell wall mannoprotein of C. albicans and radD, an arginine-inhibitable adhesin-encoding gene in F. nucleatum that is involved in interspecies coadherence. Consistent with these findings, we demonstrated that the strong coaggregation between wild-type F. nucleatum 23726 and C. albicans SN152 in an in vitro assay could be greatly inhibited by arginine and mannose. Our study also suggested a complex multifaceted mechanism underlying physical interaction between C. albicans and F. nucleatum and for the first time revealed the identity of major genetic components involved in mediating the coaggregation. These observations provide useful knowledge for developing new targeted treatments for disrupting interactions between these 2 clinically relevant pathogens. PMID:26152186

  11. Anti-biofilm Activities from Resveratrol against Fusobacterium nucleatum

    PubMed Central

    He, Zhiyan; Huang, Zhengwei; Zhou, Wei; Tang, Zisheng; Ma, Rui; Liang, Jingping

    2016-01-01

    Fusobacterium nucleatum is a Gram-negative, anaerobic bacterium that plays an important role in dental plaque biofilm formation. In this study, we evaluate the effect of resveratrol, a phytoalexin compound, on F. nucleatum biofilm formation. The effects of different concentrations of resveratrol on biofilms formed on 96-well microtiter plates at different time points were determined by the MTT assay. The structures and thicknesses of the biofilm were observed by confocal laser scanning microscopy (CLSM), and gene expression was investigated by real-time PCR. The results showed that resveratrol at sub-MIC levels can significantly decrease biofilm formation, whereas it does not affect the bacterial growth rate. It was observed by CLSM images that the biofilm was visually decreased with increasing concentrations of resveratrol. Gene expression was down regulated in the biofilm in the presence of resveratrol. Our results revealed that resveratrol can effectively inhibit biofilm formation. PMID:27458454

  12. Dipeptide utilization by the periodontal pathogens Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum.

    PubMed

    Takahashi, Nobuhiro; Sato, T

    2002-02-01

    Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum, which can frequently be isolated from periodontal pockets, preferentially utilize proteins and peptides as growth substrates. In this study, we determined the size of peptide that is preferentially utilized as a source of energy and material for cell growth by P. gingivalis, P. intermedia, P. nigrescens and F. nucleatum using various sizes of poly amino acids consisting of two to approximately 100 molecules of aspartate or glutamate. Resting cells of P. gingivalis, P. intermedia and P. nigrescens utilized aspartylaspartate, while cells of P. gingivalis and F. nucleatum utilized glutamylglutamate. The addition of aspartylaspartate to the culture medium increased the growth of P. gingivalis, P. intermedia and P. nigrescens, while the addition of glutamylglutamate promoted the growth of P. gingivalis and F. nucleatum. These results clearly indicate that dipeptides such as aspartylaspartate and glutamylglutamate can be utilized as growth substrates for P. gingivalis, P. intermedia, P. nigrescens and F. nucleatum.

  13. Mining the Proteome of Fusobacterium nucleatum subsp. nucleatum ATCC 25586 for Potential Therapeutics Discovery: An In Silico Approach

    PubMed Central

    Habib, Abdul Musaweer; Islam, Md. Saiful; Sohel, Md.; Mazumder, Md. Habibul Hasan; Sikder, Mohd. Omar Faruk

    2016-01-01

    The plethora of genome sequence information of bacteria in recent times has ushered in many novel strategies for antibacterial drug discovery and facilitated medical science to take up the challenge of the increasing resistance of pathogenic bacteria to current antibiotics. In this study, we adopted subtractive genomics approach to analyze the whole genome sequence of the Fusobacterium nucleatum, a human oral pathogen having association with colorectal cancer. Our study divulged 1,499 proteins of F. nucleatum, which have no homolog's in human genome. These proteins were subjected to screening further by using the Database of Essential Genes (DEG) that resulted in the identification of 32 vitally important proteins for the bacterium. Subsequent analysis of the identified pivotal proteins, using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Automated Annotation Server (KAAS) resulted in sorting 3 key enzymes of F. nucleatum that may be good candidates as potential drug targets, since they are unique for the bacterium and absent in humans. In addition, we have demonstrated the three dimensional structure of these three proteins. Finally, determination of ligand binding sites of the 2 key proteins as well as screening for functional inhibitors that best fitted with the ligands sites were conducted to discover effective novel therapeutic compounds against F. nucleatum. PMID:28154519

  14. Oral Fusobacterium nucleatum subsp. polymorphum binds to human salivary α-amylase.

    PubMed

    Zulfiqar, M; Yamaguchi, T; Sato, S; Oho, T

    2013-12-01

    Fusobacterium nucleatum acts as an intermediate between early and late colonizers in the oral cavity. In this study, we showed that F. nucleatum subsp. polymorphum can bind to a salivary component with a molecular weight of approximately 110 kDa and identified the protein and another major factor of 55 kDa, as salivary α-amylase by time-of-flight mass spectrometry and immuno-reactions. Salivary α-amylase is present in both monomeric and dimeric forms and we found that formation of the dimer depends on copper ions. The F. nucleatum adhered to both monomeric and dimeric salivary α-amylases, but the numbers of bacteria bound to the dimeric form were more than those bound to the monomeric form. The degree of adherence of F. nucleatum to four α-amylases from different sources was almost the same, however its binding to β-amylase was considerably decreased. Among four α-amylase inhibitors tested, acarbose and type 1 and 3 inhibitors derived from wheat flour showed significant activity against the adhesion of F.nucleatum to monomeric and dimeric amylases, however voglibose had little effect. Moreover F. nucleatum cells inhibited the enzymatic activity of salivary α-amylase in a dose-dependent manner. These results suggest that F. nucleatum plays more important and positive role as an early colonizer for maturation of oral microbial colonization.

  15. Tea polyphenols inhibit the growth and virulence properties of Fusobacterium nucleatum

    PubMed Central

    Ben Lagha, Amel; Haas, Bruno; Grenier, Daniel

    2017-01-01

    Fusobacterium nucleatum plays a key role in creating the pathogenic subgingival biofilm that initiates destructive periodontitis. It is also a common resident of the human gastrointestinal tract and has been associated with inflammatory bowel disease. The aim of the present study was to investigate the effects of green and black tea extracts as well as two of their bioactive components, EGCG and theaflavins, on the growth and virulence properties of F. nucleatum. The tea extracts and components displayed various degrees of antibacterial activity that may involve damage to the bacterial cell membrane and the chelation of iron. They also prevented biofilm formation by F. nucleatum at concentrations that did not interfere with bacterial growth. In addition, the treatment of a pre-formed F. nucleatum biofilm with the green tea extract and EGCG caused a time-dependent decrease in biofilm viability. The green and black tea extracts, EGCG, and theaflavins decreased the adherence of F. nucleatum to oral epithelial cells and matrix proteins. Moreover, these tea components also attenuated F. nucleatum-mediated hemolysis and hydrogen sulfide production, two other virulence factors expressed by this bacterium. In summary, this study showed that tea polyphenols may be of interest for treating F. nucleatum-associated disorders. PMID:28322293

  16. Fusobacterium nucleatum-Induced Impairment of Autophagic Flux Enhances the Expression of Proinflammatory Cytokines via ROS in Caco-2 Cells

    PubMed Central

    Jia, Yin-ping; Zhu, Pan; Fang, Yao; Zhang, Zhu-jun; Mao, Xu-hu; Li, Qian; Zeng, Dong-Zhu

    2016-01-01

    Fusobacterium nucleatum (F. nucleatum) plays a critical role in gastrointestinal inflammation. However, the exact mechanism by which F. nucleatum contributes to inflammation is unclear. In the present study, it was revealed that F. nucleatum could induce the production of proinflammatory cytokines (IL-8, IL-1β and TNF-α) and reactive oxygen species (ROS) in Caco-2 colorectal) adenocarcinoma cells. Furthermore, ROS scavengers (NAC or Tiron) could decrease the production of proinflammatory cytokines during F. nucleatum infection. In addition, we observed that autophagy is impaired in Caco-2 cells after F. nucleatum infection. The production of proinflammatory cytokines and ROS induced by F. nucleatum was enhanced with either autophagy pharmacologic inhibitors (3-methyladenine, bafilomycin A1) or RNA interference in essential autophagy genes (ATG5 or ATG12) in Caco-2 cells. Taken together, these results indicate that F. nucleatum-induced impairment of autophagic flux enhances the expression of proinflammatory cytokines via ROS in Caco-2 Cells. PMID:27828984

  17. Biologic Activity of Type I and Type II Fusobacterium nucleatum Isolates from Clinically Characterized Sites,

    DTIC Science & Technology

    1984-07-26

    journal of choice is the Journal of Periodontology . 1 ncl JACK W. VINCENT as COL, DC Microbiology Branch SGRD-UDZ (19 Jul 84) TO COL Jack W. Vincent...Activitity of Type I and Type II Manuscript for Publication Fusobacterium nucleatum Isolates From Clinically Chatacterized Sites. 6. PERFORMING ONG...120) were obtained from subgingival plaque samples taken from 27 clinically characterized sites utilizing a selec- tive culture medium. All isolates

  18. Association of Fusobacterium nucleatum with immunity and molecular alterations in colorectal cancer

    PubMed Central

    Nosho, Katsuhiko; Sukawa, Yasutaka; Adachi, Yasushi; Ito, Miki; Mitsuhashi, Kei; Kurihara, Hiroyoshi; Kanno, Shinichi; Yamamoto, Itaru; Ishigami, Keisuke; Igarashi, Hisayoshi; Maruyama, Reo; Imai, Kohzoh; Yamamoto, Hiroyuki; Shinomura, Yasuhisa

    2016-01-01

    The human intestinal microbiome plays a major role in human health and diseases, including colorectal cancer. Colorectal carcinogenesis represents a heterogeneous process with a differing set of somatic molecular alterations, influenced by diet, environmental and microbial exposures, and host immunity. Fusobacterium species are part of the human oral and intestinal microbiota. Metagenomic analyses have shown an enrichment of Fusobacterium nucleatum (F. nucleatum) in colorectal carcinoma tissue. Using 511 colorectal carcinomas from Japanese patients, we assessed the presence of F. nucleatum. Our results showed that the frequency of F. nucleatum positivity in the Japanese colorectal cancer was 8.6% (44/511), which was lower than that in United States cohort studies (13%). Similar to the United States studies, F. nucleatum positivity in Japanese colorectal cancers was significantly associated with microsatellite instability (MSI)-high status. Regarding the immune response in colorectal cancer, high levels of infiltrating T-cell subsets (i.e., CD3+, CD8+, CD45RO+, and FOXP3+ cells) have been associated with better patient prognosis. There is also evidence to indicate that molecular features of colorectal cancer, especially MSI, influence T-cell-mediated adaptive immunity. Concerning the association between the gut microbiome and immunity, F. nucleatum has been shown to expand myeloid-derived immune cells, which inhibit T-cell proliferation and induce T-cell apoptosis in colorectal cancer. This finding indicates that F. nucleatum possesses immunosuppressive activities by inhibiting human T-cell responses. Certain microRNAs are induced during the macrophage inflammatory response and have the ability to regulate host-cell responses to pathogens. MicroRNA-21 increases the levels of IL-10 and prostaglandin E2, which suppress antitumor T-cell-mediated adaptive immunity through the inhibition of the antigen-presenting capacities of dendritic cells and T-cell proliferation in

  19. Association of Fusobacterium nucleatum with immunity and molecular alterations in colorectal cancer.

    PubMed

    Nosho, Katsuhiko; Sukawa, Yasutaka; Adachi, Yasushi; Ito, Miki; Mitsuhashi, Kei; Kurihara, Hiroyoshi; Kanno, Shinichi; Yamamoto, Itaru; Ishigami, Keisuke; Igarashi, Hisayoshi; Maruyama, Reo; Imai, Kohzoh; Yamamoto, Hiroyuki; Shinomura, Yasuhisa

    2016-01-14

    The human intestinal microbiome plays a major role in human health and diseases, including colorectal cancer. Colorectal carcinogenesis represents a heterogeneous process with a differing set of somatic molecular alterations, influenced by diet, environmental and microbial exposures, and host immunity. Fusobacterium species are part of the human oral and intestinal microbiota. Metagenomic analyses have shown an enrichment of Fusobacterium nucleatum (F. nucleatum) in colorectal carcinoma tissue. Using 511 colorectal carcinomas from Japanese patients, we assessed the presence of F. nucleatum. Our results showed that the frequency of F. nucleatum positivity in the Japanese colorectal cancer was 8.6% (44/511), which was lower than that in United States cohort studies (13%). Similar to the United States studies, F. nucleatum positivity in Japanese colorectal cancers was significantly associated with microsatellite instability (MSI)-high status. Regarding the immune response in colorectal cancer, high levels of infiltrating T-cell subsets (i.e., CD3+, CD8+, CD45RO+, and FOXP3+ cells) have been associated with better patient prognosis. There is also evidence to indicate that molecular features of colorectal cancer, especially MSI, influence T-cell-mediated adaptive immunity. Concerning the association between the gut microbiome and immunity, F. nucleatum has been shown to expand myeloid-derived immune cells, which inhibit T-cell proliferation and induce T-cell apoptosis in colorectal cancer. This finding indicates that F. nucleatum possesses immunosuppressive activities by inhibiting human T-cell responses. Certain microRNAs are induced during the macrophage inflammatory response and have the ability to regulate host-cell responses to pathogens. MicroRNA-21 increases the levels of IL-10 and prostaglandin E2, which suppress antitumor T-cell-mediated adaptive immunity through the inhibition of the antigen-presenting capacities of dendritic cells and T-cell proliferation in

  20. Fusobacterium nucleatum and Tannerella forsythia induce synergistic alveolar bone loss in a mouse periodontitis model.

    PubMed

    Settem, Rajendra P; El-Hassan, Ahmed Taher; Honma, Kiyonobu; Stafford, Graham P; Sharma, Ashu

    2012-07-01

    Tannerella forsythia is strongly associated with chronic periodontitis, an inflammatory disease of the tooth-supporting tissues, leading to tooth loss. Fusobacterium nucleatum, an opportunistic pathogen, is thought to promote dental plaque formation by serving as a bridge bacterium between early- and late-colonizing species of the oral cavity. Previous studies have shown that F. nucleatum species synergize with T. forsythia during biofilm formation and pathogenesis. In the present study, we showed that coinfection of F. nucleatum and T. forsythia is more potent than infection with either species alone in inducing NF-κB activity and proinflammatory cytokine secretion in monocytic cells and primary murine macrophages. Moreover, in a murine model of periodontitis, mixed infection with the two species induces synergistic alveolar bone loss, characterized by bone loss which is greater than the additive alveolar bone losses induced by each species alone. Further, in comparison to the single-species infection, mixed infection caused significantly increased inflammatory cell infiltration in the gingivae and osteoclastic activity in the jaw bones. These data show that F. nucleatum subspecies and T. forsythia synergistically stimulate the host immune response and induce alveolar bone loss in a murine experimental periodontitis model.

  1. The Danger Signal Extracellular ATP Is an Inducer of Fusobacterium nucleatum Biofilm Dispersal

    PubMed Central

    Ding, Qinfeng; Tan, Kai Soo

    2016-01-01

    Plaque biofilm is the primary etiological agent of periodontal disease. Biofilm formation progresses through multiple developmental stages beginning with bacterial attachment to a surface, followed by development of microcolonies and finally detachment and dispersal from a mature biofilm as free planktonic bacteria. Tissue damage arising from inflammatory response to biofilm is one of the hallmark features of periodontal disease. A consequence of tissue damage is the release of ATP from within the cell into the extracellular space. Extracellular ATP (eATP) is an example of a danger associated molecular pattern (DAMP) employed by mammalian cells to elicit inflammatory and damage healing responses. Although, the roles of eATP as a signaling molecule in multi-cellular organisms have been relatively well studied, exogenous ATP also influences bacteria biofilm formation. Since plaque biofilms are continuously exposed to various stresses including exposure to the host damage factors such as eATP, we hypothesized that eATP, in addition to eliciting inflammation could potentially influence the biofilm lifecycle of periodontal associated bacteria. We found that eATP rather than nutritional factors or oxidative stress induced dispersal of Fusobacterium nucleatum, an organism associated with periodontal disease. eATP induced biofilm dispersal through chelating metal ions present in biofilm. Dispersed F. nucleatum biofilm, regardless of natural or induced dispersal by exogenous ATP, were more adhesive and invasive compared to planktonic or biofilm counterparts, and correspondingly activated significantly more pro-inflammatory cytokine production in infected periodontal fibroblasts. Dispersed F. nucleatum also showed higher expression of fadA, a virulence factor implicated in adhesion and invasion, compared to planktonic or biofilm bacteria. This study revealed for the first time that periodontal bacterium is capable of co-opting eATP, a host danger signaling molecule to detach

  2. Fusobacterium nucleatum induces fetal death in mice via stimulation of TLR4-mediated placental inflammatory response.

    PubMed

    Liu, Hongqi; Redline, Raymond W; Han, Yiping W

    2007-08-15

    Intrauterine infection plays a pivotal role in preterm birth (PTB) and is characterized by inflammation. Currently, there is no effective therapy available to treat or prevent bacterial-induced PTB. Using Fusobacterium nucleatum, a Gram-negative anaerobe frequently associated with PTB, as a model organism, the mechanism of intrauterine infection was investigated. Previously, it was shown that F. nucleatum induced preterm and term stillbirth in mice. Fusobacterial-induced placental infection was characterized by localized bacterial colonization, inflammation, and necrosis. In this study, F. nucleatum was shown to activate both TLR2 and TLR4 in vitro. In vivo, the fetal death rate was significantly reduced in TLR4-deficient mice (C57BL/6 TLR4(-/-) and C3H/HeJ (TLR4(d/d))), but not in TLR2-deficient mice (C57BL/6 TLR2(-/-)), following F. nucleatum infection. The reduced fetal death in TLR4-deficient mice was accompanied by decreased placental necroinflammatory responses in both C57BL/6 TLR4(-/-) and C3H/HeJ. Decreased bacterial colonization in the placenta was observed in C3H/HeJ, but not in C57BL/6 TLR4(-/-). These results suggest that inflammation, rather than the bacteria per se, was the likely cause of fetal loss. TLR2 did not appear to be critically involved, as no difference in bacterial colonization, inflammation, or necrosis was observed between C57BL/6 and C57BL/6 TLR2(-/-) mice. A synthetic TLR4 antagonist, TLR4A, significantly reduced fusobacterial-induced fetal death and decidual necrosis without affecting the bacterial colonization in the placentas. TLR4A had no bactericidal activity nor did it affect the birth outcome in sham-infected mice. TLR4A could have promise as an anti-inflammatory agent for the treatment or prevention of bacterial-induced preterm birth.

  3. High occurrence of Fusobacterium nucleatum and Clostridium difficile in the intestinal microbiota of colorectal carcinoma patients.

    PubMed

    Fukugaiti, Márcia H; Ignacio, Aline; Fernandes, Miriam R; Ribeiro Júnior, Ulysses; Nakano, Viviane; Avila-Campos, Mario J

    2015-01-01

    Colorectal carcinoma is considered the fourth leading cause of cancer deaths worldwide. Several microorganisms have been associated with carcinogenesis, including Enterococcus spp., Helicobacter pylori, enterotoxigenic Bacteroides fragilis, pathogenic E. coli strains and oral Fusobacterium. Here we qualitatively and quantitatively evaluated the presence of oral and intestinal microorganisms in the fecal microbiota of colorectal cancer patients and healthy controls. Seventeen patients (between 49 and 70 years-old) visiting the Cancer Institute of the Sao Paulo State were selected, 7 of whom were diagnosed with colorectal carcinoma. Bacterial detection was performed by qRT-PCR. Although all of the tested bacteria were detected in the majority of the fecal samples, quantitative differences between the Cancer Group and healthy controls were detected only for F. nucleatum and C. difficile. The three tested oral microorganisms were frequently observed, suggesting a need for furthers studies into a potential role for these bacteria during colorectal carcinoma pathogenesis. Despite the small number of patients included in this study, we were able to detect significantly more F. nucleatum and C. difficile in the Cancer Group patients compared to healthy controls, suggesting a possible role of these bacteria in colon carcinogenesis. This finding should be considered when screening for colorectal cancer.

  4. Genome Sequence and Analysis of the Oral Bacterium Fusobacterium nucleatum Strain ATCC 25586

    PubMed Central

    Kapatral, Vinayak; Anderson, Iain; Ivanova, Natalia; Reznik, Gary; Los, Tamara; Lykidis, Athanasios; Bhattacharyya, Anamitra; Bartman, Allen; Gardner, Warren; Grechkin, Galina; Zhu, Lihua; Vasieva, Olga; Chu, Lien; Kogan, Yakov; Chaga, Oleg; Goltsman, Eugene; Bernal, Axel; Larsen, Niels; D'Souza, Mark; Walunas, Theresa; Pusch, Gordon; Haselkorn, Robert; Fonstein, Michael; Kyrpides, Nikos; Overbeek, Ross

    2002-01-01

    We present a complete DNA sequence and metabolic analysis of the dominant oral bacterium Fusobacterium nucleatum. Although not considered a major dental pathogen on its own, this anaerobe facilitates the aggregation and establishment of several other species including the dental pathogens Porphyromonas gingivalis and Bacteroides forsythus. The F. nucleatum strain ATCC 25586 genome was assembled from shotgun sequences and analyzed using the ERGO bioinformatics suite (http://www.integratedgenomics.com). The genome contains 2.17 Mb encoding 2,067 open reading frames, organized on a single circular chromosome with 27% GC content. Despite its taxonomic position among the gram-negative bacteria, several features of its core metabolism are similar to that of gram-positive Clostridium spp., Enterococcus spp., and Lactococcus spp. The genome analysis has revealed several key aspects of the pathways of organic acid, amino acid, carbohydrate, and lipid metabolism. Nine very-high-molecular-weight outer membrane proteins are predicted from the sequence, none of which has been reported in the literature. More than 137 transporters for the uptake of a variety of substrates such as peptides, sugars, metal ions, and cofactors have been identified. Biosynthetic pathways exist for only three amino acids: glutamate, aspartate, and asparagine. The remaining amino acids are imported as such or as di- or oligopeptides that are subsequently degraded in the cytoplasm. A principal source of energy appears to be the fermentation of glutamate to butyrate. Additionally, desulfuration of cysteine and methionine yields ammonia, H2S, methyl mercaptan, and butyrate, which are capable of arresting fibroblast growth, thus preventing wound healing and aiding penetration of the gingival epithelium. The metabolic capabilities of F. nucleatum revealed by its genome are therefore consistent with its specialized niche in the mouth. PMID:11889109

  5. Evaluation of antibody level against Fusobacterium nucleatum in the serological diagnosis of colorectal cancer

    PubMed Central

    Wang, Hai-Fang; Li, Lin-Fang; Guo, Song-He; Zeng, Qiu-Yao; Ning, Fen; Liu, Wan-Li; Zhang, Ge

    2016-01-01

    Fusobacterium nucleatum (F. nucleatum, Fn) is associated with the colorectal cancer (CRC). Fn-infection could induce significant levels of serum Fn-specific antibodies in human and mice. The objective of this study was to identify Fn-infection that elicit a humoral response in patients with CRC and evaluate the diagnostic performance of serum anti-Fn antibodies. In this work, we showed the mean absorbance value of anti-Fn-IgA and -IgG in the CRC group were significantly higher than those in the benign colon disease group and healthy control group (P < 0.001). The sensitivity and specificity of ELISA for the detection of anti-Fn-IgA were 36.43% and 92.71% based on the optimal cut-off. The combination of anti-Fn-IgA and carcino-embryonic antigen (CEA) was better for diagnosing CRC (Sen: 53.10%, Spe: 96.41%; AUC = 0.848). Furthermore, combining anti-Fn-IgA with CEA and carbohydrate antigen 19-9 (CA19-9) (Sen: 40.00%, Spe: 94.22%; AUC = 0.743) had the better ability to classify CRC patients with stages I-II. These results suggested that Fn-infection elicited high level of serum anti-Fn antibodies in CRC patients, and serum anti-Fn-IgA level may be a potential diagnosing biomarker for CRC. Serum anti-Fn-IgA in combination with CEA and CA19-9 increases the sensitivity of detecting early CRC. PMID:27678333

  6. Immunobiological activities of a porin fraction isolated from Fusobacterium nucleatum ATCC 10953.

    PubMed Central

    Takada, H; Ogawa, T; Yoshimura, F; Otsuka, K; Kokeguchi, S; Kato, K; Umemoto, T; Kotani, S

    1988-01-01

    From Fusobacterium nucleatum ATCC 10953 cell envelope fraction whose inner membranes had been removed by treatment with sodium N-lauroyl sarcosinate, an outer membrane protein (37,000 Mr in a native state) was prepared by extraction with lithium dodecyl sulfate. The protein thus obtained showed distinct porin activity, namely, the ability to form hydrophilic diffusion pores by incorporation into the artificial liposome membrane. The porin fraction exhibited strong immunobiological activities in the in vitro assays: B-cell mitogenicity and polyclonal B-cell activation on murine splenocytes, stimulatory effects on guinea pig peritoneal macrophages, and enhancement of the migration of human blood monocytes. The porin fraction also exhibited immunoadjuvant activity to increase the antibody production against sheep erythrocytes in the spleen of mice that were immunized by sheep erythrocytes with porin. Although chemical analyses revealed that the test porin fraction contained a considerable amount of lipopolysaccharide (LPS) (around 12% of the fraction), the studies with LPS-nonresponding C3H/HeJ mice and on the inhibitory effects of polymyxin B strongly suggest that most of the above bioactivities are due to porin protein itself, not to coexistent LPS in the porin fraction. Images PMID:2831155

  7. Starvation response and growth in serum of Fusobacterium nucleatum, Peptostreptococcus anaerobius, Prevotella intermedia, and Pseudoramibacter alactolyticus.

    PubMed

    Brundin, Malin; Figdor, David; Sundqvist, Göran; Sjögren, Ulf

    2009-07-01

    The microbiota inhabiting the untreated root canal differ markedly from those found in post-treatment disease, yet there is limited information on the microbial characteristics distinguishing the different infections. We hypothesized that starvation survival is a key microbial property in species selection. This study analyzed starvation-survival behavior over 60 days of species representative of the untreated root canal infection: Fusobacterium nucleatum, Peptostreptococcus anaerobius, Prevotella intermedia and Pseudoramibacter alactolyticus. All species did not survive 1 day in water. In 1% serum, the 4 species could not survive beyond 2-3 weeks. They required a high initial cell density and >or=10% serum to survive the observation period. The results highlight a poor starvation-survival capacity of these 4 species compared with species prevalent in post-treatment infection, which are well equipped to endure starvation and survive in low numbers on minimal serum. These findings point to starvation-survival capacity as a selection factor for microbial participation in post-treatment disease.

  8. Antibody and T Cell Responses to Fusobacterium nucleatum and Treponema denticola in Health and Chronic Periodontitis

    PubMed Central

    Shin, Jieun; Kho, Sang-A; Choi, Yun S.; Kim, Yong C.; Rhyu, In-Chul; Choi, Youngnim

    2013-01-01

    The characteristics of the T cell response to the members of oral flora are poorly understood. We characterized the antibody and T cell responses to FadA and Td92, adhesins from Fusobacterium nucleatum, an oral commensal, and Treponema denticola, a periodontal pathogen, respectively. Peripheral blood and saliva were obtained from healthy individuals and patients with untreated chronic periodontitis (CP, n = 11 paris) and after successful treatment of the disease (n = 9). The levels of antigen-specific antibody were measured by ELISA. In plasma, IgG1 was the most abundant isotype of Ab for both Ags, followed by IgA and then IgG4. The levels of FadA-specific salivary IgA (sIgA) were higher than Td92-specific sIgA and the FadA-specific IgA levels observed in plasma. However, the periodontal health status of the individuals did not affect the levels of FadA- or Td92-specific antibody. Even healthy individuals contained FadA- and Td92-specific CD4+ T cells, as determined by the detection of intracytoplasmic CD154 after short-term in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with the antigens. Patients with CP tended to possess increased numbers of FadA- and Td92-specific CD4+ T cells but reduced numbers of Td92-specific Foxp3+CD4+ Tregs than the healthy subjects. Both FadA and Td92 induced the production of IFNγ and IL-10 but inhibited the secretion of IL-4 by PBMCs. In conclusion, F. nucleatum induced Th3 (sIgA)- and Th1 (IFNγ and IgG1)-dominant immune responses, whereas T. denticola induced a Th1 (IFNγ and IgG1)-dominant response. This IFNγ-dominant cytokine response was impaired in CP patients, and the Td92-induced IFNγ levels were negatively associated with periodontal destruction in patients. These findings may provide new insights into the homeostatic interaction between the immune system and oral bacteria and the pathogenesis of periodontitis. PMID:23335969

  9. Fap2 of Fusobacterium nucleatum is a galactose-inhibitable adhesin involved in coaggregation, cell adhesion, and preterm birth.

    PubMed

    Coppenhagen-Glazer, S; Sol, A; Abed, J; Naor, R; Zhang, X; Han, Y W; Bachrach, G

    2015-03-01

    Fusobacterium nucleatum is a common oral anaerobe involved in periodontitis that is known to translocate and cause intrauterine infections. In the oral environment, F. nucleatum adheres to a large diversity of species, facilitating their colonization and creating biological bridges that stabilize the multispecies dental biofilm. Many of these interactions (called coadherences or coaggregations) are galactose sensitive. Galactose-sensitive interactions are also involved in the binding of F. nucleatum to host cells. Hemagglutination of some F. nucleatum strains is also galactose sensitive, suggesting that a single galactose-sensitive adhesin might mediate the interaction of fusobacteria with many partners and targets. In order to identify the fusobacterial galactose-sensitive adhesin, a system for transposon mutagenesis in fusobacteria was created. The mutant library was screened for hemagglutination deficiency, and three clones were isolated. All three clones were found to harbor the transposon in the gene coding for the Fap2 outer membrane autotransporter. The three fap2 mutants failed to show galactose-inhibitable coaggregation with Porphyromonas gingivalis and were defective in cell binding. A fap2 mutant also showed a 2-log reduction in murine placental colonization compared to that of the wild type. Our results suggest that Fap2 is a galactose-sensitive hemagglutinin and adhesin that is likely to play a role in the virulence of fusobacteria.

  10. Detection of fusobacterium nucleatum and fadA adhesin gene in patients with orthodontic gingivitis and non-orthodontic periodontal inflammation.

    PubMed

    Liu, Ping; Liu, Yi; Wang, Jianning; Guo, Yang; Zhang, Yujie; Xiao, Shuiqing

    2014-01-01

    Fusobacterium nucleatum is one of the most abundant gram-negative bacilli colonizing the subgingival plaque and closely associated with periodontal disease. However it is unclear whether F. nucleatum is involved in gingival inflammation under orthodontic appliance. A novel adhesin, FadA, which is unique to oral Fusobacteria, is required for F. nucleatum binding and invasion to epithelial cells and thus may play an important role in colonization of Fusobacterium in the host. In this study, we evaluated the prevalence of F. nucleatum and its virulence factor FadA adhesion gene (fadA) in 169 subgingival biofilm samples from 55 cases of gingivitis patients with orthodontic appliances, 49 cases of gingivitis patients without orthodontic treatment, 35 cases of periodontitis patients and 30 cases of periodontally healthy people via PCR. The correlations between the F. nucleatum/fadA and gingivitis index(GI)was also analyzed. The detection rate of F. nucleatum/fadA in periodontitis group and non-orthodontic gingivitis group was higher than the other two groups (p<0.01) while it was higher in orthodontic gingivitis group than in health people (p<0.05). An obviously positive correlation was observed between the prevalence of F. nucleatum/fadA and GI. F. nucleatum carrying fadA may be more closely related to the development of gingivitis and periodontal disease compared with orthodontic gingivitis.

  11. Identification and characterization of a novel Fusobacterium nucleatum adhesin involved in physical interaction and biofilm formation with Streptococcus gordonii.

    PubMed

    Lima, Bruno P; Shi, Wenyuan; Lux, Renate

    2017-02-07

    To successfully colonize the oral cavity, bacteria must directly or indirectly adhere to available oral surfaces. Fusobacterium nucleatum plays an important role in oral biofilm community development due to its broad adherence abilities, serving as a bridge between members of the oral biofilm that cannot directly bind to each other. In our efforts to characterize the molecular mechanisms utilized by F. nucleatum to physically bind to key members of the oral community, we investigated the involvement of F. nucleatum outer membrane proteins in its ability to bind to the pioneer biofilm colonizer, Streptococcus gordonii. Here, we present evidence that in addition to the previously characterized fusobacterial adhesin RadD, the interaction between F. nucleatum ATCC 23726 and S. gordonii V288 involves a second outer membrane protein, which we named coaggregation mediating protein A (CmpA). We also characterized the role of CmpA in dual-species biofilm formation with S. gordonii V288, evaluated growth-phase-dependent as well as biofilm expression profiles of radD and cmpA, and confirmed an important role for CmpA, especially under biofilm growth conditions. Our findings underscore the complex set of specific interactions involved in physical binding and thus community integration of interacting bacterial species. This complex set of interactions could have critical implications for the formation and maturation of the oral biofilms in vivo, and could provide clues to the mechanism behind the distribution of organisms inside the human oral cavity.

  12. Inhibition of the MurA enzyme in Fusobacterium nucleatum by potential inhibitors identified through computational and in vitro approaches.

    PubMed

    Kumar, Amit; Saranathan, Rajagopalan; Prashanth, K; Tiwary, Basant K; Krishna, Ramadas

    2017-03-30

    Fusobacterium nucleatum plays a key role in several diseases such as periodontitis, gingivitis, appendicitis, and inflammatory bowel disease (IBD). The development of antibiotic resistance by this bacterium demands novel therapeutic intervention. Our recent study has reported UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA) as one of the potential target proteins in F. nucleatum. In this study, we proposed two novel MurA inhibitors through in silico screening and evaluated their mode of inhibition by in vitro experiments. It was found that MurA structural arrangement (inside-out α/β barrel) was stabilized by L/FXXXG(A) motif-based interactions. The protein was maintained in an open or substrate-free conformation due to repulsive forces between two parallelly arranged positively charged residues of domain I and II. In this conformation, we identified six best compounds that held key interactions with the substrate-binding pocket via a structure-based virtual screening of natural and chemical compound libraries. However, among these, only orientin and quercetin-3-O-d-glucuronide (Q3G) showed better interaction capability through consistent H-bond occupancy and lowest binding free energy during molecular dynamic simulations. In vitro inhibition studies evidenced the mixed and uncompetitive mode of inhibition by orientin and Q3G, respectively, with purified MurA protein. This explains the binding of orientin in both open and closed (substrate-bound) conformations of MurA, and Q3G binding in only closed conformation. Therefore, the Q3G binding mode was predicted on a MurA-substrate complex, which highlighted its constant H-bond with Cys118, a phosphoenolpyruvate (PEP) interacting residue. This suggests that Q3G may interrupt the PEP binding, thereby inhibiting the MurA activity. Thus, the current study discusses the structure of MurA and demonstrates the inhibitory action of two novel compounds.

  13. Complete genome sequences and analysis of the Fusobacterium nucleatum subspecies animalis 7-1 bacteriophage ɸFunu1 and ɸFunu2.

    PubMed

    Cochrane, Kyla; Manson McGuire, Abigail; Priest, Margaret E; Abouelleil, Amr; Cerqueira, Gustavo C; Lo, Reggie; Earl, Ashlee M; Allen-Vercoe, Emma

    2016-04-01

    Fusobacterium nucleatum is a strictly anaerobic, Gram negative bacterial species that has been associated with dental infections, pre-term labor, appendicitis, inflammatory bowel disease, and, more recently, colorectal cancer. The species is unusual in its phenotypic and genotypic heterogeneity, with some strains demonstrating a more virulent phenotype than others; however, as yet the genetic basis for these differences is not understood. Bacteriophage are known to contribute to the virulence phenotype of several bacterial species. In this work, we set out to characterize the bacteriophage associated with F. nucleatum subsp. animalis strain 7-1, a highly invasive isolate from the human gastrointestinal tract. As well, we used computational approaches to predict and compare bacteriophage signatures across available sequenced F. nucleatum genomes.

  14. Crystal Structure of FadA Adhesin from Fusobacterium nucleatum Reveals a Novel Oligomerization Motif, the Leucine Chain

    SciTech Connect

    Nithianantham, Stanley; Xu, Minghua; Yamada, Mitsunori; Ikegami, Akihiko; Shoham, Menachem; Han, Yiping W.

    2009-04-07

    Many bacterial appendages have filamentous structures, often composed of repeating monomers assembled in a head-to-tail manner. The mechanisms of such linkages vary. We report here a novel protein oligomerization motif identified in the FadA adhesin from the Gram-negative bacterium Fusobacterium nucleatum. The 2.0 {angstrom} crystal structure of the secreted form of FadA (mFadA) reveals two antiparallel {alpha}-helices connected by an intervening 8-residue hairpin loop. Leucine-leucine contacts play a prominent dual intra- and intermolecular role in the structure and function of FadA. First, they comprise the main association between the two helical arms of the monomer; second, they mediate the head-to-tail association of monomers to form the elongated polymers. This leucine-mediated filamentous assembly of FadA molecules constitutes a novel structural motif termed the 'leucine chain.' The essential role of these residues in FadA is corroborated by mutagenesis of selected leucine residues, which leads to the abrogation of oligomerization, filament formation, and binding to host cells.

  15. Wild Blueberry (Vaccinium angustifolium Ait.) Polyphenols Target Fusobacterium nucleatum and the Host Inflammatory Response: Potential Innovative Molecules for Treating Periodontal Diseases.

    PubMed

    Ben Lagha, Amel; Dudonné, Stéphanie; Desjardins, Yves; Grenier, Daniel

    2015-08-12

    Blueberries contain significant amounts of flavonoids to which a number of beneficial health effects in humans have been associated. The present study investigated the effect of a polyphenol-rich lowbush blueberry (Vaccinium angustifolium Ait.) extract on the two main etiologic components of periodontitis, a multifactorial disorder affecting the supporting structures of the teeth. Phenolic acids, flavonoids (flavonols, anthocyanins, flavan-3-ols), and procyanidins made up 16.6, 12.9, and 2.7% of the blueberry extract, respectively. The blueberry extract showed antibacterial activity (MIC = 1 mg/mL) against the periodontopathogenic bacterium Fusobacterium nucleatum. This property may result from the ability of blueberry polyphenols to chelate iron. Moreover, the blueberry extract at 62.5 μg/mL inhibited F. nucleatum biofilm formation by 87.5 ± 2.3%. Subsequently, the ability of the blueberry extract to inhibit the NF-κB signaling pathway in U937-3xκB cells was investigated. The blueberry extract dose-dependently inhibited the activation of NF-κB induced by F. nucleatum. In addition, a pretreatment of macrophages with the blueberry extract (62.5 μg/mL) inhibited the secretion of IL-1β, TNF-α, and IL-6 by 87.3 ± 1.3, 80.7 ± 5.6, and 28.2 ± 9.3%, respectively, following a stimulation with F. nucleatum. Similarly, the secretion of MMP-8 and MMP-9 was also dose-dependently inhibited. This dual antibacterial and anti-inflammatory action of lowbush blueberry polyphenols suggests that they may be promising candidates for novel therapeutic agents.

  16. Septic arthritis and osteomyelitis in a 10-year-old boy, caused by Fusobacterium nucleatum, diagnosed with PCR/16S ribosomal bacterial DNA amplification

    PubMed Central

    Kroon, Elke; Arents, Niek A; Halbertsma, Feico Jan

    2012-01-01

    A 10-year-old boy presented with an atypical non-febrile septic arthritis/osteomyelitis. He was unresponsive to routine antibiotic treatment with flucloxacillin/gentamicin as the pain and fluid collection increased. Synovial fluid cultures are negative and gram stain remained negative. Only after PCR/16S ribosomal bacterial DNA amplification a Fusobacterium nucleatum could be detected, and antibiotic therapy switched to clindamycin with rapid response. Septic osteomyelitis and arthritis are relatively rare but important infections in children needing prompt treatment, and should be considered when a child complaints about joint or bone pain without prior recent trauma. Skin bacteria are the most prevalent causative organisms, whereas Fusobacteria or other anaerobic, Gram-negative microorganisms are very seldom encountered. If cultures remain negative and the patients responds insufficiently to empiric treatment, PCR/16S ribosomal bacterial DNA amplification can be useful to detect the causative microorganisms. PMID:22605875

  17. Lethal effect of blue light-activated hydrogen peroxide, curcumin and erythrosine as potential oral photosensitizers on the viability of Porphyromonas gingivalis and Fusobacterium nucleatum

    PubMed Central

    Habiboallh, Ghanbari; Mahbobeh, Naderi Nasab; Mina, Zareian Jahromi; Majid, Zakeri; Nooshin, Arjmand

    2015-01-01

    Objectives: Recently, photodynamic therapy (PDT) has been introduced as a new modality in oral bacterial decontamination. Current research aims to evaluate the effect of photodynamic killing of visible blue light in the presence of hydrogen peroxide, curcumin and erythrosine as potential oral photosensitizers on Porphyromonas gingivalis associated with periodontal bone loss and Fusobacterium nucleatum associated with soft tissue inflammation. Materials and methods: Standard suspension of P. gingivalis and F. nucleatum were exposed to Light Emitting Diode (LED) (440–480 nm) in combination with erythrosine (22 µm), curcumin (60 µM) and hydrogen peroxide (0.3 mM) for 5 min. Bacterial samples from each treatment groups (radiation-only group, photosensitizer-only group and blue light-activated photosensitizer group) were subcultured onto the surface of agar plates. Survival of these bacteria was determined by counting the number of colony forming units (CFU) after incubation. Results: Results for antibacterial assays on P. gingivalis confirmed that curcumin, Hydrogen peroxide and erythrosine alone exerted a moderate bactericidal effect which enhanced noticeably in conjugation with visible light. The survival rate of P. gingivalis reached zero present when the suspension exposed to blue light-activated curcumin and hydrogen peroxide for 2 min. Besides, curcumin exerted a remarkable antibacterial activity against F. nucleatum in comparison with erythrosine and hydrogen peroxide (P=0.00). Furthermore, the bactericidal effect of visible light alone on P. gingivalis as black-pigmented bacteria was significant. Conclusion: Our result suggested that visible blue light in the presence of erythrosine, curcumin and hydrogen peroxide would be consider as a potential approach of PDT to kill the main gramnegative periodontal pathogens. From a clinical standpoint, this regimen could be established as an additional minimally invasive antibacterial treatment of plaque induced

  18. The Fusobacterium nucleatum major outer-membrane protein (FomA) forms trimeric, water-filled channels in lipid bilayer membranes.

    PubMed

    Kleivdal, H; Benz, R; Jensen, H B

    1995-10-01

    The pore-forming activity of the major outer-membrane protein FomA of the anaerobic Fusobacterium nucleatum was studied in artificial lipid bilayer membranes. FomA was isolated from F. nucleatum strains Fev1, ATCC 10953, and ATCC 25586 by extraction with lithium dodecyl sulfate and lithium chloride and had an apparent molecular mass of about 40 kDa. When solubilized at low temperatures, the protein ran with an apparent molecular mass of about 62 kDa on SDS/PAGE. Cross-linking experiments and two-dimensional SDS/PAGE gave evidence that the 62-kDa protein band represented the trimeric form of FomA. The protein trimers were susceptible to SDS and temperature. The stability of the porin trimers varied among the strains. The properties of the FomA channels were studied in reconstitution experiments with black lipid bilayer membranes. The F. nucleatum porins formed channels with single-channel conductances in the range 0.66-1.30 nS in M KCl. The single-channel conductance was a function of the mobilities of the ions present in the aqueous solution bathing the bilayer membrane. This means that FomA forms general diffusion channels since (a) the conductance showed a linear dependence on the salt concentration, (b) the ion selectivity was small and varied for the three strains, and (c) the channels did not exhibit any binding site for maltotriose or triglycine. The water-filled channel was voltage dependent, and conductance decrements were observed at transmembrane potentials of +/- 50 mV. The conductance decrement steps were about one-third of the total conductance of a functional unit in its fully 'open' state. This strongly suggests that the trimer is the functional unit of the porin.

  19. Fusobacterium nucleatum and Human Beta-Defensins Modulate the Release of Antimicrobial Chemokine CCL20/Macrophage Inflammatory Protein 3α ▿ †

    PubMed Central

    Ghosh, Santosh K.; Gupta, Sanhita; Jiang, Bin; Weinberg, Aaron

    2011-01-01

    Cells of the innate immune system regulate immune responses through the production of antimicrobial peptides, chemokines, and cytokines, including human beta-defensins (hBDs) and CCL20. In this study, we examined the kinetics of primary human oral epithelial cell (HOEC) production of CCL20 and hBDs in response to Fusobacterium nucleatum, a commensal bacterium of the oral cavity, which we previously showed promotes HOEC induction of hBDs. HOECs secrete maximal levels of CCL20 at 6 h, following stimulation by F. nucleatum cell wall (FnCW). The kinetics of CCL20 release is distinct from that of hBD-2 and -3, which peaks after 24 h and 48 h of FnCW stimulation, respectively. FnCW-induced release of CCL20 by HOECs requires both transcriptional and translational activation. Release of CCL20 by HOECs is inhibited by brefeldin A, suggesting that it is secreted through a vesicle transport pathway. Other epithelium-derived agents that FnCW induces, such as hBD-2, hBD-3, tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), are also able to release CCL20. By focusing on mitogen-activated protein kinases, we show that both extracellular signal-regulated kinase 1/2 and p38, but not JNK, are required for hBD-, TNF-α-, and IL-1β-induced secretion of CCL20 by HOECs. The ability of FnCW and its induced hBDs to produce proinflammatory cytokines and CCL20 suggests the broad role of F. nucleatum and human antimicrobial peptides in primary immune responses elicited by oral epithelium. PMID:21911466

  20. Bactericidal effect of visible light in the presence of erythrosine on Porphyromonas gingivalis and Fusobacterium nucleatum compared with diode laser, an in vitro study

    PubMed Central

    Habiboallah, Ghanbari; Mahdi, Zakeri; Mahbobeh, Naderi Nasab; Mina, Zareian Jahromi; Sina, Faghihi

    2014-01-01

    Objectives: Recently, photodynamic therapy (PDT) has been introduced as a new modality in oral bacterial decontamination. Besides, the ability of laser irradiation in the presence of photosensitizing agent to lethal effect on oral bacteria is well documented. Current research aims to evaluate the effect of photodynamic killing of visible blue light in the presence of plaque disclosing agent erythrosine as photosensitizer on Porphyromonas gingivalis associated with periodontal bone loss and Fusobacterium nucleatum associated with soft tissue inflammation, comparing with the near-infrared diode laser. Materials and methods: Standard suspension of P. gingivalis and F. nucleatum were exposed to Light Emitting Diode (LED) (440–480 nm) used to photopolymerize composite resine dental restoration in combination with erythrosine (22 µm) up to 5 minutes. Bacterial sample were also exposed to a near-infrared diode laser (wavelength, 830 nm), using identical irradiation parameters for comparison. Bacterial samples from each treatment groups (radiation-only group, erythrosine-only group and light or laser with erythrosine group) were subcultured onto the surface of agar plates. Survival of these bacteria was determined by counting the number of colony forming units (CFU) after incubation. Results: Exposure to visible blue light and diode laser in conjugation with erythrosine significantly reduced both species examined viability, whereas erythrosine-treated samples exposed to visible light suggested a statically meaningful differences comparing to diode laser. In addition, bactericidal effect of visible light or diode laser alone on P. gingivalis as black-pigmented bacteria possess endogenous porphyrins was noticeably. Conclusion: Our result suggested that visible blue light source in the presence of plaque disclosing agent erythrosine could can be consider as potential approach of PDT to kill the main gram-negative periodontal pathogens. From a clinical standpoint, this

  1. Periodontal pathogens Porphyromonas gingivalis and Fusobacterium nucleatum promote tumor progression in an oral-specific chemical carcinogenesis model

    PubMed Central

    Gallimidi, Adi Binder; Fischman, Stuart; Revach, Brurya; Bulvik, Raanan; Maliutina, Alina; Rubinstein, Ariel M.

    2015-01-01

    Oral squamous cell carcinoma (OSCC) is a lethal disease whose incidence is increasing. Epidemiologic studies demonstrate an association between periodontitis and oral cancer, and periodontal pathogens are implicated in the pathogenesis of numerous disorders, including rheumatoid arthritis, cardiovascular diseases, diabetes and gastrointestinal malignancies. Nevertheless, a causal role for periodontal pathogens in OSCC has not been shown, partly due to the lack of an appropriate animal model. Here, utilizing a newly-established murine model of periodontitis-associated oral tumorigenesis, we report that chronic bacterial infection promotes OSCC, and that augmented signaling along the IL-6-STAT3 axis underlies this effect. Our results indicate that periodontal pathogens P. gingivalis and F. nucleatum stimulate tumorigenesis via direct interaction with oral epithelial cells through Toll-like receptors. Furthermore, oral pathogens stimulate human OSCC proliferation and induce expression of key molecules implicated in tumorigenesis. To the best of our knowledge, these findings represent the first demonstration of a mechanistic role for oral bacteria in chemically induced OSCC tumorigenesis. These results are highly relevant for the design of effective prevention and treatment strategies for OSCC. PMID:26158901

  2. The Fusobacterium nucleatum Outer Membrane Protein RadD Is an Arginine-Inhibitable Adhesin Required for Inter-Species Adherence and the Structured Architecture of Multi-Species Biofilm

    PubMed Central

    Kaplan, Christopher W.; Lux, Renate; Haake, Susan Kinder; Shi, Wenyuan

    2009-01-01

    Summary A defining characteristic of the suspected periodontal pathogen Fusobacterium nucleatum is its ability to adhere to a plethora of oral bacteria. This distinguishing feature is suggested to play an important role in oral biofilm formation and pathogenesis, with fusobacteria proposed to serve as central “bridging organisms” in the architecture of the oral biofilm bringing together species which would not interact otherwise. Previous studies indicate that these bacterial interactions are mediated by galactose- or arginine-inhibitable adhesins although genetic evidence for the role and nature of these proposed adhesins remains elusive. To characterize these adhesins at the molecular level, the genetically transformable F. nucleatum strain ATCC 23726 was screened for adherence properties, and arginine inhibitable adhesion was evident, while galactose-inhibitable adhesion was not detected. Six potential arginine binding proteins were isolated from the membrane fraction of F. nucleatum ATCC 23726 and identified via mass spectroscopy as members of the outer membrane family of proteins in F. nucleatum. Inactivation of the genes encoding these six candidates for arginine-inhibitable adhesion and two additional homologues revealed that only a mutant derivative carrying an insertion in Fn1526 (now designated as radD) demonstrated significantly decreased co-aggregation with representatives of the Gram-positive “early oral colonizers”. Lack of the 350 kDa outer membrane protein encoded by radD resulted in the failure to form the extensive structured biofilm observed with the parent strain when grown in the presence of Streptococcus sanguinis ATCC 10556. These findings indicate that radD is responsible for arginine-inhibitable adherence of F. nucleatum and provides definitive molecular evidence that F. nucleatum adhesins play a vital role in inter-species adherence and multispecies biofilm formation. PMID:19007407

  3. One-step purification and porin transport activity of the major outer membrane proteins P2 from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis.

    PubMed

    Kattner, Christof; Pfennig, Sabrina; Massari, Paola; Tanabe, Mikio

    2015-03-01

    Bacterial porins are major outer membrane proteins that function as essential solute transporters between the bacteria and the extracellular environment. Structural features of porins are also recognized by eukaryotic cell receptors involved in innate and adaptive immunity. To better investigate the function of porins, proper refolding is necessary following purification from inclusion bodies [1, 2]. Using a single-step size exclusion chromatographic method, we have purified three major porins from pathogenic bacteria, the OmpP2 (P2) from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis, at high yield and report their unique solute transport activity with size exclusion limit. Furthermore, we have optimized their purification method and achieved improvement of their thermostability for facilitating functional and structural analyses.

  4. Intra- and Interspecies Regulation of Gene Expression by Actinobacillus actinomycetemcomitans LuxS

    PubMed Central

    Fong, Karen P.; Chung, Whasun O.; Lamont, Richard J.; Demuth, Donald R.

    2001-01-01

    The cell density-dependent control of gene expression is employed by many bacteria for regulating a variety of physiological functions, including the generation of bioluminescence, sporulation, formation of biofilms, and the expression of virulence factors. Although periodontal organisms do not appear to secrete acyl-homoserine lactone signals, several species, e.g., Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, have recently been shown to secrete a signal related to the autoinducer II (AI-2) of the signal system 2 pathway in Vibrio harveyi. Here, we report that the periodontal pathogen Actinobacillus actinomycetemcomitans expresses a homolog of V. harveyi luxS and secretes an AI-2-like signal. Cell-free conditioned medium from A. actinomycetemcomitans or from a recombinant Escherichia coli strain (E. coli AIS) expressing A. actinomycetemcomitans luxS induced luminescence in V. harveyi BB170 >200-fold over controls. AI-2 levels peaked in mid-exponential-phase cultures of A. actinomycetemcomitans and were significantly reduced in late-log- and stationary-phase cultures. Incubation of early-log-phase A. actinomycetemcomitans cells with conditioned medium from A. actinomycetemcomitans or from E. coli AIS resulted in a threefold induction of leukotoxic activity and a concomitant increase in leukotoxin polypeptide. In contrast, no increase in leukotoxin expression occurred when cells were exposed to sterile medium or to conditioned broth from E. coli AIS−, a recombinant strain in which luxS was insertionally inactivated. A. actinomycetemcomitans AI-2 also induced expression of afuA, encoding a periplasmic iron transport protein, approximately eightfold, suggesting that LuxS-dependent signaling may play a role in the regulation of iron acquisition by A. actinomycetemcomitans. Finally, A. actinomycetemcomitans AI-2 added in trans complemented a luxS knockout mutation in P. gingivalis by modulating the expression of the lux

  5. The structure of the LPS O-chain of Fusobacterium nucleatum strain 25586 containing two novel monosaccharides, 2-acetamido-2,6-dideoxy-l-altrose and a 5-acetimidoylamino-3,5,9-trideoxy-gluco-non-2-ulosonic acid.

    PubMed

    Vinogradov, Evgeny; St Michael, Frank; Cox, Andrew D

    2017-02-22

    Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. Recently, it has been gaining attention as a potential causative agent for colorectal cancer and is strongly linked with pregnancy complications including pre-term and still births. Little is known about the virulence factors of this organism, and thus we have initiated studies to examine the bacterium's surface glycochemistry. We isolated lipopolysaccharide (LPS) from F. nucleatum strain 25586 and purified and performed structural analysis on the O-antigen polysaccharide. The polysaccharide contained two novel sugars, 2-acetamido-2,6-dideoxy-l-altrose (l-6dAltNAc) and a 5-acetimidoylamino-3,5,9-trideoxy-gluco-non-2-ulosonic acid (Non5Am), which was tentatively assigned the l-glycero-l-gluco configuration. The polysaccharide was found to have a trisaccharide repeating unit, which is phosphorylated with phosphocholine (PCho), and the following structure was established: -[-4-β-Nonp5Am-4-α-l-6dAltpNAc3PCho-3-β-d-QuipNAc-]- We propose the trivial name 'fusaminic acid' for the novel nonulosonic acid. It is the first occurrence of a 9-deoxynonulosonic acid with a hydroxyl group at C-7, which is occupied by an amino group in all monosaccharides of this class described so far.

  6. Reaction of Human Sera from Juvenile Periodontitis, Rapidly Progressive Periodontitis, and Adult Periodontitis Patients with Selected Periodontopathogens,

    DTIC Science & Technology

    1984-11-13

    Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans (Y-4) serving as antigens. Increased i ii ~. DD I 1473 EDITION OF I NOV 6 , IS OBSOLETE...Bacteroides ging1valis, Capnocytophaga (Bacteroides) ochraceus, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans (Y-4) serving as...thio-dependent collagenolytic activity has been identified in the culture filtrate of B. gingivalis. 3’ Actinobacillus actinomycetemcomitans (Aa) has

  7. Factors affecting antimicrobial susceptibility of Fusobacterium species.

    PubMed Central

    Rowland, M D; Del Bene, V E; Lewis, J W

    1987-01-01

    Fifteen clinical isolates of Fusobacterium species were studied to determine their quality of growth on five agar media, their susceptibility to penicillin, cephalothin, cefoxitin, and cefotaxime, the inoculum effect, and the presence of L forms and beta-lactamase. Wilkins-Chalgren agar supported confluent growth best, but Fusobacterium nucleatum exhibited poor growth on all agar media. Most isolates exhibited poor reproducibility of MIC results with repeated agar dilution testing. However, most isolates were susceptible to all antibiotics at the breakpoint concentrations. No inoculum effect was observed, but preparation of an inoculum at a 0.5 McFarland nephelometric standard produced a lower than expected number of CFU (10(6) CFU) in some isolates. L forms were frequently seen. No beta-lactamase was found. The variability in MICs seen with beta-lactam antibiotics was not found when clindamycin was tested. MIC studies with Fusobacterium spp. may be complicated by poor growth on agar media, poor reproducibility, and small inoculum size. PMID:3494743

  8. 5S ribosomal ribonucleic acid sequences in Bacteroides and Fusobacterium: evolutionary relationships within these genera and among eubacteria in general

    NASA Technical Reports Server (NTRS)

    Van den Eynde, H.; De Baere, R.; Shah, H. N.; Gharbia, S. E.; Fox, G. E.; Michalik, J.; Van de Peer, Y.; De Wachter, R.

    1989-01-01

    The 5S ribosomal ribonucleic acid (rRNA) sequences were determined for Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides capillosus, Bacteroides veroralis, Porphyromonas gingivalis, Anaerorhabdus furcosus, Fusobacterium nucleatum, Fusobacterium mortiferum, and Fusobacterium varium. A dendrogram constructed by a clustering algorithm from these sequences, which were aligned with all other hitherto known eubacterial 5S rRNA sequences, showed differences as well as similarities with respect to results derived from 16S rRNA analyses. In the 5S rRNA dendrogram, Bacteroides clustered together with Cytophaga and Fusobacterium, as in 16S rRNA analyses. Intraphylum relationships deduced from 5S rRNAs suggested that Bacteroides is specifically related to Cytophaga rather than to Fusobacterium, as was suggested by 16S rRNA analyses. Previous taxonomic considerations concerning the genus Bacteroides, based on biochemical and physiological data, were confirmed by the 5S rRNA sequence analysis.

  9. Fusobacterium canifelinum sp. nov., from the oral cavity of cats and dogs.

    PubMed

    Conrads, Georg; Citron, Diane M; Mutters, Reinier; Jang, Spencer; Goldstein, Ellie J C

    2004-08-01

    Fourteen strains of Gram-negative, anaerobic, fluoroquinolone-resistant, non-sporulating rods were isolated from various infections in cats and dogs, as well as from wounds in humans after cat- or dog-bites. These strains were characterized by sequencing of the 16S-23S rDNA internal transcribed spacer (ITS) regions, 16S rDNA, DNA-DNA hybridization, phylogenetic analysis, and phenotypic tests. The results indicate that the novel strains belong to a distinct species, closely related to Fusobacterium nucleatum. The species Fusobacterium canifelinum sp. nov. is proposed, with strain ATCC BAA 689T as the type strain.

  10. Pyogenic liver abscess caused by Fusobacterium in a 21-year-old immunocompetent male

    PubMed Central

    Ahmed, Zohair; Bansal, Saurabh K; Dhillon, Sonu

    2015-01-01

    A 21-year-old male with no significant past medical history, presented with right upper quadrant (RUQ) abdominal pain along with fevers and chills. Lab work revealed leukocytosis, anemia, and slightly elevated alkaline phosphatase. Viral serology for hepatitis B virus, hepatitis C virus, and human immunodeficiency virus were negative and he was immunocompetent. Computed tomography imaging revealed hepatic abscesses, the largest measuring 9.5 cm. Empiric antibiotics were started and percutaneous drains were placed in the abscesses. Anaerobic cultures from the abscesses grew Fusobacterium nucleatum. This is a gram negative anaerobic bacteria; a normal flora of the oral cavity. Fusobacterium is most commonly seen in Lemiere’s disease, which is translocation of oral bacteria to the internal jugular vein causing a thrombophlebitis and subsequent spread of abscesses. Our patient did not have Lemiere’s, and is the first case described of fusobacterium pyogenic liver abscess in a young immunocompetent male with good oral hygiene. This case was complicated by sepsis, empyema, and subsequent abscesses located outside the liver. These abscesses’ have the propensity to flare abruptly and can be fatal. This case not only illustrates fusobacterium as a rare entity for pyogenic liver abscess, but also the need for urgent diagnosis and treatment. It is incumbent on physicians to diagnose and drain any suspicious hepatic lesions. While uncommon, such infections may develop without any overt source and can progress rapidly. Prompt drainage with antibiotic therapy remains the cornerstone of therapy. PMID:25834342

  11. Pyogenic liver abscess caused by Fusobacterium in a 21-year-old immunocompetent male.

    PubMed

    Ahmed, Zohair; Bansal, Saurabh K; Dhillon, Sonu

    2015-03-28

    A 21-year-old male with no significant past medical history, presented with right upper quadrant (RUQ) abdominal pain along with fevers and chills. Lab work revealed leukocytosis, anemia, and slightly elevated alkaline phosphatase. Viral serology for hepatitis B virus, hepatitis C virus, and human immunodeficiency virus were negative and he was immunocompetent. Computed tomography imaging revealed hepatic abscesses, the largest measuring 9.5 cm. Empiric antibiotics were started and percutaneous drains were placed in the abscesses. Anaerobic cultures from the abscesses grew Fusobacterium nucleatum. This is a gram negative anaerobic bacteria; a normal flora of the oral cavity. Fusobacterium is most commonly seen in Lemiere's disease, which is translocation of oral bacteria to the internal jugular vein causing a thrombophlebitis and subsequent spread of abscesses. Our patient did not have Lemiere's, and is the first case described of fusobacterium pyogenic liver abscess in a young immunocompetent male with good oral hygiene. This case was complicated by sepsis, empyema, and subsequent abscesses located outside the liver. These abscesses' have the propensity to flare abruptly and can be fatal. This case not only illustrates fusobacterium as a rare entity for pyogenic liver abscess, but also the need for urgent diagnosis and treatment. It is incumbent on physicians to diagnose and drain any suspicious hepatic lesions. While uncommon, such infections may develop without any overt source and can progress rapidly. Prompt drainage with antibiotic therapy remains the cornerstone of therapy.

  12. Actinobacillus actinomycetemcomitans endocarditis.

    PubMed Central

    Affias, S.; West, A.; Stewart, J. W.; Haldane, E. V.

    1978-01-01

    Two patients had infective endocarditis due to Actinobacillus actinomycetemcomitans. One, a 52-year-old woman with a prosthetic aortic valve, was successfully treated with carbenicillin and gentamicin. The other, a 47-year old man with calcific aortic valve disease, required emergency valvectomy and prosthetic valve replacement and responded to a combination of penicillin and gentamicin. PMID:647545

  13. 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

  14. Isolation of a Novel Bacteriophage Specific for the Periodontal Pathogen Fusobacterium nucleatum▿

    PubMed Central

    Machuca, Pamela; Daille, Leslie; Vinés, Enrique; Berrocal, Liliana; Bittner, Mauricio

    2010-01-01

    Fusobacterium nucleatum is a periodontal pathogen that has been directly associated with the development and progression of periodontal disease, a widespread pathology that affects the support tissues of the tooth. We isolated a new bacteriophage (FnpΦ02) that specifically infects this bacterium. Transmission electron microscopy showed that the virion is composed of an icosahedral head and a segmented tail. The size of the phage genome was estimated to be approximately 59 kbp of double-stranded DNA. The morphological features and the genetic characteristics suggest that FnpΦ02 is part of the Siphoviridae family. Using one-step growth and adsorption experiments, the latent period, burst size, and adsorption rate were estimated to be 15 h, 100 infectious units per cell, and 7.5 × 10−10 ml min−1, respectively. A small fragment of phage DNA was cloned and sequenced, showing 93% nucleotide identity with the phage PA6 of Propionibacterium acnes and amino acid identity with fragments of two proteins (Gp3 and Gp4) of this phage. To our knowledge, FnpΦ02 is the first phage described to infect Fusobacterium nucleatum and provides the base for future exploration of phages in the control of periodontal disease. PMID:20851973

  15. 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.

  16. Evolutionary Divergence of Aggregatibacter actinomycetemcomitans.

    PubMed

    Kittichotirat, W; Bumgarner, R E; Chen, C

    2016-01-01

    Gram-negative facultative Aggregatibacter actinomycetemcomitans is an oral pathogen associated with periodontitis. The genetic heterogeneity among A. actinomycetemcomitans strains has been long recognized. This study provides a comprehensive genomic analysis of A. actinomycetemcomitans and the closely related nonpathogenic Aggregatibacter aphrophilus. Whole genome sequencing by Illumina MiSeq platform was performed for 31 A. actinomycetemcomitans and 2 A. aphrophilus strains. Sequence similarity analysis shows a total of 3,220 unique genes across the 2 species, where 1,550 are core genes present in all genomes and 1,670 are variable genes (accessory genes) missing in at least 1 genome. Phylogenetic analysis based on 397 concatenated core genes distinguished A. aphrophilus and A. actinomycetemcomitans. The latter was in turn divided into 5 clades: clade b (serotype b), clade c (serotype c), clade e/f (serotypes e and f), clade a/d (serotypes a and d), and clade e' (serotype e strains). Accessory genes accounted for 14.1% to 23.2% of the A. actinomycetemcomitans genomes, with a majority belonging to the category of poorly characterized by Cluster of Orthologous Groups classification. These accessory genes were often organized into genomic islands (n = 387) with base composition biases, suggesting their acquisitions via horizontal gene transfer. There was a greater degree of similarity in gene content and genomic islands among strains within clades than between clades. Strains of clade e' isolated from human were found to be missing the genomic island that carries genes encoding cytolethal distending toxins. Taken together, the results suggest a pattern of sequential divergence, starting from the separation of A. aphrophilus and A. actinomycetemcomitans through gain and loss of genes and ending with the divergence of the latter species into distinct clades and serotypes. With differing constellations of genes, the A. actinomycetemcomitans clades may have evolved

  17. A bacteriocin of Actinobacillus actinomycetemcomitans.

    PubMed Central

    Hammond, B F; Lillard, S E; Stevens, R H

    1987-01-01

    An inhibitory factor from Actinobacillus actinomycetemcomitans Y4 was isolated, and its properties indicated that it was a bacteriocin (actinobacillicin). The bacteriocin was active against Streptococcus sanguis strains, Streptococcus uberis (FDC1), and Actinomyces viscosus T14 as well as other strains of A. actinomycetemcomitans, but not against other crevicular bacteria, including other streptococci and actinomycetes. The activity of this bacteriocin was inhibited by pronase, trypsin, and heat (45 min at 56 degrees C) but not by DNase, RNase, phospholipase, exposure to UV light, or low pH (1.0 to 6.5). Although actinobacillicin markedly inhibited glycolysis in S. sanguis, the primary mechanism of its bactericidal action appears to be alterations in cell permeability, with the resultant leakage of RNA, DNA, and other essential intracellular macromolecules. These findings provide an ecologic explanation for the reciprocal growth relationship between A. actinomycetemcomitans and S. sanguis/Actinomyces viscosus observed in localized juvenile periodontitis. Images PMID:3818090

  18. Severe forefoot infection complicated by Fusobacterium russii.

    PubMed

    Ullrich, Elisabeth; Grisold, Andrea J; Feierl, Gebhard; Lumenta, David B; Leitner, Eva

    2016-12-01

    We present the first case of a complicated foot infection caused by Fusobacterium russii in Austria. F. russii is highly associated with mammals such as cats and dogs. Our case underlines the difficulties in isolation and identification of anaerobes and the pitfalls in antimicrobial treatment of polymicrobial infections.

  19. Fusobacterium and Enterobacteriaceae: Important players for CRC?

    PubMed Central

    Allen-Vercoe, Emma; Jobin, Christian

    2014-01-01

    The gut microbiota plays an essential role in regulating intestinal homeostasis through its capacity to modulate various biological activities ranging from barrier, immunity and metabolic function. Not surprisingly, microbial dysbiosis is associated with numerous intestinal disorders including inflammatory bowel diseases (IBD) and colorectal cancer (CRC). In this piece, we will review recent evidence that gut microbial dysbiosis can influence intestinal disease, including colitis and CRC. We will discuss the biological events implicated in the development of microbial dysbiosis and the emergence of CRC-associated microorganisms, focusing on E.coli and F. nucleatum. Finally, the mechanisms by which E.coli and F. nucleatum exert potentially carcinogenic effects on the host will be reviewed. PMID:24972311

  20. In vitro antimicrobial susceptibility of Actinobacillus actinomycetemcomitans.

    PubMed Central

    Slots, J; Evans, R T; Lobbins, P M; Genco, R J

    1980-01-01

    The agar dilution technique was used for determination of the antibiotic susceptibilities of 57 oral isolates and 2 nonoral isolates of Actinobacillus actinomycetemcomitans. Tetracycline, minocycline, and chloramphenicol inhibited more than 96% of the strains tested at a concentration of less than or equal to 2 micrograms/ml; 89% of the strains were inhibited by 2 micrograms of carbenicillin per ml. The other antimicrobial agents tested were less active. Approximately 10% of the A. actinomycetemcomitans strains were resistant to ampicillin, erythromycin, and penicillin G at concentrations of 32 to 64 micrograms/ml. These data suggest that tetracycline and minocycline may be valuable drugs in the treatment of A. actinomycetemcomitans infections. PMID:6903116

  1. Selective medium for isolation of Actinobacillus actinomycetemcomitans.

    PubMed Central

    Slots, J

    1982-01-01

    A selective medium, TSBV (tryptic soy-serum-bacitracin-vancomycin) agar, was developed for the isolation of Actinobacillus actinomycetemcomitans, TSBV agar contained (per liter) 40 g of tryptic soy agar, 1 g of yeast extract, 100 ml of horse serum. 75 mg of bacitracin, and 5 mg of vancomycin. The TSBV medium suppressed most oral species and permitted significantly higher recovery of A. actinomycetemcomitans than nonselective blood agar medium. The distinct colonial morphology and positive catalase reaction of A. actinomycetemcomitans easily distinguished this bacterium from Haemophilus aphrophilus, Capnocytophaga species, and a few other contaminating organisms. With the TSBV medium, even modestly equipped laboratories will be able to isolate and identify A. actinomycetemcomitans from clinical specimens. Images PMID:7068837

  2. Immunosuppressive properties of Actinobacillus actinomycetemcomitans leukotoxin.

    PubMed Central

    Rabie, G; Lally, E T; Shenker, B J

    1988-01-01

    Actinobacillus actinomycetemcomitans produces a leukotoxin that kills human polymorphonuclear cells (PMNs) and monocytes but not lymphocytes. In this study, we examined A. actinomycetemcomitans leukotoxin for its ability to alter human peripheral blood lymphocyte (HPBL) responsiveness. After a 90-min exposure to the leukotoxin, all monocytes were killed and HPBL responsiveness to mitogens and antigens was significantly inhibited. The ability of the leukotoxin to inhibit HPBL responses was not surprising, since monocytes and macrophages are required for many lymphocyte functions. However, we were unable to totally restore HPBL responsiveness when adherent autologous monocytes were added back to cultures of leukotoxin-treated lymphocytes. These studies demonstrate that A. actinomycetemcomitans leukotoxin may also exert nonlethal effects directly on lymphocytes. Furthermore, impaired lymphocyte function did not appear to be the result of indirect effects of products released by dying monocytes. Although it is not clear how A. actinomycetemcomitans acts to cause disease, several investigators have proposed that impaired host defenses may play a pivotal role. Several studies have demonstrated defects in PMN, monocyte, and lymphocyte function in patients with periodontal disease. These findings, along with the data presented in this paper, support the hypothesis that patients who harbor A. actinomycetemcomitans could suffer from local or systemic immune suppression. The effects of this suppression may be to enhance the pathogenicity of A. actinomycetemcomitans itself or that of some other opportunistic organism. PMID:3335399

  3. Identification and characterization of the major cell envelope proteins of oral strains of Actinobacillus actinomycetemcomitans.

    PubMed Central

    Di Rienzo, J M; Spieler, E L

    1983-01-01

    The major cell envelope protein compositions of seven Actinobacillus actinomycetemcomitans strains of human origin were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major envelope polypeptides were homogeneous, in relation to molecular weight, in all of the strains that were examined. The characterization of the five major proteins, designated Env1 through Env5, in the leukotoxic strain Y4 revealed that proteins Env2 to -5 may reside in the outer membrane as suggested by differential detergent extractions and 125I-labeling experiments. The proteins did not demonstrate covalent or ionic interactions with the peptidoglycan; however, one protein, Env2, displayed heat-modifiable properties, having apparent molecular weights of 32,000 and 45,000 when heated in sodium dodecyl sulfate at 50 and 100 degrees C, respectively. The protein composition of the extracellular "bleb" material, normally released by strain Y4, was determined, and proteins Env1 to -4 were the predominant protein species found. A comparison of the cell envelope proteins of strain Y4 with those of other members of the human oral flora, including species within the genera Capnocytophaga, Bacteroides, and Fusobacterium, revealed distinct differences on the basis of molecular size and heat-modifiable properties. However, the membrane proteins of Haemophilus aphrophilus showed a remarkable degree of homology with those of A. actinomycetemcomitans. Images PMID:6401694

  4. 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

  5. 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.

  6. 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

  7. Transformation of Actinobacillus actinomycetemcomitans by electroporation, utilizing constructed shuttle plasmids.

    PubMed Central

    Sreenivasan, P K; LeBlanc, D J; Lee, L N; Fives-Taylor, P

    1991-01-01

    Actinobacillus actinomycetemcomitans, a periodontal pathogen, has been strongly implicated in human periodontal disease. Advances in the molecular analysis of A. actinomycetemcomitans virulence factors have been limited due to the unavailability of systems for genetic transfer, transposon mutagenesis, and gene complementation. Slow progress can be traced almost exclusively to the lack of gene vector systems and methods for the introduction of DNA into A. actinomycetemcomitans. An electrotransformation system that allowed at least five strains of A. actinomycetemcomitans to be transformed with stable shuttle plasmids which efficiently replicated in both Escherichia coli and A. actinomycetemcomitans was developed. One plasmid, a potential shuttle vector designated pDL282, is 5.7 kb in size, has several unique restriction enzyme sites, and codes for resistance to spectinomycin and ampicillin. E. coli and A. actinomycetemcomitans were transformed with equal efficiencies of approximately 10(5) transformants per micrograms of DNA. Similar transformation efficiencies were obtained whether the plasmid DNA was isolated from A. actinomycetemcomitans or E. coli. In addition, frozen competent cells of A. actinomycetemcomitans yielded comparable efficiencies of transformation. Restriction enzyme analysis of pDL282 isolated after transformation confirmed the presence of intact donor plasmids. A plasmid isolated from A. pleuropneumoniae was also capable of transforming some isolates of A. actinomycetemcomitans, although generally at a lower frequency. The availability of these shuttle plasmids and an efficient transformation procedure should significantly facilitate the molecular analysis of virulence factors of A. actinomycetemcomitans. PMID:1937823

  8. Fusobacterium necrophorum Pharyngitis Complicated by Lemierre's Syndrome

    PubMed Central

    Nenci, Gabriele; Boccadori, Costanza; Mangani, Valerio; Oggioni, Roberto

    2016-01-01

    We report the case of an 18-year-old woman who was referred to our outpatient clinic because of a 2-week history of sore throat, high fever, and neck tenderness unresponsive to a 7-day amoxicillin/clavulanic acid course. Infectious mononucleosis was initially suspected, but an extremely high value of procalcitonin and clinical deterioration suggested a bacterial sepsis, prompting the patient admission to our internal medicine ward. Blood cultures were positive for Fusobacterium necrophorum. CT scan detected a parapharyngeal abscess, a right internal jugular vein thrombosis, and multiple bilateral lung abscesses, suggesting the diagnosis of Lemierre's syndrome. The patient was treated with a 2-week course of metronidazole and meropenem with a gradual clinical recovery. She was thereafter discharged home with metronidazole and amoxicillin/clavulanic acid for 14 days and a 3-month course of enoxaparin, experiencing an uneventful recovery. The present case highlights the importance of taking into consideration the Lemierre's syndrome whenever a pharyngotonsillitis has a severe and unusual course. PMID:28050168

  9. Fusobacterium necrophorum Pharyngitis Complicated by Lemierre's Syndrome.

    PubMed

    Faraone, Antonio; Fortini, Alberto; Nenci, Gabriele; Boccadori, Costanza; Mangani, Valerio; Oggioni, Roberto

    2016-01-01

    We report the case of an 18-year-old woman who was referred to our outpatient clinic because of a 2-week history of sore throat, high fever, and neck tenderness unresponsive to a 7-day amoxicillin/clavulanic acid course. Infectious mononucleosis was initially suspected, but an extremely high value of procalcitonin and clinical deterioration suggested a bacterial sepsis, prompting the patient admission to our internal medicine ward. Blood cultures were positive for Fusobacterium necrophorum. CT scan detected a parapharyngeal abscess, a right internal jugular vein thrombosis, and multiple bilateral lung abscesses, suggesting the diagnosis of Lemierre's syndrome. The patient was treated with a 2-week course of metronidazole and meropenem with a gradual clinical recovery. She was thereafter discharged home with metronidazole and amoxicillin/clavulanic acid for 14 days and a 3-month course of enoxaparin, experiencing an uneventful recovery. The present case highlights the importance of taking into consideration the Lemierre's syndrome whenever a pharyngotonsillitis has a severe and unusual course.

  10. Monoclonal antibodies to leukotoxin of Actinobacillus actinomycetemcomitans.

    PubMed Central

    DiRienzo, J M; Tsai, C C; Shenker, B J; Taichman, N S; Lally, E T

    1985-01-01

    Hybridoma cell lines which produce monoclonal antibodies to a leukotoxin from Actinobacillus actinomycetemcomitans were prepared. The monoclonal antibodies were selected for their ability to neutralize the cytotoxic activity of the leukotoxin and recognize the toxin on nitrocellulose blots. The antibodies belonged to either the immunoglobulin G1 (IgG1) or IgG2 subclass and differed in their ability to bind to the leukotoxin on nitrocellulose blots. However, only slight differences in neutralization titers were observed. Use of the monoclonal antibodies revealed that polymyxin B-extracted or osmotic shock-released leukotoxin could be separated into several high-molecular-weight polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis with the monoclonal antibodies also demonstrated that the leukotoxin was present in eight oral strains of A. actinomycetemcomitans that had been previously classified by a biological assay as leukotoxic. The availability of these monoclonal antibodies should facilitate and expand studies concerning the role of the leukotoxin in the pathogenicity of A. actinomycetemcomitans. Images PMID:3965404

  11. Complete Genome Sequence of Aggregatibacter actinomycetemcomitans Strain IDH781

    PubMed Central

    May, Anthony C.; Ehrlich, Rachel L.; Balashov, Sergey; Ehrlich, Garth D.; Shanmugam, Mayilvahanan; Fine, Daniel H.; Ramasubbu, Narayanan

    2016-01-01

    We report here the complete genomic sequence and methylome of Aggregatibacter actinomycetemcomitans strain IDH781. This rough strain is used extensively as a model organism to characterize localized aggressive periodontitis pathogenesis, the basic biology and oral cavity colonization of A. actinomycetemcomitans, and its interactions with other members of the oral microbiome. PMID:27834722

  12. Requirements for invasion of epithelial cells by Actinobacillus actinomycetemcomitans.

    PubMed Central

    Sreenivasan, P K; Meyer, D H; Fives-Taylor, P M

    1993-01-01

    Actinobacillus actinomycetemcomitans, an oral bacterium implicated in human periodontal disease, was recently demonstrated to invade cultured epithelial cells (D. H. Meyer, P. K. Sreenivasan, and P. M. Fives-Taylor, Infect. Immun. 59:2719-2726, 1991). This report characterizes the requirements for invasion of KB cells by A. actinomycetemcomitans. The roles of bacterial and host factors were investigated by using selective agents that influence specific bacterial or host cell functions. Inhibition of bacterial protein synthesis decreased invasion, suggesting the absence of a preformed pool of proteins involved in A. actinomycetemcomitans invasion. Inhibition of bacterial and eukaryotic energy synthesis also decreased invasion, confirming that A. actinomycetemcomitans invasion is an active process. Bacterial adherence to KB cells was indicated by scanning electron microscopy of infected KB cells. Further, the addition of A. actinomycetemcomitans-specific serum to the bacterial inoculum reduced invasion substantially, suggesting a role for bacterial attachment in invasion. Many of the adherent bacteria invaded the epithelial cells under optimal conditions. Inhibitors of receptor-mediated endocytosis inhibited invasion by A. actinomycetemcomitans. Like that of many facultatively intracellular bacteria, A. actinomycetemcomitans invasion was not affected by eukaryotic endosomal acidification. These are the first published observations describing the requirements for epithelial cell invasion by a periodontopathogen. They demonstrate that A. actinomycetemcomitans utilizes a mechanism similar to those used by many but not all invasive bacteria to gain entry into eukaryotic cells. Images PMID:8454326

  13. Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in young Chinese adults.

    PubMed

    Mombelli, A; Gmür, R; Frey, J; Meyer, J; Zee, K Y; Tam, J O; Lo, E C; Di Rienzo, J; Lang, N P; Corbet, E F

    1998-08-01

    The aim of this study was to determine the presence or absence of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in young Chinese adults and to examine the A. actinomycetemcomitans isolates from positive subjects with regard to the serotype distribution, presence of the leukotoxin gene lktA and the promoter for the leukotoxin operon as well as the incidence of phage Aa phi 23. Sixty subjects, working in a knitting factory in the Province of Guangzhou, People's Republic of China, were investigated. Subgingival microbial samples were taken from both upper first molars. They were cultured both anaerobically and in 5% CO2. P. gingivalis was found in 33 subjects. On average, it constituted 7% of the total anaerobic cultivable counts. A. actinomycetemcomitans was detected in 37 subjects of which seven yielded counts > 10(5). Twenty-one subjects were positive for both organisms. A. actinomycetemcomitans serotype a was found in 9 subjects, serotype c was found in 23 and serotype e in 5. A. actinomycetemcomitans serotypes b and d were not detected in any subjects. Presence of the leukotoxin gene lktA was demonstrated for all A. actinomycetemcomitans isolates; however, none of the A. actinomycetemcomitans strains from the present study had a deletion in the promoter region of the leukotoxin operon. The results of this investigation show a high frequency of the putative periodontal pathogens P. gingivalis and A. actinomycetemcomitans and corroborate the concept that there is variation in virulence and pathogenic potential among isolates from different subjects.

  14. Les infections à Fusobacterium chez l’enfant

    PubMed Central

    Arane, Karen; Goldman, Ran D.

    2016-01-01

    Résumé Question Une otite moyenne aiguë chez un patient de 2 ans qui fréquente ma clinique a dégénéré en mastoïdite avec forte fièvre et les résultats de culture se sont révélés positifs pour le Fusobacterium. Que dois-je faire ensuite? Réponse Le Fusobacterium est un genre de bactéries anaérobies. Quoique les infections à Fusobacterium soient rares, elles peuvent devenir graves si elles ne sont pas traitées rapidement. Le traitement approprié est une antibiothérapie combinée associant une β-lactamine (pénicilline, céphalosporine) et un agent contre les microbes anaérobies (métronidazole, clindamycine). Il faut parfois une intervention chirurgicale pour la mastoïdite, comme le drainage d’abcès ou l’insertion d’un tube de ventilation. Le traitement tardif d’une infection causée par le Fusobacterium peut entraîner de sérieuses complications, dont le syndrome de Lemierre. Il faudrait une surveillance étroite de l’enfant en milieu hospitalier. PMID:27737993

  15. Association of Fusobacterium species in pancreatic cancer tissues with molecular features and prognosis.

    PubMed

    Mitsuhashi, Kei; Nosho, Katsuhiko; Sukawa, Yasutaka; Matsunaga, Yasutaka; Ito, Miki; Kurihara, Hiroyoshi; Kanno, Shinichi; Igarashi, Hisayoshi; Naito, Takafumi; Adachi, Yasushi; Tachibana, Mami; Tanuma, Tokuma; Maguchi, Hiroyuki; Shinohara, Toshiya; Hasegawa, Tadashi; Imamura, Masafumi; Kimura, Yasutoshi; Hirata, Koichi; Maruyama, Reo; Suzuki, Hiromu; Imai, Kohzoh; Yamamoto, Hiroyuki; Shinomura, Yasuhisa

    2015-03-30

    Recently, bacterial infection causing periodontal disease has attracted considerable attention as a risk factor for pancreatic cancer. Fusobacterium species is an oral bacterial group of the human microbiome. Some evidence suggests that Fusobacterium species promote colorectal cancer development; however, no previous studies have reported the association between Fusobacterium species and pancreatic cancer. Therefore, we examined whether Fusobacterium species exist in pancreatic cancer tissue. Using a database of 283 patients with pancreatic ductal adenocarcinoma (PDAC), we tested cancer tissue specimens for Fusobacterium species. We also tested the specimens for KRAS, NRAS, BRAF and PIK3CA mutations and measured microRNA-21 and microRNA-31. In addition, we assessed epigenetic alterations, including CpG island methylator phenotype (CIMP). Our data showed an 8.8% detection rate of Fusobacterium species in pancreatic cancers; however, tumor Fusobacterium status was not associated with any clinical and molecular features. In contrast, in multivariate Cox regression analysis, compared with the Fusobacterium species-negative group, we observed significantly higher cancer-specific mortality rates in the positive group (p = 0.023). In conclusion, Fusobacterium species were detected in pancreatic cancer tissue. Tumor Fusobacterium species status is independently associated with a worse prognosis of pancreatic cancer, suggesting that Fusobacterium species may be a prognostic biomarker of pancreatic cancer.

  16. Activation of rat B lymphocytes by Actinobacillus actinomycetemcomitans.

    PubMed Central

    Yoshie, H; Taubman, M A; Ebersole, J L; Olson, C L; Smith, D J; Pappo, J

    1985-01-01

    We examined the lymphoproliferative responses of cervical lymphocytes and splenocytes of homozygous (rnu/rnu) congenitally athymic nude and normal heterozygous (rnu/+) Rowett rats to whole cells of Actinobacillus actinomycetemcomitans, a suspected periodontal disease pathogen. Previously sensitized cells from immunized only, infected only, or immunized and infected, normal rats demonstrated proliferation in response to formalinized A. actinomycetemcomitans, but cells from nude rats did not proliferate. The maximum antigenic response was observed at day 5 of culture. A. actinomycetemcomitans caused cervical lymphocytes and splenocytes from untreated naive normal and nude rats to undergo increased DNA synthesis at day 2 of culture. Highly enriched nonsensitized spleen T cells prepared on a nylon wool column did not respond to A. actinomycetemcomitans, whereas enriched nonsensitized B cells proliferated. Differences in response were probably not attributable to contributions from macrophages in the T- or B-cell populations, since macrophage percentages were approximately the same in both preparations. T-cell reconstitution of nude rats with neonatal thymus cells from rnu/+rats resulted in partial recovery of T-cell function but had no effect on the mitogenic response to A. actinomycetemcomitans. It is suggested that the antigenic responses to A. actinomycetemcomitans are dependent on T cells and that A. actinomycetemcomitans cells have mitogenic activity for B cells. The potential importance of these findings in periodontal disease is discussed. PMID:3871196

  17. Electron microscopy of phages in serotypes of Actinobacillus actinomycetemcomitans.

    PubMed

    Olsen, I; Namork, E; Myhrvold, V

    1993-12-01

    Actinobacillus actinomycetemcomitans, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica and Pasteurella multocida strains were examined by transmission electron microscopy for the presence of bacteriophages. Phages were detected in serotype a (SUNY 75) and e (UOH 1705) and in the fresh clinical isolates UOH Q1243 and UOH Q1247 of A. actinomycetemcomitans. Phages were not found in serotype b, c and d strains of A. actinomycetemcomitans, in the fresh clinical isolate UOH Q1244 of this species or in old strains (including reference strains) of related species from the Actinobacillus-Haemophilus-Pasteurella group.

  18. The cell envelope proteome of Aggregatibacter actinomycetemcomitans.

    PubMed

    Smith, K P; Fields, J G; Voogt, R D; Deng, B; Lam, Y-W; Mintz, K P

    2015-04-01

    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 27% of the predicted open reading frames 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. A total of 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, whereas 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.

  19. 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.

  20. 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.

  1. AI-2 of Aggregatibacter actinomycetemcomitans inhibits Candida albicans biofilm formation.

    PubMed

    Bachtiar, Endang W; Bachtiar, Boy M; Jarosz, Lucja M; Amir, Lisa R; Sunarto, Hari; Ganin, Hadas; Meijler, Michael M; Krom, Bastiaan P

    2014-01-01

    Aggregatibacter actinomycetemcomitans, a Gram-negative bacterium, and Candida albicans, a polymorphic fungus, are both commensals of the oral cavity but both are opportunistic pathogens that can cause oral diseases. A. actinomycetemcomitans produces a quorum-sensing molecule called autoinducer-2 (AI-2), synthesized by LuxS, that plays an important role in expression of virulence factors, in intra- but also in interspecies communication. The aim of this study was to investigate the role of AI-2 based signaling in the interactions between C. albicans and A. actinomycetemcomitans. A. actinomycetemcomitans adhered to C. albicans and inhibited biofilm formation by means of a molecule that was secreted during growth. C. albicans biofilm formation increased significantly when co-cultured with A. actinomycetemcomitans luxS, lacking AI-2 production. Addition of wild-type-derived spent medium or synthetic AI-2 to spent medium of the luxS strain, restored inhibition of C. albicans biofilm formation to wild-type levels. Addition of synthetic AI-2 significantly inhibited hypha formation of C. albicans possibly explaining the inhibition of biofilm formation. AI-2 of A. actinomycetemcomitans is synthesized by LuxS, accumulates during growth and inhibits C. albicans hypha- and biofilm formation. Identifying the molecular mechanisms underlying the interaction between bacteria and fungi may provide important insight into the balance within complex oral microbial communities.

  2. Aggregatibacter Actinomycetemcomitans – A Tooth Killer?

    PubMed Central

    Ummer, Fajar; Dhivakar, C.P

    2014-01-01

    Strong evidence is available on Aggregatibacter actinomycetemcomitans (A.a) on its role as the causative agent of localised juvenile periodontitis (LJP), a disease characterised by rapid destruction of the tooth-supporting tissues. This organism possesses a large number of virulence factors with a wide range of activities which enable it to colonise the oral cavity, invade periodontal tissues, evade host defences, initiate connective tissue destruction and interfere with tissue repair. Adhesion to epithelial and tooth surfaces is dependent on the presence of surface proteins and structures such as microvesicles and fimbriae. Invasion has been demonstrated in vivo and in vitro. The organism has a number of means of evading host defences which include: (i) production of leukotoxin; (ii) producing immunosuppressive factors; (iv) secreting proteases capable of cleaving IgG; and (v) producing Fc-binding. PMID:25302290

  3. Evidence that extracellular components function in adherence of Actinobacillus actinomycetemcomitans to epithelial cells.

    PubMed Central

    Meyer, D H; Fives-Taylor, P M

    1993-01-01

    Extracellular microvesicles and a highly proteinaceous polymer associated with a leukotoxin-producing strain, Actinobacillus actinomycetemcomitans SUNY 75, were shown to increase adherence of other weakly adherent A. actinomycetemcomitans strains to KB epithelial cells. Images PMID:8406899

  4. Cloning and expression of the leukotoxin gene from Actinobacillus actinomycetemcomitans.

    PubMed Central

    Kolodrubetz, D; Dailey, T; Ebersole, J; Kraig, E

    1989-01-01

    The leukotoxin produced by Actinobacillus actinomycetemcomitans has been implicated in the etiology of juvenile periodontitis. To initiate a genetic analysis of the role of this protein in disease, we have cloned the leukotoxin gene in Escherichia coli. Recombinant colonies carrying toxin gene sequences were isolated by screening a genomic A. actinomycetemcomitans library with a DNA probe for the leukotoxin gene from a related bacterium, Pasteurella haemolytica. To demonstrate that the cloned A. actinomycetemcomitans DNA contained a functional leukotoxin gene, protein extracts of E. coli containing the A. actinomycetemcomitans clone were tested directly for leukotoxic activity against human cell lines in chromium release assays. A construct containing the entire cloned region produced a functional toxin. No cytotoxicity was seen when extracts from cells containing plasmids with deletions in the putative coding region were used. Furthermore, the toxin produced by the cloned gene has the same target cell specificity as the leukotoxin extracted directly from A. actinomycetemcomitans. These results indicate that sequences encoding a functional leukotoxin have been cloned and are expressed in E. coli. Southern blot analysis of DNA from leukotoxin-producing (Lkt+) and non-leukotoxin-producing (Lkt-) strains indicated that the Lkt- strain also contained a copy of the gene. Images PMID:2707855

  5. A Rare Case of Fusobacterium Necrophorum Liver Abscesses

    PubMed Central

    Hannoodi, Faris; Sabbagh, Hussam; Kulairi, Zain; Kumar, Sarwan

    2017-01-01

    Liver abscesses are an uncommon disease that can present with vague symptoms. Fusobacterium necrophorum causing liver abscesses is a rare condition and only a few cases have been reported. An 88-year-old female presented to her primary care physician with one week of fevers, night sweats, chills, fatigue and vague right upper quadrant abdominal pain. She denied nausea, vomiting, constipation, diarrhea and unintentional weight loss. A computed tomography scan of the abdomen showed two liver abscesses in the right lobe as well as extensive diverticulosis. Percutaneous drainage was performed and draining catheters were placed in the abscesses. Culture of the abscess fluid grew Fusobacterium necrophorum. She was treated with ceftriaxone and metronidazole as per sensitivities. Rare cases of F. necrophorum hepatic abscesses have been published. The source of infection described in reported cases included hematogenous spread from dental caries/peritonsillar abscess and those involving the gastrointestinal tract resulting from inflammation of the bowel wall or from inflamed diverticuli via the portal circulation. In one study, thirteen cases of liver abscess due to F. necrophorum were studied, and two of these cases had diverticular disease without inflammation. PMID:28243433

  6. [Role of Actinobacillus actinomycetemcomitans in human infection].

    PubMed

    Giglio, C; Aránguiz, V; Giglio, M S; Fernández, A

    1990-04-01

    Actinobacillus actinomycetemcomitans (AA), is a cocobacillus thin and small, non motile, uncapsulate and capnophilic. AA, is: one of the species encountered in the mouth's comensal flora being able to be isolated in gingival crevices culture and oral mucosa in a 20% of the healthy population. An important number of pathogenic factors make it well equipped, to protect itself from host's defense mechanisms, and to destroy the periodontal tissue. Between the most important we find lipopolisacarides and leucotoxines which promote tisular invasion and destructive qualities of this microorganism. Since 1912, there are numerous reports of infectious process associated to it, between which we find: endocarditis in native and prothesic valve, soft tissues abscess, pneumonia, brain's abscess, urethritis, vertebral osteomielitis, thyroid's abscess, pericarditis and periodontal juvenile illness, being this one in which its isolation is more frequent. In vitro, AA is very susceptible to tetracicline. This antibiotic reaches high concentrations in gingival crevices, has significant affinity to the alveolar bone and contributes to protect the collagen. These special feature make them the election drug in periodontal disease produced by this microorganism.

  7. Update on Actinobacillus Actinomycetemcomitans and Porphyromonas gingivalis in human periodontal disease.

    PubMed

    Slots, J

    1999-10-01

    Actinobacillus actinomycetemcomitans is an important pathogen of periodontitis in young individuals. Porphyromonas gingivalis is a major pathogen of severe adult periodontitis. A. actinomycetemcomitans and P. gingivalis can be transmitted from family member to family member and may cause periodontitis in the recipient individual. In the USA, A. actinomycetemcomitans occurs more frequently in Hispanics and Asians than in Caucasians. P. gingivalis is more common in Hispanics, Asians and Blacks than in Caucasians. A. actinomycetemcomitans and P. gingivalis strains differ in genotype, serotype, toxin and enzyme production, and cellular invasiveness. Variation in virulence may help explain differing clinical outcomes of periodontal A. actinomycetemcomitans and P. gingivalis infections. A. actinomycetemcomitans and P. gingivalis cannot be eradicated from the great majority of deep periodontal pockets by mechanical debridement alone. A. actinomycetemcomitans may be removed from subgingival sites by adjunctive systemic amoxicillin-metronidazole or other appropriate antibiotic therapies. Subgingival eradication of P. gingivalis may require periodontal surgery as well as antibiotic therapy.

  8. Association between periodontal condition and subgingival microbiota in women during pregnancy: a longitudinal study

    PubMed Central

    BORGO, Priscila Viola; RODRIGUES, Viviane Aparecida Arenas; FEITOSA, Alfredo Carlos Rodrigues; XAVIER, Karla Correa Barcelos; AVILA-CAMPOS, Mario Julio

    2014-01-01

    Objectivo In this study, the gingival conditions and the quantitative detection for Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia in pregnant women were determined. Material and Methods Quantitative determinations of periodontal bacteria by using a SyBr green system in women during pregnancy were performed. Women at the 2nd and 3rd trimesters of pregnancy and non-pregnant women were included in this study. A. actinomycetemcomitans was observed in high numbers in women at the 2nd and 3rd trimesters of pregnancy with a significant difference (p<0.05). F. nucleatum and P. intermedia were also observed in high levels. Results and Conclusion Our results show that pregnant women are more susceptible to gingivitis, and the presence of A. actinomycetemcomitans in subgingival biofilm might be taken into account for the treatment of periodontal disease. PMID:25591021

  9. Adhesion of Actinobacillus actinomycetemcomitans to a human oral cell line.

    PubMed Central

    Mintz, K P; Fives-Taylor, P M

    1994-01-01

    Two quantitative, rapid assays were developed to study the adhesion of Actinobacillus actinomycetemcomitans, an oral bacterium associated with periodontal disease, to human epithelial cells. The human oral carcinoma cell line KB was grown in microtiter plates, and adherent bacteria were detected by an enzyme-linked immunosorbent assay with purified anti-A. actinomycetemcomitans serum and horseradish peroxidase-conjugated secondary antibody or [3H]thymidine-labeled bacteria. Adhesion was found to be time dependent and increased linearly with increasing numbers of bacteria added. Variation in the level of adhesion was noted among strains of A. actinomycetemcomitans. Adhesion was not significantly altered by changes in pH (from pH 5 to 9) but was sensitive to sodium chloride concentrations greater than 0.15 M. Pooled human saliva was inhibitory for adhesion when bacteria were pretreated with saliva before being added to the cells. Pretreatment of the KB cells with saliva did not inhibit adhesion. Protease treatment of A. actinomycetemcomitans reduced adhesion of the bacteria to KB cells. The data are consistent with the hypothesis that a protein(s) is required for bacterial adhesion and that host components may play a role in modulating adhesion to epithelial cells. Images PMID:8063383

  10. Avidity of antibody responses to Actinobacillus actinomycetemcomitans in periodontitis.

    PubMed Central

    O'Dell, D S; Ebersole, J L

    1995-01-01

    We designed a study to examine the serum IgG antibody avidity characteristics in: (i) normal subjects (N); (ii) Actinobacillus actinomycetemcomitans-infected adult periodontitis (AP Aa+); (iii) A. actinomycetemcomitans-infected localized juvenile periodontitis (LJP Aa+); and (iv) AP subjects (AP) with various antibody patterns and disease presentation. Although there were significant elevations in antibody levels for AP Aa+ and LJP Aa+ patients compared with AP and normal patients (P < 0.0001), there were no significant differences in the avidity indices (AI). Correlations of antibody levels to avidity revealed that functional activity of the antibody as measured by avidity was independent of antibody levels. Increasing antibody levels correlated with an increase in the number of infected sites, yet there was a trend for A1 to decrease with increased infection. Avidity indices for all patient groups did not appear to show a strong biologic relationship to plaque; however, in AP Aa+ and LJP Aa+ patients there was a generally positive relationship between avidity and bleeding on probing or pocket depth. In AP Aa+ and LJP Aa+ patients, and in AP patients there was a positive relationship of avidity through a threshold of approximately 8 active disease sites. This study hypothesized that antibody avidity to A. actinomycetemcomitans could help to explain the relationship between the active host response and chronic infection with this pathogen. The results provide evidence that both antibody levels and avidity may contribute to the variation in host resistance to infection and disease associated with A. actinomycetemcomitans. PMID:7648712

  11. Oxidative and nonoxidative killing of Actinobacillus actinomycetemcomitans by human neutrophils.

    PubMed Central

    Miyasaki, K T; Wilson, M E; Brunetti, A J; Genco, R J

    1986-01-01

    Actinobacillus actinomycetemcomitans is a facultative gram-negative microorganism which has been implicated as an etiologic agent in localized juvenile periodontitis and in subacute bacterial endocarditis and abscesses. Although resistant to serum bactericidal action and to oxidant injury mediated by superoxide anion (O2-) and hydrogen peroxide (H2O2), this organism is sensitive to killing by the myeloperoxidase-hydrogen peroxide-chloride system (K.T. Miyasaki, M.E. Wilson, and R.J. Genco, Infect. Immun. 53:161-165, 1986). In this study, we examined the sensitivity of A. actinomycetemcomitans to killing by intact neutrophils under aerobic conditions, under anaerobic conditions, and under aerobic conditions in the presence of the heme-protein inhibitor sodium cyanide. Intact neutrophils killed opsonized A. actinomycetemcomitans under aerobic and anaerobic conditions, and the kinetics of these reactions indicated that both oxidative and nonoxidative mechanisms were operative. Oxidative mechanisms contributed significantly, and most of the killing attributable to oxidative mechanisms was inhibited by sodium cyanide, which suggested that the myeloperoxidase-hydrogen peroxide-chloride system participated in the oxidative process. We conclude that human neutrophils are capable of killing A. actinomycetemcomitans by both oxygen-dependent and oxygen-independent pathways, and that most oxygen-dependent killing requires myeloperoxidase activity. PMID:3013778

  12. Azithromycin kills invasive Aggregatibacter actinomycetemcomitans in gingival epithelial cells.

    PubMed

    Lai, Pin-Chuang; Walters, John D

    2013-03-01

    Aggregatibacter actinomycetemcomitans invades periodontal pocket epithelium and is therefore difficult to eliminate by periodontal scaling and root planing. It is susceptible to azithromycin, which is taken up by many types of mammalian cells. This led us to hypothesize that azithromycin accumulation by gingival epithelium could enhance the killing of intraepithelial A. actinomycetemcomitans. [(3)H]azithromycin transport by Smulow-Glickman gingival epithelial cells and SCC-25 oral epithelial cells was characterized. To test our hypothesis, we infected cultured Smulow-Glickman cell monolayers with A. actinomycetemcomitans (Y4 or SUNY 465 strain) for 2 h, treated them with gentamicin to eliminate extracellular bacteria, and then incubated them with azithromycin for 1 to 4 h. Viable intracellular bacteria were released, plated, and enumerated. Azithromycin transport by both cell lines exhibited Michaelis-Menten kinetics and was competitively inhibited by l-carnitine and several other organic cations. Cell incubation in medium containing 5 μg/ml azithromycin yielded steady-state intracellular concentrations of 144 μg/ml in SCC-25 cells and 118 μg/ml in Smulow-Glickman cells. Azithromycin induced dose- and time-dependent intraepithelial killing of both A. actinomycetemcomitans strains. Treatment of infected Smulow-Glickman cells with 0.125 μg/ml azithromycin killed approximately 29% of the intraepithelial CFU of both strains within 4 h, while treatment with 8 μg/ml azithromycin killed ≥82% of the CFU of both strains (P < 0.05). Addition of carnitine inhibited the killing of intracellular bacteria by azithromycin (P < 0.05). Thus, human gingival epithelial cells actively accumulate azithromycin through a transport system that facilitates the killing of intraepithelial A. actinomycetemcomitans and is shared with organic cations.

  13. Interaction of oral bacteria with gingival epithelial cell multilayers.

    PubMed

    Dickinson, B C; Moffatt, C E; Hagerty, D; Whitmore, S E; Brown, T A; Graves, D T; Lamont, R J

    2011-06-01

    Primary gingival epithelial cells were cultured in multilayers as a model for the study of interactions with oral bacteria associated with health and periodontal disease. Multilayers maintained at an air-liquid interface in low-calcium medium displayed differentiation and cytokeratin properties characteristic of junctional epithelium. Multilayers were infected with fluorescently labeled Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum or Streptococcus gordonii, and bacterial association was determined by confocal microscopy and quantitative image analysis. Porphyromonas gingivalis invaded intracellularly and spread from cell to cell; A. actinomycetemcomitans and F. nucleatum remained extracellular and showed intercellular movement through the multilayer; whereas S. gordonii remained extracellular and predominantly associated with the superficial cell layer. None of the bacterial species disrupted barrier function as measured by transepithelial electrical resistance. P. gingivalis did not elicit secretion of proinflammatory cytokines. However, A. actinomycetemcomitans and S. gordonii induced interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6 and IL-8 secretion; and F. nucleatum stimulated production of IL-1β and TNF-α. Aggregatibacter actinomycetemcomitans, F. nucleatum and S. gordonii, but not P. gingivalis, increased levels of apoptosis after 24 h infection. The results indicate that the organisms with pathogenic potential were able to traverse the epithelium, whereas the commensal bacteria did not. In addition, distinct host responses characterized the interaction between the junctional epithelium and oral bacteria.

  14. Aggregatibacter actinomycetemcomitans Invasion Induces Interleukin-1β Production Through Reactive Oxygen Species and Cathepsin B.

    PubMed

    Okinaga, Toshinori; Ariyoshi, Wataru; Nishihara, Tatsuji

    2015-06-01

    Interleukin-1 (IL-1) cytokines, IL-1α, IL-1β, and IL-18 play a crucial role in inflammatory responses in a variety of diseases including periodontitis. In this study, the periodontopathic bacterial pathogen, Aggregatibacter actinomycetemcomitans, induced cell death and cytokine release in macrophages. Cell viability was reduced by A. actinomycetemcomitans invasion using (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay. The production of IL-1β in A. actinomycetemcomitans-invaded macrophage cells was detected by real-time reverse transcriptase-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. Treatment with a caspase-1 inhibitor and silencing of the caspase-1 gene had no effect on IL-1β secretion induced by A. actinomycetemcomitans invasion. Pattern recognition receptor, NLRP3 was upregulated in A. actinomycetemcomitans-invaded macrophages. However, NLRP3 knockdown had no effect on the secretion of IL-1β in A. actinomycetemcomitans-invaded RAW 264 cells. In addition, A. actinomycetemcomitans invasion induced the generation of reactive oxygen species (ROS) and the release of cathepsin B in RAW 264 cells. Interestingly, CA074-Me, a cathepsin B inhibitor, and N-Acetyl-l-cysteine, a ROS inhibitor, prevented the production of IL-1β induced by A. actinomycetemcomitans. Taken together, these results suggest A. actinomycetemcomitans induce IL-1β production in RAW 264 cells through the production of ROS and cathepsin B, but not through the NLRP3/caspase-1 pathway.

  15. The presence of phage-infected Actinobacillus actinomycetemcomitans in localized juvenile periodontitis patients.

    PubMed

    Preus, H R; Olsen, I; Namork, E

    1987-11-01

    Electron microscopy revealed 2 different types of bacteriophages isolated from Actinobacillus actinomycetemcomitans colonizing exclusively diseased sites in 4 patients with localized juvenile periodontitis (LJP). All sites infected with phage were undergoing periodontal destruction, as judged from consecutive routine radiographs. The phages isolated had a wide host range as assessed from their ability to infect a series of reference strains of A. actinomycetemcomitans. A 5th patient harboured non-infected A. actinomycetemcomitans in a surgically treated site which had undergone no bone destruction during the last 12 months. The present findings suggested that the pathogenic potential of A. actinomycetemcomitans in LJP may increase due to phage infection.

  16. Aggregatibacter actinomycetemcomitans infection mimicking lung cancer: a case report.

    PubMed

    Matzumura-Kuan, Melissa; Jennings, Jeffrey

    2014-09-01

    Pulmonary infections can mimic a pulmonary neoplasm. Multiple organisms, including bacteria, viruses, and fungi, can present with similar clinical, radiographic, and surgical findings as neoplastic processes. Because treatment and the prognosis are completely different, an accurate diagnosis is crucial, and lung biopsy is usually required. Aggregatibacter actinomycetemcomitans is part of the normal oral flora and is a rare cause of invasive infection due to hematogenous dissemination or aspiration, particularly infective endocarditis. We present a case of A. actinomycetemcomitans and Actinomyces co-infection that presented as a mediastinal mass, with surgical findings similar to lung malignancy but with biopsy and culture showing an infectious origin. After antibiotic treatment, follow-up images showed resolution of the mass.

  17. Actinomycetemcomitin: a new bacteriocin produced by Aggregatibacter (Actinobacillus) actinomycetemcomitans.

    PubMed

    Lima, Francisca Lúcia; de Carvalho, Maria Auxiliadora Roque; Apolônio, Ana Carolina Morais; Bemquerer, Marcelo Porto; Santoro, Marcelo Matos; Oliveira, Jamil Silvano; Alviano, Celuta Sales; Farias, Luiz de Macêdo

    2008-02-01

    Aggregatibacter (Actinobacillus) actinomycetemcomitans P(7-20) strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30-60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45 degrees C, and after treatment with proteolytic enzymes such as trypsin, alpha-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.

  18. 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.

  19. Leukotoxic activity of Aggregatibacter actinomycetemcomitans and periodontal attachment loss.

    PubMed

    Höglund Åberg, Carola; Haubek, Dorte; Kwamin, Francis; Johansson, Anders; Claesson, Rolf

    2014-01-01

    Aggregatibacter actinomycetemcomitans is a Gram-negative periodontitis-associated bacterium that expresses a toxin that selectively affects leukocytes. This leukotoxin is encoded by an operon belonging to the core genome of this bacterial species. Variations in the expression of the leukotoxin have been reported, and a well-characterized specific clonal type (JP2) of this bacterium with enhanced leukotoxin expression has been isolated. In particular, the presence of the JP2 genotype significantly increases the risk for the progression of periodontal attachment loss (AL). Based on these findings we hypothesized that variations in the leukotoxicity are linked to disease progression in infected individuals. In the present study, the leukotoxicity of 239 clinical isolates of A. actinomycetemcomitans was analysed with different bioassays, and the genetic peculiarities of the isolates were related to their leukotoxicity based on examination with molecular techniques. The periodontal status of the individuals sampled for the presence of A. actinomycetemcomitans was examined longitudinally, and the importance of the observed variations in leukotoxicity was evaluated in relation to disease progression. Our data show that high leukotoxicity correlates with an enhanced risk for the progression of AL. The JP2 genotype isolates were all highly leukotoxic, while the isolates with an intact leukotoxin promoter (non-JP2 genotypes) showed substantial variation in leukotoxicity. Genetic characterization of the non-JP2 genotype isolates indicated the presence of highly leukotoxic genotypes of serotype b with similarities to the JP2 genotype. Based on these results, we conclude that A. actinomycetemcomitans harbours other highly virulent genotypes besides the previously described JP2 genotype. In addition, the results from the present study further highlight the importance of the leukotoxin as a key virulence factor in aggressive forms of periodontitis.

  20. Detection and strain identification of Actinobacillus actinomycetemcomitans by nested PCR.

    PubMed Central

    Leys, E J; Griffen, A L; Strong, S J; Fuerst, P A

    1994-01-01

    By using PCR, Actinobacillus actinomycetemcomitans strains were identified directly from plaque samples without the need to isolate or culture bacteria. DNA fragments were generated by a nested, two-step PCR amplification of the ribosomal spacer region between the 16S and 23S rRNA genes. For the first amplification, primers homologous to sequences common to all bacterial species were used. This was followed by a second amplification with primers specific to A. actinomycetemcomitans. The ribosomal DNA spacer region was amplified from as few as 10 bacterial cells within a total population of 10(8) cells (0.00001%), and cross-reactivity between species was not observed. DNA fragments specific for Porphyromonas gingivalis were generated from the same samples by using a P. gingivalis-specific primer, and equivalent sensitivity and specificity were observed. A. actinomycetemcomitans was detected in 60% and P. gingivalis was detected in 79% of 52 subjects tested. Sequence analysis of the spacer region DNA fragment for A. actinomycetemcomitans gave precise strain identification, producing unique sequences for seven reference strains and identification of nine plaque-derived isolates. A phylogenetic tree based on quantitative sequence relationships was constructed. Two-step PCR amplification directly from plaque samples combined with sequence analysis of the ribosomal DNA spacer region provides a sensitive assay for detection and strain identification of multiple species directly from a single plaque sample. This simplified approach provides a practical method for large-scale studies on the transmission and pathogenicity of periodontitis-associated bacteria. Images PMID:8051258

  1. Identification of an immunoglobulin Fc receptor of Actinobacillus actinomycetemcomitans.

    PubMed Central

    Mintz, K P; Fives-Taylor, P M

    1994-01-01

    Actinobacillus actinomycetemcomitans expresses proteins that bind to the Fc portion of immunoglobulins. The immunoglobulin Fc receptors on the surface of A. actinomycetemcomitans were detected by the binding of biotinylated human or murine Fc molecules to strain SUNY 465 adsorbed to the bottom of microtiter wells. Biotinylated Fc binding was inhibited by unlabeled Fc molecules and human plasma. Fc receptors were identified by the binding of biotinylated Fc molecules to bacterial membrane proteins separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose. Multiple bands were identified, and the major Fc-binding protein was determined to be a heat-modifiable protein. This protein migrated with approximate molecular weights of 25,000 and 32,000 (unheated and heated, respectively). Amino-terminal sequence analysis of this protein revealed a sequence identical to the heat-modifiable protein described for A. actinomycetemcomitans ATCC 43718. This protein sequence exhibits significant homology with the N termini of outer membrane protein A (OmpA) of Escherichia coli and related OmpA-like proteins from other gram-negative bacteria. Images PMID:7927715

  2. Porphyromonas gingivalis gingipain is involved in the detachment and aggregation of Aggregatibacter actinomycetemcomitans biofilm.

    PubMed

    Haraguchi, A; Miura, M; Fujise, O; Hamachi, T; Nishimura, F

    2014-06-01

    Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are major periodontal pathogens that cause several types of periodontal disease. Our previous study suggested that P. gingivalis gingipains secreted in the subgingival environment are related to the detachment of A.actinomycetemcomitans biofilms. However, it remains unclear whether arginine-specific cysteine proteinase (Rgp) and lysine-specific proteinase (Kgp) play different roles in the detachment of A. actinomycetemcomitans biofilm. The aim of this study was to investigate possible disruptive roles of Kgp and Rgp in the aggregation and attachment of A. actinomycetemcomitans. While P. gingivalis ATCC33277 culture supernatant has an ability to decrease autoaggregation and coaggregation of A. actinomycetemcomitans cells, neither the boiled culture supernatant of ATCC33277 nor the culture supernatant of KDP136 showed this ability. The addition of KYT-1 and KYT-36, specific inhibitors of Rgp and Kgp, respectively, showed no influence on the ability of P. gingivalis culture supernatant. The result of gelatin zymography suggested that other proteases processed by gingipains mediated the decrease of A. actinomycetemcomitans aggregations. We also examined the biofilm-destructive effect of gingipains by assessing the detachment of A. actinomycetemcomitans from polystyrene surfaces. Scanning electron microscope analysis indicated that A. actinomycetemcomitans cells were detached by P. gingivalis Kgp. The quantity of A. actinomycetemcomitans in biofilm was decreased in co-culture with P. gingivalis. However, this was not found after the addition of KYT-36. These findings suggest that Kgp is a critical component for the detachment and decrease of A. actinomycetemcomitans biofilms.

  3. In vitro efficacy of diallyl sulfides against the periodontopathogen Aggregatibacter actinomycetemcomitans.

    PubMed

    Velliyagounder, Kabilan; Ganeshnarayan, Krishnaraj; Velusamy, Senthil Kumar; Fine, Daniel H

    2012-05-01

    The in vitro antibacterial effects of diallyl sulfide (DAS) against the Gram-negative periodontopathogen Aggregatibacter actinomycetemcomitans, the key etiologic agent of the severe form of localized aggressive periodontitis and other nonoral infections, were studied. A. actinomycetemcomitans was treated with garlic extract, allicin, or DAS, and the anti-A. actinomycetemcomitans effects of the treatment were evaluated. Garlic extract, allicin, and DAS significantly inhibited the growth of A. actinomycetemcomitans (greater than 3 log; P < 0.01) compared to control cells. Heat inactivation of the garlic extracts significantly reduced the protein concentration; however, the antimicrobial effect was retained. Purified proteins from garlic extract did not exhibit antimicrobial activity. Allicin lost all its antimicrobial effect when it was subjected to heat treatment, whereas DAS demonstrated an antimicrobial effect similar to that of the garlic extract, suggesting that the antimicrobial activity of garlic extract is mainly due to DAS. An A. actinomycetemcomitans biofilm-killing assay performed with DAS showed a significant reduction in biofilm cell numbers, as evidenced by both confocal microscopy and culture. Scanning electron microscopy (SEM) analysis of DAS-treated A. actinomycetemcomitans biofilms showed alterations of colony architecture indicating severe stress. Flow cytometry analysis of OBA9 cells did not demonstrate apoptosis or cell cycle arrest at therapeutic concentrations of DAS (0.01 and 0.1 μg/ml). DAS-treated A. actinomycetemcomitans cells demonstrated complete inhibition of glutathione (GSH) S-transferase (GST) activity. However, OBA9 cells, when exposed to DAS at similar concentrations, showed no significant differences in GST activity, suggesting that DAS-induced GST inhibition might be involved in A. actinomycetemcomitans cell death. These findings demonstrate that DAS exhibits significant antibacterial activity against A. actinomycetemcomitans and

  4. Serum antibody in Actinobacillus actinomycetemcomitans-infected patients with periodontal disease.

    PubMed Central

    Ebersole, J L; Sandoval, M N; Steffen, M J; Cappelli, D

    1991-01-01

    This study was designed to (i) delineate the characteristics of serum antibody responses to Actinobacillus actinomycetemcomitans in patients with periodontitis who are infected with A. actinomycetemcomitans; irrespective of disease classification; (ii) assess the relationship of the elevated antibody levels to colonization of the oral cavity by A. actinomycetemcomitans; and (iii) describe the serotype distribution of A. actinomycetemcomitans and antibodies to the microorganism in infected patients with various clinical classifications. To compare the levels of various isotype-specific antibodies to the different antigens, studies were performed that allowed quantitation of each isotype-specific antibody in a human reference standard. By using this reference standard, it was shown that the levels of immunoglobulin G (IgG), IgM, and IgA responses to A. actinomycetemcomitans were similar among the infected patients, irrespective of disease classification. Also, we demonstrated that the serum antibody response to serotype b was quantitatively greater in all isotypes. Our findings indicate that b was the most frequent A. actinomycetemcomitans serotype detected in the patients and appears to be capable of initiating a substantial serum IgG antibody response that may contain cross-reactive antibodies to other serotypes of A. actinomycetemcomitans. Generally, in cases in which the response to a single serotype was elevated, only that type of A. actinomycetemcomitans was detected in the plaque. Individuals exhibiting elevated antibodies to multiple serotypes were most consistently colonized by the serotype b microorganism. This study represents the first report detailing the distribution of IgG subclass antibodies to A. actinomycetemcomitans in periodontal disease. The results demonstrated that the primary responses of patients with periodontitis to A. actinomycetemcomitans were of the IgG1 and IgG3 subclasses, which is consistent with elicited responses to protein antigens

  5. In Vitro Efficacy of Diallyl Sulfides against the Periodontopathogen Aggregatibacter actinomycetemcomitans

    PubMed Central

    Ganeshnarayan, Krishnaraj; Velusamy, Senthil Kumar; Fine, Daniel H.

    2012-01-01

    The in vitro antibacterial effects of diallyl sulfide (DAS) against the Gram-negative periodontopathogen Aggregatibacter actinomycetemcomitans, the key etiologic agent of the severe form of localized aggressive periodontitis and other nonoral infections, were studied. A. actinomycetemcomitans was treated with garlic extract, allicin, or DAS, and the anti-A. actinomycetemcomitans effects of the treatment were evaluated. Garlic extract, allicin, and DAS significantly inhibited the growth of A. actinomycetemcomitans (greater than 3 log; P < 0.01) compared to control cells. Heat inactivation of the garlic extracts significantly reduced the protein concentration; however, the antimicrobial effect was retained. Purified proteins from garlic extract did not exhibit antimicrobial activity. Allicin lost all its antimicrobial effect when it was subjected to heat treatment, whereas DAS demonstrated an antimicrobial effect similar to that of the garlic extract, suggesting that the antimicrobial activity of garlic extract is mainly due to DAS. An A. actinomycetemcomitans biofilm-killing assay performed with DAS showed a significant reduction in biofilm cell numbers, as evidenced by both confocal microscopy and culture. Scanning electron microscopy (SEM) analysis of DAS-treated A. actinomycetemcomitans biofilms showed alterations of colony architecture indicating severe stress. Flow cytometry analysis of OBA9 cells did not demonstrate apoptosis or cell cycle arrest at therapeutic concentrations of DAS (0.01 and 0.1 μg/ml). DAS-treated A. actinomycetemcomitans cells demonstrated complete inhibition of glutathione (GSH) S-transferase (GST) activity. However, OBA9 cells, when exposed to DAS at similar concentrations, showed no significant differences in GST activity, suggesting that DAS-induced GST inhibition might be involved in A. actinomycetemcomitans cell death. These findings demonstrate that DAS exhibits significant antibacterial activity against A. actinomycetemcomitans and

  6. 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

  7. Interleukin-1-like activity in capsular material from Haemophilus actinomycetemcomitans.

    PubMed Central

    Harvey, W; Kamin, S; Meghji, S; Wilson, M

    1987-01-01

    This paper describes the activity of a bacterial surface component (capsular material, CM) in biological assays for interleukin-1 (IL-1). CM from the periodontal pathogen Haemophilus actinomycetemcomitans was tested in the following in vitro assays: mouse thymocyte proliferation (LAF assay), stimulation of collagenase and prostaglandin (PG) E2 synthesis by articular chondrocytes, and stimulation of PGE2 synthesis by fibroblasts. In all these assays, CM gave a response similar to an IL-1 preparation. This ability to mimic IL-1 suggests an important role for CM in both cell-mediated immunity and connective tissue destruction in localized juvenile periodontitis (LJP). PMID:3032779

  8. Sustained mitogen-activated protein kinase activation with Aggregatibacter actinomycetemcomitans causes inflammatory bone loss.

    PubMed

    Dunmyer, J; Herbert, B; Li, Q; Zinna, R; Martin, K; Yu, H; Kirkwood, K L

    2012-10-01

    Aggregatibacter actinomycetemcomitans is a gram-negative facultative capnophile involved in pathogenesis of aggressive forms of periodontal disease. In the present study, we interrogated the ability of A. actinomycetemcomitans to stimulate innate immune signaling and cytokine production and established that A. actinomycetemcomitans causes bone loss in a novel rat calvarial model. In vitro studies indicated that A. actinomycetemcomitans stimulated considerable production of soluble cytokines, tumor necrosis factor-α, interleukin-6 and interleukin-10 in both primary bone marrow-derived macrophages and NR8383 macrophages. Immunoblot analysis indicated that A. actinomycetemcomitans exhibits sustained activation of all major mitogen-activated protein kinase (MAPK) pathways, as well as the negative regulator of MAPK signaling, MAPK phosphatase-1 (MKP-1), for at least 8 h. In a rat calvarial model of inflammatory bone loss, high and low doses of formalin-fixed A. actinomycetemcomitans were microinjected into the supraperiosteal calvarial space for 1-2 weeks. Histological staining and micro-computed tomography of rat calvariae revealed a significant increase of inflammatory and fibroblast infiltrate and increased bone resorption as measured by total lacunar pit formation. From these data, we provide new evidence that fixed whole cell A. actinomycetemcomitans stimulation elicits a pro-inflammatory host response through sustained MAPK signaling, leading to enhanced bone resorption within the rat calvarial bone.

  9. Aggregatibacter actinomycetemcomitans, a potent immunoregulator of the periodontal host defense system and alveolar bone homeostasis.

    PubMed

    Herbert, B A; Novince, C M; Kirkwood, K L

    2016-06-01

    Aggregatibacter actinomycetemcomitans is a perio-pathogenic bacteria that has long been associated with localized aggressive periodontitis. The mechanisms of its pathogenicity have been studied in humans and preclinical experimental models. Although different serotypes of A. actinomycetemcomitans have differential virulence factor expression, A. actinomycetemcomitans cytolethal distending toxin (CDT), leukotoxin, and lipopolysaccharide (LPS) have been most extensively studied in the context of modulating the host immune response. Following colonization and attachment in the oral cavity, A. actinomycetemcomitans employs CDT, leukotoxin, and LPS to evade host innate defense mechanisms and drive a pathophysiologic inflammatory response. This supra-physiologic immune response state perturbs normal periodontal tissue remodeling/turnover and ultimately has catabolic effects on periodontal tissue homeostasis. In this review, we have divided the host response into two systems: non-hematopoietic and hematopoietic. Non-hematopoietic barriers include epithelium and fibroblasts that initiate the innate immune host response. The hematopoietic system contains lymphoid and myeloid-derived cell lineages that are responsible for expanding the immune response and driving the pathophysiologic inflammatory state in the local periodontal microenvironment. Effector systems and signaling transduction pathways activated and utilized in response to A. actinomycetemcomitans will be discussed to further delineate immune cell mechanisms during A. actinomycetemcomitans infection. Finally, we will discuss the osteo-immunomodulatory effects induced by A. actinomycetemcomitans and dissect the catabolic disruption of balanced osteoclast-osteoblast-mediated bone remodeling, which subsequently leads to net alveolar bone loss.

  10. 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

  11. Quantitative proteomics reveal distinct protein regulations caused by Aggregatibacter actinomycetemcomitans within subgingival biofilms.

    PubMed

    Bao, Kai; Bostanci, Nagihan; Selevsek, Nathalie; Thurnheer, Thomas; Belibasakis, Georgios N

    2015-01-01

    Periodontitis is an infectious disease that causes the inflammatory destruction of the tooth-supporting (periodontal) tissues, caused by polymicrobial biofilm communities growing on the tooth surface. Aggressive periodontitis is strongly associated with the presence of Aggregatibacter actinomycetemcomitans in the subgingival biofilms. Nevertheless, whether and how A. actinomycetemcomitans orchestrates molecular changes within the biofilm is unclear. The aim of this work was to decipher the interactions between A. actinomycetemcomitans and other bacterial species in a multi-species biofilm using proteomic analysis. An in vitro 10-species "subgingival" biofilm model, or its derivative that included additionally A. actinomycetemcomitans, were anaerobically cultivated on hydroxyapatite discs for 64 h. When present, A. actinomycetemcomitans formed dense intra-species clumps within the biofilm mass, and did not affect the numbers of the other species in the biofilm. Liquid chromatography-tandem mass spectrometry was used to identify the proteomic content of the biofilm lysate. A total of 3225 and 3352 proteins were identified in the biofilm, in presence or absence of A. actinomycetemcomitans, respectively. Label-free quantitative proteomics revealed that 483 out of the 728 quantified bacterial proteins (excluding those of A. actinomycetemcomitans) were accordingly regulated. Interestingly, all quantified proteins from Prevotella intermedia were up-regulated, and most quantified proteins from Campylobacter rectus, Streptococcus anginosus, and Porphyromonas gingivalis were down-regulated in presence of A. actinomycetemcomitans. Enrichment of Gene Ontology pathway analysis showed that the regulated groups of proteins were responsible primarily for changes in the metabolic rate, the ferric iron-binding, and the 5S RNA binding capacities, on the universal biofilm level. While the presence of A. actinomycetemcomitans did not affect the numeric composition or absolute protein

  12. Quantitative Proteomics Reveal Distinct Protein Regulations Caused by Aggregatibacter actinomycetemcomitans within Subgingival Biofilms

    PubMed Central

    Bao, Kai; Bostanci, Nagihan; Selevsek, Nathalie; Thurnheer, Thomas; Belibasakis, Georgios N.

    2015-01-01

    Periodontitis is an infectious disease that causes the inflammatory destruction of the tooth-supporting (periodontal) tissues, caused by polymicrobial biofilm communities growing on the tooth surface. Aggressive periodontitis is strongly associated with the presence of Aggregatibacter actinomycetemcomitans in the subgingival biofilms. Nevertheless, whether and how A. actinomycetemcomitans orchestrates molecular changes within the biofilm is unclear. The aim of this work was to decipher the interactions between A. actinomycetemcomitans and other bacterial species in a multi-species biofilm using proteomic analysis. An in vitro 10-species “subgingival” biofilm model, or its derivative that included additionally A. actinomycetemcomitans, were anaerobically cultivated on hydroxyapatite discs for 64 h. When present, A. actinomycetemcomitans formed dense intra-species clumps within the biofilm mass, and did not affect the numbers of the other species in the biofilm. Liquid chromatography-tandem mass spectrometry was used to identify the proteomic content of the biofilm lysate. A total of 3225 and 3352 proteins were identified in the biofilm, in presence or absence of A. actinomycetemcomitans, respectively. Label-free quantitative proteomics revealed that 483 out of the 728 quantified bacterial proteins (excluding those of A. actinomycetemcomitans) were accordingly regulated. Interestingly, all quantified proteins from Prevotella intermedia were up-regulated, and most quantified proteins from Campylobacter rectus, Streptococcus anginosus, and Porphyromonas gingivalis were down-regulated in presence of A. actinomycetemcomitans. Enrichment of Gene Ontology pathway analysis showed that the regulated groups of proteins were responsible primarily for changes in the metabolic rate, the ferric iron-binding, and the 5S RNA binding capacities, on the universal biofilm level. While the presence of A. actinomycetemcomitans did not affect the numeric composition or absolute

  13. 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

  14. Fusobacterium necrophorum findings in Denmark from 2010 to 2014 using data from the Danish microbiology database.

    PubMed

    Bank, Steffen; Jensen, Anders; Nielsen, Hanne Merete; Kristensen, Lena Hagelskjaer; Voldstedlund, Marianne; Prag, Jørgen

    2016-12-01

    Fusobacterium necrophorum findings in Denmark and estimation of the incidence of F. necrophorum bacteraemia was described using data from the nationwide Danish microbiology database (MiBa). All microbiological reports on any Fusobacterium species in Denmark were extracted for a period of 5 years from 2010 to 2014 from MiBa and from the local department of clinical microbiology. The overall incidence of F. necrophorum bacteraemia from 2010 to 2014 was 2.8 cases per million/year vs 9.4 in the age group 15-24 years. F. necrophorum was rare in blood cultures from children and middle-aged patients and then raised again. However, 48 of 232 cases of Fusobacterium bacteraemia were not identified to species level, so the incidences of F. necrophorum bacteraemia may be underestimated in our study. F. necrophorum was found in throat swabs in the age group between 13 and 40 years and in otitis media in children below 2 years in those departments which performed anaerobic culture. The incidence of F. necrophorum bacteraemia found was comparable to earlier reported figures for Lemierre's syndrome. Fusobacterium bacteraemia should always be identified to species level.

  15. Effects of Aggregatibacter actinomycetemcomitans leukotoxin on endothelial cells.

    PubMed

    Dietmann, Anelia; Millonig, Alban; Combes, Valery; Couraud, Pierre-Olivier; Kachlany, Scott C; Grau, Georges E

    2013-01-01

    Aggregatibacter actinomycetemcomitans is a human pathogen that produces leukotoxin (LtxA) as a major virulence factor. In this study the effect of LtxA on microvascular endothelial cell viability and phenotype was studied. High doses of single LtxA treatment (500 ng/ml to 5 μg/ml) significantly and irreversibly decreased cell proliferation and induced apoptosis, as assessed by tetrazolium salt and annexin V assay, respectively. Apoptosis was partially inhibited by the pan-caspase inhibitor, z-VAD-fmk. LtxA caused a cell cycle arrest in the G2/M phase after 72 h. Between 500 ng/ml and 5 μg/ml, after long- or short-term stimulation LtxA increased the expression of ICAM-1 and VCAM-1, as well as the percentages of endothelial cells expressing these adhesion molecules. Thus, A. actinomycetemcomitans LtxA has substantial pro-inflammatory effects on human brain endothelial cells by upregulation of ICAM-1 and VCAM-1. Furthermore, LtxA in higher concentration was found to decrease proliferation and induces apoptosis in microvascular endothelial cells.

  16. Identification of Actinobacillus actinomycetemcomitans by leukotoxin gene-specific hybridization and polymerase chain reaction assays.

    PubMed Central

    Tønjum, T; Haas, R

    1993-01-01

    Eleven strains of Actinobacillus actinomycetemcomitans isolated from cases of systemic infections, local abscesses, and periodontitis were identified by genetic assays using the leukotoxin gene as the target. We have developed a polymerase chain reaction (PCR) assay, based on the leukotoxin structural gene of this pathogen, which clearly identified all tested strains of A. actinomycetemcomitans and separated them from the closely related Haemophilus aphrophilus as well as other bacterial species. Furthermore, DNA-DNA hybridization was performed with the cloned partial leukotoxin structural gene (lktA) as a probe, which again clearly distinguished A. actinomycetemcomitans from H. aphrophilus, parts of the normal oral flora, and species harboring RTX (repeats in toxin) family-related cytotoxins. The PCR fragment amplified from the leukotoxin structural gene gave results similar to those given by the cloned leukotoxin gene when used as a probe in hybridization experiments. The hybridization and PCR assays described here are fundamental improvements for the identification of A. actinomycetemcomitans. Images PMID:8349764

  17. 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

  18. Serology of oral Actinobacillus actinomycetemcomitans and serotype distribution in human periodontal disease.

    PubMed Central

    Zambon, J J; Slots, J; Genco, R J

    1983-01-01

    Actinobacillus actinomycetemcomitans from the human oral cavity was serologically characterized with rabbit antisera to the type strain NCTC 9710; a number of reference strains, including Y4, ATCC 29522, ATCC 29523, ATCC 29524, NCTC 9709; and our own isolates representative of each of 10 biotypes. Using immunoabsorbed antisera, we identified three distinct serotypes by immunodiffusion and indirect immunofluorescence. Serotype a was represented by ATCC 29523 and SUNYaB 75; serotype b was represented by ATCC 29522 and Y4; and serotype c was represented by NCTC 9710 and SUNYaB 67. Indirect immunofluorescence revealed no reaction between the three A. actinomycetemcomitans serotype-specific antisera and 62 strains representing 23 major oral bacterial species. Distinct from the serotype antigens were at least one A. actinomycetemcomitans species common antigen and an antigen shared with other Actinobacillus species, Haemophilus aphrophilus, and Haemophilus paraphrophilus. All serotype a A. actinomycetemcomitans strains failed to ferment xylose, whereas all serotype b organisms fermented xylose. Serotype c included xylose-positive as well as xylose-negative strains. A total of 301 isolates of A. actinomycetemcomitans from the oral cavity of 74 subjects were serologically categorized by indirect immunofluorescence with serotype-specific rabbit antisera. Each patient harbored only one serotype of A. actinomycetemcomitans. Fourteen healthy subjects, five diabetics, and seventeen adult periodontitis patients exhibited serotypes a and b in approximately equal frequency, whereas serotype c was found less frequently. In contrast, in 29 localized juvenile periodontitis patients, the incidence of serotype b was approximately two times higher than that of serotypes a or c, suggesting a particularly high periodontopathic potential of A. actinomycetemcomitans serotype b strains. In subjects infected with A. actinomycetemcomitans, serum antibodies were detected to the serotype

  19. 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.

  20. Actinobacillus actinomycetemcomitans adheres to human gingival fibroblasts and modifies cytoskeletal organization.

    PubMed

    Gutiérrez-Venegas, Gloria; Kawasaki-Cárdenas, Perla; Garcés, Carla Portillo; Román-Alvárez, Patricia; Barajas-Torres, Carolina; Contreras-Marmolejo, Luis Arturo

    2007-09-01

    Adherence of Actinobacillus actinomycetemcomitans to human gingival fibroblast cells induces cytoskeletal reorganization. A. actinomycetemcomitans is considered a pathogenic bacteria involved in localized aggressive periodontitis. Studies with epithelial cells have shown an adherent capacity of bacteria that is increased under anaerobic conditions. For adherence to take place, there is a need for interaction between extracellular vesicles and bacterial fimbriae. However, molecular events associated with the adherence process are still unknown. The aim of this study was to investigate whether A. actinomycetemcomitans adherence to human gingival fibroblasts promotes cytoskeletal reorganization. Adherence was determined with light microscopy and scanning electron microscopy. For F-actin visualization, cells were treated with fluorescein-isothiocyanate-phalloidin and samples were examined with epifluorescence optics. Fluorescent was recorded on Kodak T-Max 400 film. We showed that A. actinomycetemcomitans adheres to human gingival fibroblast primary cultures, this property stimulating an increase in the intracellular calcium levels. In human gingival fibroblast primary cultures, we observed that maximal A. actinomycetemcomitans adherence took place 1.5h after culture infection occurred and remained for 6h. The adherence was associated with morphologic alterations and an increased in the intracellular calcium levels. These experiments suggest that A. actinomycetemcomitans adherence cause morphological alterations, induce actin stress fibers and recruitment of intracellular calcium levels.

  1. Studies of leukotoxin from Actinobacillus actinomycetemcomitans using the promyelocytic HL-60 cell line.

    PubMed Central

    Zambon, J J; DeLuca, C; Slots, J; Genco, R J

    1983-01-01

    The promyelocytic HL-60 cell line was examined for susceptibility to leukotoxin from Actinobacillus actinomycetemcomitans. Strains of A. actinomycetemcomitans which caused lysis of human peripheral blood polymorphonuclear leukocytes also lysed HL-60 cells as determined by release of intracellular lactate dehydrogenase. The killing of HL-60 cells by A. actinomycetemcomitans was dose dependent and temperature dependent, reached maximal levels after 45 min of incubation, and was inhibited by rabbit antisera to A. actinomycetemcomitans. Of 100 oral isolates of A. actinomycetemcomitans from 55 subjects, 16% from 11 healthy subjects, 43% from 13 adult periodontitis patients, 75% from 4 insulin-dependent diabetics, 66% from 2 generalized juvenile periodontitis patients, and 55% from 25 localized juvenile periodontitis patients produced leukotoxin. The same subject could harbor both leukotoxin-producing and -nonproducing isolates. The significantly higher proportion of leukotoxin-producing isolates in the disease groups compared with the healthy group is consistent with the hypothesis that leukotoxin from A. actinomycetemcomitans is an important virulence factor in the pathogenesis of certain forms of periodontal disease. PMID:6572616

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

    PubMed

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

    2008-10-01

    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.

  3. 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

  4. 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

  5. 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

  6. NLRP3 inflammasome is required for apoptosis of Aggregatibacter actinomycetemcomitans-infected human osteoblastic MG63 cells.

    PubMed

    Zhao, Panyu; Liu, Junchao; Pan, Chunling; Pan, Yaping

    2014-09-01

    Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is a Gram-negative bacterium which is implicated in the pathogenesis of human periodontal disease and in particular aggressive periodontitis. Virulence factors from A. actinomycetemcomitans have been shown to induce apoptosis of osteoblasts, however, the underlying mechanisms of the induction of apoptosis are poorly understood. In the present study, the infection of A. actinomycetemcomitans in human osteoblastic MG63 cells was established. Accordingly, A. actinomycetemcomitans infection enhanced significant apoptosis of MG63 cells. We found that both expression levels of NLRP3 and ASC were increased dramatically after MG63 cell cultures exposed to A. actinomycetemcomitans. Moreover, the secretion of mature interleukin-1β (IL-1β) and IL-18 were extensively induced in A. actinomycetemcomitans-infected cells as compared with non-invasion group of MG63 cell cultures, indicating the activation of the NLRP3 inflammasome during infection. Finally, we found that the knockdown expression of NLRP3 by specific small interfering RNA (siRNA) attenuated apoptosis of A. actinomycetemcomitans-infected MG63 cells. Our data suggest that A. actinomycetemcomitans promotes apoptosis of human osteoblasts at least partially through the NLRP3 inflammasome.

  7. The Relationship Between Fusobacterium Species and Other Flora in Mixed Infection

    DTIC Science & Technology

    1986-01-01

    Pseudonionas aeruginosa were studied. Electronmicrographs showed the presence of a thin mucopolysaccharide wall before and after inoculation into mice in 12...hacteriun isolates before inoculation showed at thin necrop/wrin and two of thle three F. 14ritrnll, dense caIpsule of mucopolysaccharide (fig. 1) in Elect...x 784 ())idcw lehce a’d lau ~ ~ PATHOGENICITY OF FUSOBACTERIUM SPP. 97 presence of a mucopolysaccharide layer. Three of the last dose was 5.6 ± 2-2

  8. [Brain abscess due to Fusobacterium necrophorum in a patient with convulsion and no signs of meningitis].

    PubMed

    Shimohata, Mitsuteru; Naruse, Satoshi; Kawasaki, Satoshi; Watanabe, Yumiko; Koyama, Miyako; Ito, Yasushi; Tanaka, Hajime

    2012-01-01

    Here, we report brain abscess due to Fusobacterium necrophorum (F. necrophorum) in a 78-year-old healthy man. He developed convulsion and did not have any signs of meningitis. Although the brain magnetic resonance imaging findings of the left occipital lobe were typical of a brain abscess, his cerebrospinal fluid examination revealed only slight pleocytosis and mild increase in protein levels. Thus, it was difficult to rule out the possibility of metastatic brain tumor; the patient's condition was provisionally diagnosed as symptomatic epilepsy secondary to brain abscess. His convulsion disappeared soon after administration of antiepileptic, antibacterial, and steroid agents. A craniotomy was performed to evacuate the abscess, and F. necrophorum was identified by culturing the abscess contents. After the operation, he was treated with appropriate antibacterial agents, which resulted in resolution of the brain abscess. Although Fusobacterium species are gram-negative anaerobic bacilli commensal of the human oropharynx, we need to recognize that Fusobacterium species can be a primary pathogen causing brain abscesses and may leave residual neurological sequelae without early appropriate treatment.

  9. Actinobacillus actinomycetemcomitans and localized juvenile periodontitis. Clinical, microbiologic and histologic studies.

    PubMed

    Christersson, L A

    1993-01-01

    The present studies examined Actinobacillus actinomycetemcomitans and its role in localized juvenile periodontitis (LJP). The distribution of the bacteria was studied in healthy normals, patients with adult periodontitis, diabetics, and those with LJP. Over 95% of the LJP patients harbored A. actinomycetemcomitans, whereas only 17% of healthy subjects, 21% of adult periodontitis patients, and 5% of diabetics were positive. All members of a LJP family harboring the organism yielded isolates of the same biotype and serotype. The transmission of the bacteria was studied after transfer of the bacteria, with periodontal probes from infected to healthy gingival sites, within the oral cavity of LJP patients. Newly colonized gingival sites, 50% of those involved, became free of A. actinomycetemcomitans after only 3 weeks. A purposely forceful inoculation contributed to a more predictable colonization (89%), but only prolonged the colonization with one week. Treatment of LJP lesions with scaling and root planing resulted in minimal clinical and microbiological changes during a 16 week follow-up period. However, gingival curettage and modified Widman flap surgery suppressed A. actinomycetemcomitans in 75% and 89% of the sites, and resulted in resolution of periodontal pocket depth and gain in attachment level. Gingival tissue specimens, from 35 LJP sites, 3 control sites, and one monkey biopsy, were studied to verify the hypothesis of gingival infiltration of A. actinomycetemcomitans. Bacteria were identified immunohistologically with rabbit antisera serospecific to the three A. actinomycetemcomitans serotypes. Positive staining was observed in the tissue from all but one LJP patient. Twenty-eight (80%) lesions were positive for A. actinomycetemcomitans antigens in the gingival connective tissue, often with antigens located both between and within cells. The specimen from a culture positive control demonstrated no signs of invasion, similar to the monkey specimen

  10. In vitro susceptibilities of Actinobacillus actinomycetemcomitans to a number of antimicrobial combinations.

    PubMed Central

    Pavicić, M J; van Winkelhoff, A J; de Graaff, J

    1992-01-01

    The in vitro susceptibilities of Actinobacillus actinomycetemcomitans to 14 antimicrobial combinations were studied by using the checkerboard titration technique. The results, expressed as the range of the fractional inhibitory concentration indices, were as follows: for metronidazole or its hydroxymetabolite combined with cefixime, 0.2 to 0.6; for moxalactam, 0.2 to 0.6; for penicillin G, 0.3 to 0.6; for tobramycin, 0.8 to 2.0; for erythromycin, 0.8 to 1.7; for ciprofloxacin, 0.2 to 0.6; for tetracycline, 0.8 to 1.2. Our observations indicated that the beta-lactam antibiotics as well as ciprofloxacin act synergistically with both metronidazole and its hydroxymetabolite against A. actinomycetemcomitans. Synergistic interactions were independent of the individual MICs of the antibiotics tested. Erythromycin, tobramycin, and tetracycline combined with either metronidazole or its hydroxymetabolite showed additive to indifferent effects against the five strains of A. actinomycetemcomitans, with the fractional inhibitory concentration indices ranging from 0.8 to 2.0. A. actinomycetemcomitans was found to be highly susceptible to ciprofloxacin (MIC of ciprofloxacin for 90% of strains tested, 0.010 micrograms/ml) and cefixime (MIC of cefixime for 90% of strains tested, 0.8 micrograms/ml). The results indicate that in patients who are allergic to penicillin, cefixime and ciprofloxacin may be useful alternative antibiotics in combination with metronidazole for the treatment of A. actinomycetemcomitans-associated periodontitis. PMID:1482130

  11. Opsonic antibody activity against Actinobacillus actinomycetemcomitans in patients with rapidly progressive periodontitis.

    PubMed Central

    Sjöström, K; Darveau, R; Page, R; Whitney, C; Engel, D

    1992-01-01

    Actinobacillus actinomycetemcomitans has been closely associated with early-onset, severe periodontitis, and such patients often have serum immunoglobulin G (IgG) antibodies reactive with antigens of this gram-negative pathogen. We examined the functionality and potential importance of these antibodies. The opsonic activity against A. actinomycetemcomitans of sera from 30 patients with rapidly progressive periodontitis (RPP) and from 28 periodontally normal subjects was tested by using polymorphonuclear leukocyte (PMN) chemiluminescence and bactericidal assays. Peak chemiluminescence values correlated strongly with killing observed in the PMN-dependent bactericidal assay (r = 0.88; P < 0.001). Neither the mean IgG titer nor the mean peak chemiluminescence differed significantly between the two groups. However, when the relationship between chemiluminescence and titer was examined, regression analysis showed that antibodies present in low-titer normal sera were significantly more effective at opsonizing A. actinomycetemcomitans than antibodies present in low-titer RPP patient sera (P = 0.04). Thus, periodontally normal individuals may be better able than RPP patients to clear A. actinomycetemcomitans in early stages of colonization, and anti-A. actinomycetemcomitans antibodies in RPP patients may be relatively ineffective in preventing infection by this organism. PMID:1398993

  12. Presence of bacteriophage Aa phi 23 correlates with the population genetic structure of Actinobacillus actinomycetemcomitans.

    PubMed

    Haubek, D; Willi, K; Poulsen, K; Meyer, J; Kilian, M

    1997-02-01

    Several bacteriophages associated with the oral bacterium Actinobacillus actinomycetemcomitans have been identified. Lysogeny might affect the virulence of this bacterium, which has been implicated in the etiology of juvenile and adult periodontitis. We have determined the presence of bacteriophage Aa phi 23-related DNA sequences among 185 A. actinomycetemcomitans strains belonging to 2 well-characterized collections and have related the findings to the population genetic structure of the collections. 2 cloned Aa phi 23-specific DNA probes were used in Southern blot hybridization experiments to detect homologous sequences in whole-cell DNA of the strains. DNA from 65 (35%) of the 185 strains hybridized to either of the DNA probes. The majority (74%) of the hybridizing strains showed an identical hybridization pattern, indicating presence of phage Aa phi 23. Whole-cell DNA from the remaining hybridizing strains hybridized to the probes with different patterns, indicating that DNA sequences related to but different from phage Aa phi 23 occur in these strains. The majority (81%) of the strains which harbored phage Aa phi 23 were of serotype a, whereas serotype d strains appeared to be resistant to infection with this phage. There was a clear correlation between hybridization patterns and genetic subdivisions based on our previous population genetic analyses of A. actinomycetemcomitans. However, there was no significant correlation between occurrence of Aa phi 23 among A. actinomycetemcomitans strains and the periodontal status of the patients from whom the isolates were obtained, suggesting that this bacteriophage does not significantly influence the virulence of A. actinomycetemcomitans.

  13. 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

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

    2013-09-01

    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

  14. 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

  15. Structural proteins of the Actinobacillus actinomycetemcomitans bacteriophage phi Aa.

    PubMed

    Stevens, R H; Hammond, B F; Fine, D H

    1990-08-01

    øAa is an A1 morphotype bacteriophage which infects certain strains of Actinobacillus actinomycetemcomitans. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of dissociated, purified phi Aa particles revealed 7 major structural proteins (P1-P7) ranging in size from 17.5 to 52.7 kilodaltons (Kd). Treatment of the intact phage particles with 67% dimethyl sulfoxide (DMSO) resulted in the separation of the virion head and tail subunits. Purification of the head subunits was accomplished by sucrose density gradient centrifugation of the DMSO-treated phage particles. The purified head subunits were composed of a single protein having an electrophoretic mobility which corresponded to a 39.5 Kd protein (P3) of the intact virus. Raising the pH of a purified phi Aa suspension to 12.7 disrupted the head subunits, as well as the tail tube and tail fibers, releasing intact contractile tail sheaths. The tail sheaths were collected by centrifugation. The purified tail sheaths were analyzed by SDS-PAGE and were found to be composed of two proteins (P1 and P2) having molecular weights of 52.7 and 41.2 Kd respectively. The location of each of the 4 remaining major structural proteins in the phi Aa virion remains to be determined.

  16. 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

  17. The heat-modifiable outer membrane protein of Actinobacillus actinomycetemcomitans: relationship to OmpA proteins.

    PubMed Central

    Wilson, M E

    1991-01-01

    The outer membrane of Actinobacillus actinomycetemcomitans contains a 29-kDa protein which exhibits heat modifiability on sodium dodecyl sulfate-polyacrylamide gels and represents a major target for immunoglobulin G antibody in sera of periodontitis patients colonized by this organism. In the present study, the N-terminal amino acid sequence of the 29-kDa outer membrane protein was determined and compared with reported sequences for other known proteins. The heat-modifiable outer membrane protein of A. actinomycetemcomitans was found to exhibit significant N-terminal homology with the OmpA proteins of other gram-negative bacteria. Moreover, this protein reacted with antiserum raised against the purified OmpA protein of Escherichia coli K-12. Whether the heat-modifiable OMP of A. actinomycetemcomitans also shares functional properties of OmpA proteins, particularly with respect to bacteriophage receptor activity, is presently under investigation. Images PMID:2050416

  18. Evidence for invasion of a human oral cell line by Actinobacillus actinomycetemcomitans.

    PubMed Central

    Meyer, D H; Sreenivasan, P K; Fives-Taylor, P M

    1991-01-01

    Actinobacillus actinomycetemcomitans, an oral bacterial species associated with periodontal disease, was found to invade human cell lines. Invasion was demonstrated by recovery of viable organisms from gentamicin-treated KB cell monolayers and by light and electron microscopy. Internalization occurred through a cytochalasin D-sensitive process. Invasion efficiencies of some A. actinomycetemcomitans strains were comparable to those of invasive members of the family Enterobacteriaceae. Differences in invasiveness were correlated with bacterial colonial morphology. Smooth variants invaded more proficiently than rough variants. A. actinomycetemcomitans can undergo a smooth-to-rough colonial morphology shift which results in the loss of invasiveness. Coordinated regulation of genes involved in the rough-to-smooth phenotypic transitions may play a role in the episodic nature of periodontal disease. Images PMID:1855989

  19. NADPH Oxidase Contributes to Resistance against Aggregatibacter actinomycetemcomitans-Induced Periodontitis in Mice.

    PubMed

    Bast, Antje; Kubis, Helen; Holtfreter, Birte; Ribback, Silvia; Martin, Heiner; Schreiner, Helen C; Dominik, Malte J; Breitbach, Katrin; Dombrowski, Frank; Kocher, Thomas; Steinmetz, Ivo

    2017-02-01

    Aggregatibacter actinomycetemcomitans is a Gram-negative commensal bacterium of the oral cavity which has been associated with the pathogenesis of periodontitis with severe alveolar bone destruction. The role of host factors such as reactive oxygen and nitrogen intermediates in periodontal A. actinomycetemcomitans infection and progression to periodontitis is still ill-defined. Therefore, this study aimed to analyze the role of NADPH oxidase and inducible nitric oxide synthase (iNOS) in a murine model of A. actinomycetemcomitans-induced periodontitis. NADPH oxidase-deficient (gp91(phox) knockout [KO]), iNOS-deficient (iNOS KO), and C57BL/6 wild-type mice were orally infected with A. actinomycetemcomitans and analyzed for bacterial colonization at various time points. Alveolar bone mineral density and alveolar bone volume were quantified by three-dimensional micro-computed tomography, and the degree of tissue inflammation was calculated by histological analyses. At 5 weeks after infection, A. actinomycetemcomitans persisted at significantly higher levels in the murine oral cavities of infected gp91(phox) KO mice than in those of iNOS KO and C57BL/6 mice. Concomitantly, alveolar bone mineral density was significantly lower in all three infected groups than in uninfected controls, but with the highest loss of bone density in infected gp91(phox) KO mice. Only infected gp91(phox) KO mice revealed significant loss of alveolar bone volume and enhanced inflammatory cell infiltration, as well as an increased number of osteoclasts. Our results indicate that NADPH oxidase is important to control A. actinomycetemcomitans infection in the murine oral cavity and to prevent subsequent alveolar bone destruction and osteoclastogenesis.

  20. Subclass and molecular form of immunoglobulin A antibodies to Actinobacillus actinomycetemcomitans in juvenile periodontitis.

    PubMed Central

    Brown, T A; Byres, L; Gardner, M; Van Dyke, T E

    1991-01-01

    Patients with juvenile periodontitis frequently have elevated levels of serum immunoglobulin A (IgA) antibodies to antigens of Actinobacillus actinomycetemcomitans. IgA occurs in two subclasses, IgA1 and IgA2, and in monomeric and polymeric forms. Because IgA1 is susceptible to cleavage by IgA1 proteases produced by microorganisms found at mucosal sites and in the gingival crevice, we wished to determine the IgA subclass distribution of antibodies to antigens of A. actinomycetemcomitans. The molecular form was examined because it may indicate the origin of the IgA and because the form differs in acute and chronic infections. There is also evidence that monomeric and polymeric IgA have different biological functions. Serum was taken from patients with juvenile periodontitis before and at intervals during and after initiation of therapy. IgA subclass distribution was determined against a sonic extracts of A. actinomycetemcomitans ATCC 2952a (serotype b) by using monoclonal anti-subclass reagents in an enzyme-linked immunosorbent assay. To determine the molecular form of the antibodies, sera were separated by high-performance liquid chromatography on a size-exclusion column. Fractions were assayed for antibody activity by the enzyme-linked immunosorbent assay, and described above. The results of the subclass analysis of the sera indicated that while both IgA1 and IgA2 antibodies to A. actinomycetemcomitans sonic extract are often found before, during, and after treatment, IgA1 antibodies dominated the response. There was a predominance of monomeric IgA1 antibodies to A. actinomycetemcomitans sonic extracts in most samples before, during, and after treatment. The monomeric form is consistent with what is seen in other chronic infections. The predominance of IgA1 antibodies implies that any protective effects of the IgA response to A. actinomycetemcomitans could be compromised by microbial IgA1 proteases. PMID:1997415

  1. 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

  2. Phenotypic changes in nonfimbriated smooth strains of Aggregatibacter actinomycetemcomitans grown in low-humidity solid medium.

    PubMed

    Pei, Zhenhua; Niu, Zhongying; Shi, Shenggen; Shi, Liang; Tang, Chuhua

    2013-04-01

    Aggregatibacter actinomycetemcomitans is the primary etiologic agent of localized aggressive periodontitis. In vitro, it can undergo fimbriated rough to nonfimbriated smooth phenotypic transition, accompanied by an increase in invasive ability and a decrease in adhesive ability. No opposite direction phenotypic transition was reported. To better understand its pathogenicity, the authors studied the morphological changes of nonfimbriated smooth strains induced by growth environmental humidity. Transmission electron microscopy was used to identify fimbriae expression change. It was found that the lower medium humidity, the more fimbriae reexpressed. In conclusion, the smooth strain of A. actinomycetemcomitans can reexpress the fimbriae in lower humidity environment.

  3. Actinobacillus actinomycetemcomitans strains Y4 and N27 adhere to hydroxyapatite by distinctive mechanisms.

    PubMed Central

    Kagermeier, A S; London, J

    1985-01-01

    Actinobacillus actinomycetemcomitans strains Y4 and N27 absorb to spheroidal hydroxyapatite in roughly the same numbers per milligram of substrate and with the same tenacity as two previously tested Cytophaga species. Although the two strains of A. actinomycetemcomitans exhibited similar affinities and number of binding sites for SHA, their response to enzyme treatment and heating were very different. The capacity of strain Y4 to attach to spheroidal hydroxyapatite was diminished by treatment with proteases and phospholipases and was unaffected by neuraminidase, while strain N27 was unaffected by proteases and phospholipases and lost its binding capabilities when treated with neuraminidase. Images PMID:3972445

  4. Aggregatibacter actinomycetemcomitans lipopolysaccharide affects human gingival fibroblast cytoskeletal organization.

    PubMed

    Gutiérrez-Venegas, Gloria; Contreras-Marmolejo, Luis Arturo; Román-Alvárez, Patricia; Barajas-Torres, Carolina

    2008-04-01

    The cytoskeleton is a dynamic structure that plays a key role in maintaining cell morphology and function. This study investigates the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the dynamics and organization of actin, tubulin, vimentin, and vinculin proteins in human gingival fibroblasts (HGF). A time-dependent study showed a noticeable change in actin architecture after 1.5 h of incubation with LPS (1 microg/ml) with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 24 h. When 0.01-10 microg/ml of LPS was added to human gingival fibroblast cultures, cells acquired a round, flat shape and gradually developed cytoplasmic ruffling. Lipopolysaccharides extracted from Aggregatibacter actinomycetemcomitans periodontopathogenic bacteria promoted alterations in F-actin stress fibres of human gingival cells. Normally, human gingival cells have F-actin fibres that are organized in linear distribution throughout the cells, extending along the cell's length. LPS-treated cells exhibited changes in cytoskeletal protein organization, and F-actin was reorganized by the formation of bundles underneath and parallel to the cell membrane. We also found the reorganization of the vimentin network into vimentin bundling after 1.5 h of treatment. HGF cells exhibited diffuse and granular gamma-tubulin stain. There was no change in LPS-treated HGF. However, vinculin plaques distributed in the cell body diminished after LPS treatment. We conclude that the dynamic and structured organization of cytoskeletal filaments and actin assembly in human gingival fibroblasts is altered by LPS treatment and is accompanied by a decrease in F-actin pools.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. Differential regulation of the leukotoxin operon in highly leukotoxic and minimally leukotoxic strains of Actinobacillus actinomycetemcomitans.

    PubMed Central

    Hritz, M; Fisher, E; Demuth, D R

    1996-01-01

    The expression of the leukotoxin (ltx) operon varies significantly among Actinobacillus actinomycetemcomitans strains. The dual promoters driving ltx expression in the highly toxic strain JP2 have been previously characterized (J. M. Brogan, E. T. Lally, K. Poulsen, M. Kilian, and D. R. Demuth, Infect. Immun. 62:501-508, 1994), and genetic analyses of A. actinomycetemcomitans suggest that highly toxic strains like JP2 arose from minimally toxic strains, presumably by deletion of a 530-bp domain within the ltx promoter region (K. Poulsen, E. Theilade, E.T. Lally, D. R. Demuth, and M. Kilian, Microbiology 140:2049-2060, 1994). However, the ltx promoter of minimally toxic A. actinomycetemcomitans strains has not been well characterized. In this study, deletion and primer extension analyses showed that the ltx promoter of A. actinomycetemcomitans 652 is situated approximately 150 bp upstream of the ltxC gene and initiates transcription 138 nucleotides upstream of ltxC. In contrast to strain JP2, only a single promoter appears to drive ltx expression in 652. The 652 promoter resides within the 530-bp region that is absent from the JP2 promoter sequence, suggesting that the specific sequences controlling ltx expression differ in highly toxic and minimally toxic A. actinomycetemcomitans strains. In addition, ltx expression in strain 652 was shown to be induced three- to fourfold when cells were grown under anaerobic conditions. The induction of whole cell leukotoxicity, was accompanied by increases in the levels of Ltx polypeptide and the steady-state levels of ltx mRNA, suggesting that regulation occurred at the level of transcription. In contrast, the levels of leukotoxicity, Ltx polypeptide, and fix mRNA in strain JP2 were unaffected by anaerobic growth. These results suggest that the ltx operon is differentially regulated in highly toxic and minimally toxic A. actinomycetemcomitans strains and that the sequences controlling the oxygen-dependent regulation of ltx

  11. Serotype b of Aggregatibacter actinomycetemcomitans increases osteoclast and memory T-lymphocyte activation.

    PubMed

    Melgar-Rodríguez, S; Díaz-Zúñiga, J; Alvarez, C; Rojas, L; Monasterio, G; Carvajal, P; Escobar, A; Sanz, M; Vernal, R

    2016-04-01

    During periodontitis, alveolar bone resorption is associated with activation of T helper type 17 (Th17) lymphocytes and receptor activator of nuclear factor-κB ligand (RANKL) -induced osteoclasts. We previously reported that serotype b of Aggregatibacter actinomycetemcomitans has a higher capacity to trigger Th17-type differentiation and function in activated T lymphocytes and its lipopolysaccharide is a more potent immunogen compared with the other serotypes. This study aimed to investigate whether serotype b of A. actinomycetemcomitans induces higher Th17-associated RANKL production, RANKL-induced osteoclast activation, and antigen-specific memory T lymphocyte proliferation. On naive CD4(+) T lymphocytes stimulated with autologous dendritic cells primed with different A. actinomycetemcomitans serotypes, RANKL production, T-bet, GATA-3, RORC2 and Foxp3 expression, RORC2/RANKL intracellular double-expression, TRAP(+) osteoclast activation, and bone resorption were quantified. The frequency of proliferating memory T lymphocytes in response to A. actinomycetemcomitans serotypes was determined in periodontitis and healthy subjects. Naive CD4(+) T lymphocytes stimulated by serotype b-primed dendritic cells elicited higher levels of RANKL, RORC2, TRAP(+) osteoclasts, and bone resorption than the same cells stimulated with the other serotypes. RANKL positively correlated and co-expressed with RORC2. Memory T lymphocytes responding to serotype b were more frequently detected in periodontitis patients than healthy subjects. These results indicate that serotype b of A. actinomycetemcomitans is associated with higher production of RANKL and these increased levels are associated with Th17 lymphocyte induction, osteoclast activation, and bone resorption.

  12. Immunoglobulin G subclass response of localized juvenile periodontitis patients to Actinobacillus actinomycetemcomitans Y4 lipopolysaccharide.

    PubMed Central

    Wilson, M E; Hamilton, R G

    1992-01-01

    Sera from patients with localized juvenile periodontitis (LJP) often contain markedly elevated immunoglobulin G (IgG) antibody titers to serospecific determinants of the lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans. The objective of the present study was to define the subclass distribution of the IgG antibody response of LJP patients to this key cell envelope antigen. IgG subclass antibody responses to A. actinomycetemcomitans LPS were quantified in an enzyme-linked immunosorbent assay with human IgG subclass-restricted monoclonal antibodies. Serum antibody concentrations were calculated by heterologous interpolation of a dose-response curve constructed by using human-mouse chimeric antibodies. Sixteen of 17 LJP serum samples tested contained significantly greater concentrations of IgG2 than IgG1 antibodies reactive toward A. actinomycetemcomitans LPS. Geometric mean antibody concentrations of IgG1 and IgG2 were 7.8 and 136.5 micrograms/ml, respectively, among LJP patients with elevated IgG titers to LPS (94% of whom were black). However, both IgG1 and IgG2 antibody concentrations were significantly greater than the corresponding values obtained from sera from LJP patients with low IgG titers to LPS. Among LJP patients with elevated IgG titers to A. actinomycetemcomitans LPS, serum IgG2 concentration and total IgG concentration were also significantly elevated compared with both low-titered LJP sera and sera from periodontally healthy race-matched controls. The results of this study indicate that the humoral response of a predominantly black population of LJP patients to A. actinomycetemcomitans includes the production of LPS-reactive IgG antibodies which are primarily of the IgG2 subclass. PMID:1563768

  13. Effect of adoptive transfer of cloned Actinobacillus actinomycetemcomitans-specific T helper cells on periodontal disease.

    PubMed Central

    Yamashita, K; Eastcott, J W; Taubman, M A; Smith, D J; Cox, D S

    1991-01-01

    Previously we isolated several Actinobacillus actinomycetemcomitans-specific T-cell clones from the spleens and lymph nodes of immunized Rowett rats. These clones were characterized as W3/13+, W3/25+, OX8-, and OX22-, suggesting a T helper (Th) phenotype. In the current experiments, 10(6) cells from a single A. actinomycetemcomitans-specific clone (A3) were adoptively transferred to a group (AaTh; n = 13) of normal heterozygous rats (rnu/+) at 28 days of age. A second group received no T cells (AaNT; n = 15), and a third group also received no T cells (NAaNT, n = 11). Beginning 1 day after transfer, the first and second groups were infected orally with A. actinomycetemcomitans for 5 consecutive days. The presence of infection was confirmed immediately after challenge and after 5 months, when the experiments were ended. Significantly higher numbers of lymphocytes were recovered from the gingival tissues of the first group than from those of either of the other groups. Also, this group showed significantly elevated (P less than 0.01) serum immunoglobulin G and immunoglobulin M antibody to A. actinomycetemcomitans in an enzyme-linked immunosorbent assay when compared with both other groups. Bone loss was significantly lower (P less than 0.01) in recipients of A. actinomycetemcomitans-specific cloned cells when compared with the other infected group and was approximately equal to the bone loss of the uninfected group. These results are consistent with the hypothesis that T-cell regulation can affect periodontal disease. In this regulation, T helper cells appear to interfere with periodontal bone loss. PMID:1825991

  14. The conserved carboxyl domain of MorC, an inner membrane protein of Aggregatibacter actinomycetemcomitans, is essential for membrane function.

    PubMed

    Smith, K P; Voogt, R D; Ruiz, T; Mintz, K P

    2016-02-01

    Morphogenesis protein C (MorC) of Aggregatibacter actinomycetemcomitans is important for maintaining the membrane morphology and integrity of the cell envelope of this oral pathogen. The MorC sequence and operon organization were found to be conserved in Gammaproteobacteria, based on a bioinformatic analysis of 435 sequences from representative organisms. Functional conservation of MorC was investigated using an A. actinomycetemcomitans morC mutant as a model system to express MorC homologs from four phylogenetically diverse representatives of the Gammaproteobacteria: Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, and Moraxella catarrhalis. The A. actinomycetemcomitans strains expressing the homologous proteins were assessed for sensitivity to bile salts, leukotoxin secretion, autoaggregation and membrane morphology. MorC from the most closely related organism (H. influenzae) was functionally identical to MorC from A. actinomycetemcomitans. However, the genes from more distantly related organisms restored some but not all A. actinomycetemcomitans mutant phenotypes. In addition, deletion mutagenesis indicated that the most conserved portion of the protein, the C-terminus DUF490 domain, was necessary to maintain the integrity of the membrane. Deletion of the last 10 amino acids of this domain of the A. actinomycetemcomitans MorC protein was sufficient to disrupt membrane stability and leukotoxin secretion. The data suggest that the MorC sequence is functionally conserved across Gammaproteobacteria and the C-terminus of the protein is essential for maintaining membrane physiology.

  15. Mitogen-Activated Protein Kinase 2 Signaling Shapes Macrophage Plasticity in Aggregatibacter actinomycetemcomitans-Induced Bone Loss.

    PubMed

    Herbert, Bethany A; Steinkamp, Heidi M; Gaestel, Matthias; Kirkwood, Keith L

    2017-01-01

    Aggregatibacter actinomycetemcomitans is associated with aggressive periodontal disease, which is characterized by inflammation-driven alveolar bone loss. A. actinomycetemcomitans activates the p38 mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase 2 (MK2) stress pathways in macrophages that are involved in host responses. During the inflammatory process in periodontal disease, chemokines are upregulated to promote recruitment of inflammatory cells. The objective of this study was to determine the role of MK2 signaling in chemokine regulation during A. actinomycetemcomitans pathogenesis. Utilizing a murine calvarial model, Mk2(+/+) and Mk2(-/-) mice were treated with live A. actinomycetemcomitans bacteria at the midsagittal suture. MK2 positively regulated the following macrophage RNA: Emr1 (F4/80), Itgam (CD11b), Csf1r (M-CSF Receptor), Itgal (CD11a), Tnf, and Nos2 Additionally, RNA analysis revealed that MK2 signaling regulated chemokines CCL3 and CCL4 in murine calvarial tissue. Utilizing the chimeric murine air pouch model, MK2 signaling differentially regulated CCL3 and CCL4 in the hematopoietic and nonhematopoietic compartments. Bone resorption pits in calvaria, observed by micro-computed tomography, and osteoclast formation were decreased in Mk2(-/-) mice compared to Mk2(+/+) mice after A. actinomycetemcomitans treatment. In conclusion, these data suggest that MK2 in macrophages contributes to regulation of chemokine signaling during A. actinomycetemcomitans-induced inflammation and bone loss.

  16. Role of exopolysaccharide in Aggregatibacter actinomycetemcomitans-induced bone resorption in a rat model for periodontal disease.

    PubMed

    Shanmugam, Mayilvahanan; Gopal, Prerna; El Abbar, Faiha; Schreiner, Helen C; Kaplan, Jeffrey B; Fine, Daniel H; Ramasubbu, Narayanan

    2015-01-01

    Aggregatibacter actinomycetemcomitans a causative agent of periodontal disease in humans, forms biofilm on biotic and abiotic surfaces. A. actinomycetemcomitans biofilm is heterogeneous in nature and is composed of proteins, extracellular DNA and exopolysaccharide. To explore the role played by the exopolysaccharide in the colonization and disease progression, we employed genetic reduction approach using our rat model of A. actinomycetemcomitans-induced periodontitis. To this end, a genetically modified strain of A. actinomycetemcomitans lacking the pga operon was compared with the wild-type strain in the rat infection model. The parent and mutant strains were primarily evaluated for bone resorption and disease. Our study showed that colonization, bone resorption/disease and antibody response were all elevated in the wild-type fed rats. The bone resorption/disease caused by the pga mutant strain, lacking the exopolysaccharide, was significantly less (P < 0.05) than the bone resorption/disease caused by the wild-type strain. Further analysis of the expression levels of selected virulence genes through RT-PCR showed that the decrease in colonization, bone resorption and antibody titer in the absence of the exopolysaccharide might be due to attenuated levels of colonization genes, flp-1, apiA and aae in the mutant strain. This study demonstrates that the effect exerted by the exopolysaccharide in A. actinomycetemcomitans-induced bone resorption has hitherto not been recognized and underscores the role played by the exopolysaccharide in A. actinomycetemcomitans-induced disease.

  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. [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

  19. Identification of a novel bacterial outer membrane interleukin-1Β-binding protein from Aggregatibacter actinomycetemcomitans.

    PubMed

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

    2013-01-01

    Aggregatibacter actinomycetemcomitans 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

  20. Aggregatibacter actinomycetemcomitans: virulence of its leukotoxin and association with aggressive periodontitis.

    PubMed

    Åberg, Carola Höglund; Kelk, Peyman; Johansson, Anders

    2015-01-01

    Periodontitis is an infection-induced inflammatory disease that causes loss of the tooth supporting tissues. Much focus has been put on comparison of the microbial biofilm in the healthy periodontium with the diseased one. The information arising from such studies is limited due to difficulties to compare the microbial composition in these two completely different ecological niches. A few longitudinal studies have contributed with information that makes it possible to predict which individuals who might have an increased risk of developing aggressive forms of periodontitis, and the predictors are either microbial or/and host-derived factors. The most conspicuous condition that is associated with disease risk is the presence of Aggregatibacter actinomycetemcomitans at the individual level. This Gram-negative bacterium has a great genetic variation with a number of virulence factors. In this review we focus in particular on the leukotoxin that, based on resent knowledge, might be one of the most important virulence factors of A. actinomycetemcomitans.

  1. Extraction and isolation of a leukotoxin from Actinobacillus actinomycetemcomitans with polymyxin B.

    PubMed Central

    Tsai, C C; Shenker, B J; DiRienzo, J M; Malamud, D; Taichman, N S

    1984-01-01

    A leukotoxin from Actinobacillus actinomycetemcomitans was isolated by a procedure that includes polymyxin B extraction, ion-exchange chromatography, and gel filtration chromatography. The procedure resulted in the recovery of 48% of the toxin with a 99-fold increase in specific activity. The isolated toxin has a molecular mass of 180,000 daltons by gel filtration and 115,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It retains all the major biological characteristics previously documented for crude leukotoxin preparations, including susceptibility to heat and proteolytic enzymes and neutralization by sera from patients with juvenile periodontitis. The isolated leukotoxin destroys human but not rat or guinea pig polymorphonuclear leukocytes and has no apparent effect on human erythrocytes. The availability of the A. actinomycetemcomitans leukotoxin should facilitate studies on its chemistry and mode of action as well as its role in the pathogenesis of human periodontal disease. Images PMID:6319288

  2. 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

  3. A gene cluster for the synthesis of serotype g-specific polysaccharide antigen in Aggregatibacter actinomycetemcomitans.

    PubMed

    Tsuzukibashi, Osamu; Saito, Masanori; Kobayashi, Taira; Umezawa, Koji; Nagahama, Fumio; Hiroi, Takachika; Hirasawa, Masatomo; Takada, Kazuko

    2014-04-01

    Aggregatibacter actinomycetemcomitans is an important pathogen related to aggressively progressive periodontal breakdown in adolescents and adults. The species can be divided into six serotypes (a-f) according to their surface carbohydrate antigens. Recently, a new serotype g of A. actinomycetemcomitans was proposed. The aim of the present study was to sequence the gene cluster associated with the biosynthesis of the serotype g-specific polysaccharide antigen and develop serotype-specific primers for PCR assay to identify serotype g strains of A. actinomycetemcomitans. The serotype-specific polysaccharide (SSPS) gene cluster of the NUM-Aa 4039 strain contained 21 genes in 21,842-bp nucleotides. The similarity of the SSPS gene cluster sequence was 96.7 % compared with that of the serotype e strain. Seventeen serotype g genes showed more than 90 % homology both in nucleotide and amino acids to the serotype e strain. Three additional genes with 1,579 bp in NUM-Aa 4039 were inserted into the corresponding ORF13 of the serotype e strain. The serotype g-specific primers were designed from the insertion region of NUM-Aa 4039. Serotypes of the a-f strains were not amplified by serotype-specific g primers; only NUM-Aa 4039 showed an amplicon band. The NUM-Aa 4039 strain was three genes in the SSPS gene cluster different from those of serotype e strain. The specific primers derived from these different regions are useful for identification and distribution of serotype g strain among A. actinomycetemcomitans from clinical samples.

  4. The cytolethal distending toxin of Aggregatibacter actinomycetemcomitans inhibits macrophage phagocytosis and subverts cytokine production.

    PubMed

    Ando-Suguimoto, Ellen Sayuri; da Silva, Maike Paulino; Kawamoto, Dione; Chen, Casey; DiRienzo, Joseph M; Mayer, Marcia Pinto Alves

    2014-03-01

    Aggregatibacter actinomycetemcomitans is an important periodontal pathogen that can participate in periodontitis and other non-oral infections. The cytolethal distending toxin (Cdt) is among the virulence factors produced by this bacterium. The Cdt is also secreted by several mucosa-associated Gram-negative pathogens and may play a role in perpetuating the infection by modulating the immune response. Although the toxin targets a wide range of eukaryotic cell types little is known about its activity on macrophages which play a key part in alerting the rest of the immune system to the presence of pathogens and their virulence factors. In view of this, we tested the hypothesis that the A. actinomycetemcomitans Cdt (AaCdt) disrupts macrophage function by inhibiting phagocytic activity as well as affecting the production of cytokines. Murine macrophages were co-cultured with either wild-type A. actinomycetemcomitans or a Cdt(-) mutant. Viable counts and qPCR showed that phagocytosis of the wild-type strain was significantly reduced relative to that of the Cdt(-) mutant. Addition of recombinant Aa(r)Cdt to co-cultures along with the Cdt(-) mutant diminished the phagocytic activity similar to that observed with the wild type strain. High concentrations of Aa(r)Cdt resulted in decreased phagocytosis of fluorescent bioparticles. Nitric oxide production was modulated by the presence of Cdt and the levels of IL-1β, IL-12 and IL-10 were increased. Production of TNF-α did not differ in the co-culture assays but was increased by the presence of Aa(r)Cdt. These data suggest that the Cdt may modulate macrophage function in A. actinomycetemcomitans infected sites by impairing phagocytosis and modifying the pro-inflammatory/anti-inflammatory cytokine balance.

  5. Immune suppression induced by Actinobacillus actinomycetemcomitans: effects on immunoglobulin production by human B cells.

    PubMed Central

    Shenker, B J; Vitale, L A; Welham, D A

    1990-01-01

    Actinobacillus actinomycetemcomitans produces an immunosuppressive factor (ISF) which has been shown to suppress mitogen- and antigen-induced DNA, RNA, and protein synthesis in human T lymphocytes. In this study, we examined purified A. actinomycetemcomitans ISF for its ability to alter immunoglobulin production by human B cells. The ISF caused a dose-dependent inhibition of pokeweed mitogen (PWM)-induced immunoglobulin G (IgG) and IgM production. Preexposure to ISF was not required to achieve maximal inhibition of immunoglobulin synthesis, as previously observed for its effect on T-cell activation. Nevertheless, the ISF appeared to act by irreversibly affecting the early stages of cell activation. While PWM-induced immunoglobulin production is under the influence of T-regulatory circuits, it appears that the ISF interacts directly with B cells. First, ISF failed to alter either the synthesis of interleukin-2 (IL-2) or the expression of IL-2 receptors on T cells. Second, experiments in which individual purified populations of cells were exposed to ISF, washed, and placed back into tissue culture indicated that when all cells (i.e., T cells, B cells, and monocytes) were exposed to ISF, significant suppression was observed. However, when only one cell population was treated with ISF, suppression of both IgG and IgM synthesis was observed only when the B-cell-enriched population was exposed to ISF. These results in conjunction with our earlier findings suggest that the ISF functions via the activation of a regulatory subpopulation of B lymphocytes, which in turn either directly or indirectly (via suppressor T cells) downregulate both B- and T-cell responsiveness. Furthermore, it is hypothesized that patients who harbor A. actinomycetemcomitans could suffer from local or systemic immune suppression. This suppression may enhance the pathogenicity of A. actinomycetemcomitans itself or that of some other opportunistic organism. Images PMID:2254014

  6. Actinobacillus actinomycetemcomitans serotype e--biotypes, genetic diversity and distribution in relation to periodontal status.

    PubMed

    Doğan, B; Saarela, M H; Jousimies-Somer, H; Alaluusua, S; Asikainen, S

    1999-04-01

    Actinobacillus actinomycetemcomitans isolates from 356 individuals were screened for identification of serotype e in order to investigate its distribution in relation to periodontal status. From subjects with serotype e, 1-6 isolates per subject (n = 61) were genotyped using arbitrarily primed-polymerase chain reaction (AP-PCR) and apaH gene polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis to determine the genetic heterogeneity within the serotype. Furthermore, one serotype e strain per subject was tested for fermentation of 8 carbohydrates for biotyping. Among patients with adult periodontitis (n = 219), localized juvenile periodontitis (n = 55) and other forms of early-onset periodontitis (n = 18) serotypes b, a and c, respectively, were the most frequently detected serotypes. Non-periodontitis subjects (n = 64) were predominantly colonized with serotype c. Serotype e was found in 30 (14%) adult periodontitis patients, 2 (11%) early-onset periodontitis patients and in 5 (8%) non-periodontitis individuals, but in none of the 55 localized juvenile periodontitis patients. AP-PCR distinguished 3 and apaH gene PCR-RFLP analysis 2 genotypes among the 61 A. actinomycetemcomitans serotype e isolates, one genotype per subject. The AP-PCR genotypes 1 and 3 represented the apaH genotype 1 and the AP-PCR genotype 2 the apaH genotype 2. On the basis of variable fermentation of galactose and xylose, 3 biotypes among A. actinomycetemcomitans serotype e were established. Contrary to the absence of A. actinomycetemcomitans serotype e in localized juvenile periodontitis patients, its detection frequency was comparable among other forms of periodontitis and periodontal health. Clinical serotype e isolates form at least 2 genetic types and 3 biotypes.

  7. DNA analysis of temperate bacteriophage Aa(phi)23 isolated from actinobacillus actinomycetemcomitans.

    PubMed

    Willi, K; Meyer, J

    1998-05-01

    The DNA of the temperate bacteriophage Aaphi23 isolated from the oral bacterium Actinobacillus actinomycetemcomitans was examined structurally both in the phage head and in the prophage. The DNA in phage particles comprises 44 kb linear molecules with a terminal redundancy of 1.6 kb, which represent circular permutations. Thus, DNA is packaged into phage heads by the headful mechanism. The Aaphi23 prophage is integrated into the host chromosome.

  8. 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

  9. 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

  10. 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

  11. Essential oils and isolated compounds from Lippia alba leaves and flowers: antimicrobial activity and osteoclast apoptosis.

    PubMed

    Juiz, Paulo José Lima; Lucchese, Angelica Maria; Gambari, Roberto; Piva, Roberta; Penolazzi, Letizia; Di Ciano, Martina; Uetanabaro, Ana Paula Trovatti; Silva, Franceli; Avila-Campos, Mario Julio

    2015-01-01

    In the present study, essential oils extracted from the leaves and flowers of Lippia alba (Mill.) N.E.Br. (L. alba) were analyzed for their antimicrobial activity and their effects on osteoclasts. The periodontal pathogens, Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans; ATCC 43717), Fusobacterium nucleatum (F. nucleatum; ATCC 25586) and Porphyromonas gingivalis (P. gingivalis); ATCC 33277) were used in antimicrobial activity assays for determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), whereas Bacteroides fragilis (B. fragilis; ATCC 25285) was used as the control microorganism. Osteoclast (OC) apoptosis was assessed by TUNEL assay and Fas receptor expression was detected by immunocytochemistry. The analysis of antimicrobial activity revealed that P. gingivalis had the lowest MIC values, whereas A. actinomycetemcomitans had the highest. L. alba essential oils were found to be toxic to human cells, although the compounds, carvone, limonene and citral, were non-toxic and induced apoptosis in the OCs. This study demonstrates that L. alba has potential biotechnological application in dentistry. In fact periodontal disease has a multifactorial etiology, and the immune response to microbial challenge leads to osteoclast activation and the resorption of the alveolar bone, resulting in tooth loss.

  12. 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.

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

    PubMed Central

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

    2013-01-01

    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. PMID:23353051

  14. Human Infection with Fusobacterium necrophorum (Necrobacillosis), with a Focus on Lemierre's Syndrome†

    PubMed Central

    Riordan, Terry

    2007-01-01

    Summary: Human infection with Fusobacterium necrophorum usually involves F. necrophorum subsp. funduliforme rather than F. necrophorum subsp. necrophorum, which is a common pathogen in animals. Lemierre's syndrome, or postanginal sepsis, is the most common life-threatening manifestation. Tonsillitis is followed by septic thrombophlebitis of the internal jugular vein and then a septicemia with septic emboli in lungs and other sites. Recent evidence suggests that F. necrophorum can be limited to the throat and cause persistent or recurrent tonsillitis. F. necrophorum is unique among non-spore-forming anaerobes, first for its virulence and association with Lemierre's syndrome as a monomicrobial infection and second because it seems probable that it is an exogenously acquired infection. The source of infection is unclear; suggestions include acquisition from animals or human-to-human transmission. Approximately 10% of published cases are associated with infectious mononucleosis, which may facilitate invasion. Recent work suggests that underlying thrombophilia may predispose to internal jugular vein thrombophlebitis. Lemierre's syndrome was relatively common in the preantibiotic era but seemed to virtually disappear with widespread use of antibiotics for upper respiratory tract infection. In the last 15 years there has been a rise in incidence, possibly related to restriction in antibiotic use for sore throat. PMID:17934077

  15. Nucleotide sequence of the leukotoxin gene from Actinobacillus actinomycetemcomitans: homology to the alpha-hemolysin/leukotoxin gene family.

    PubMed Central

    Kraig, E; Dailey, T; Kolodrubetz, D

    1990-01-01

    The leukotoxin produced by Actinobacillus actinomycetemcomitans has been implicated in the etiology of localized juvenile periodontitis. To initiate a genetic analysis into the role of this protein in disease, we have cloned its gene, lktA. We now present the complete nucleotide sequence of the lktA gene from A. actinomycetemcomitans. When the deduced amino acid sequence of the leukotoxin protein was compared with those of other proteins, it was found to be homologous to the leukotoxin from Pasteurella haemolytica and to the alpha-hemolysins from Escherichia coli and Actinobacillus pleuropneumoniae. Each alignment showed at least 42% identity. As in the other organisms, the lktA gene of A. actinomycetemcomitans was linked to another gene, lktC, which is thought to be involved in the activation of the leukotoxin. The predicted LktC protein was related to the leukotoxin/hemolysin C proteins from the other bacteria, since they shared a minimum of 49% amino acid identity. Surprisingly, although actinobacillus species are more closely related to pasteurellae than to members of the family Enterobacteriaciae, LktA and LktC from A. actinomycetemcomitans shared significantly greater sequence identity with the E. coli alpha-hemolysin proteins than with the P. haemolytica leukotoxin proteins. Despite the overall homology to the other leukotoxin/hemolysin proteins, the LktA protein from A. actinomycetemcomitans has several unique properties. Most strikingly, it is a very basic protein with a calculated pI of 9.7; the other toxins have estimated pIs around 6.2. The unusual features of the A. actinomycetemcomitans protein are discussed in light of the different species and target-cell specificities of the hemolysins and the leukotoxins. Images PMID:2318535

  16. Quantitative PCR studies of Aggregatibacter actinomycetemcomitans and Treponema denticola/Tanerella forsythensis Complex as Etiological Factors of Chronic Periodontitis.

    PubMed

    Yanushevich, O O; Ayvazova, R A; Shibaeva, A V; Rebrikov, D V; Trubnikova, E V; Kudykina, Yu K; Zylnikova, M V; Zaripova, R S; Shevelev, A B

    2016-02-01

    Real-time quantitative PCR (Dentofl or kit) was used to detect DNA of periodontal pathogens in specimens from 92 patients with chronic periodontitis and from a control sample of 12 normal subjects. A bimodal distribution of patients by periodontium colonization with A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythensis, and T. denticola was demonstrated. A new approach to interpretation of the results of quantitative evaluation of periodontal pathogens, including the notion of pathological colonization level, led to classification of all cases with chronic generalized periodontitis into 3 groups: associated with A. actinomycetemcomitans, with T. forsythensis/T. denticola complex, and cases of uncertain genesis.

  17. Antigens of Actinobacillus actinomycetemcomitans recognized by patients with juvenile periodontitis and periodontally normal subjects.

    PubMed Central

    Sims, T J; Moncla, B J; Darveau, R P; Page, R C

    1991-01-01

    Most juvenile periodontitis patients respond to infection by Actinobacillus actinomycetemcomitans by producing serum antibodies. Specific antigens inducing the humoral immune response have not been identified, nor has the role of the resulting antibodies in disease progression been determined. Adsorbed and unadsorbed sera from juvenile periodontitis patients and normal subjects were analyzed by enzyme-linked immunosorbent assay and Western blots (immunoblots), using digested and undigested bacterial sonicates and French pressure cell fractions to determine the biochemical class, cross-reactivity, and cellular location of the antigens in different A. actinomycetemcomitans serotypes. Antigens detected by using high-titer sera included the following: (i) serotype-specific nonprotein material located on the cell surface, (ii) soluble-fraction proteins showing highly variable antibody binding, (iii) cross-reactive proteins, and (iv) a protein present in soluble and cell wall fractions and immunopositive for all sera tested. In addition, one apparently nonprotein component that was enriched in the cell wall fraction was observed. Sera with high immunoglobulin G titers to one, two, three, or none of the three A. actinomycetemcomitans serotypes were observed. There was a high degree of variation from one patient to another in the humoral immune response to serotype-specific and cross-reactive antigens. As demonstrated by whole-cell adsorption experiments, the serotype-specific surface antigen accounted for approximately 72 to 90% of the total antibody-binding activity for sera with titers greater than 100-fold above background, while cross-reactive antigen accounted for less than 28%. Antibody binding the whole-cell sonicate for high-titer sera was inhibited 90% by lipopolysaccharide from the same serotype, strongly suggesting that lipopolysaccharide is the immunodominant antigen class. Images PMID:1705243

  18. Regulation of leukotoxin in leukotoxic and nonleukotoxic strains of Actinobacillus actinomycetemcomitans.

    PubMed Central

    Spitznagel, J; Kraig, E; Kolodrubetz, D

    1991-01-01

    Actinobacillus actinomycetemcomitans is a gram-negative bacterium that has been implicated in the etiology of several forms of periodontitis, especially localized juvenile periodontitis. A potent leukotoxin (Lkt) is produced by most A. actinomycetemcomitans isolates from patients with periodontal disease, but some isolates are leukotoxin nonproducing (Lkt-). The molecular bases for the differences in leukotoxin expression are being explored to clarify the role of leukotoxin in pathogenesis. We have previously cloned the leukotoxin structural gene, lktA, from the leukotoxin-producing (Lkt+) strain JP2 and have shown that it is linked to three other genes, lktB, lktC, and lktD, whose gene products are thought to be required for activation and localization of the leukotoxin. These genes have now been used in Southern blot analysis to demonstrate that Lkt- strains, like Lkt+ strains, contain all four genes of the lkt gene cluster. While restriction fragment length polymorphisms were detected, they did not correlate with toxin phenotype. RNA blot analysis demonstrated that Lkt+ strains produced two transcripts, one 9.3 kb in length and the other 4.3 kb. They encode lktCABD and lktCA. respectively. Lkt- strains contained significantly lower levels of the 4.3-kb transcript with no discernible 9.3-kb message. The leukotoxic activity of the A. actinomycetemcomitans strains, measured by chromium release assays, correlated with the lkt RNA content. Therefore, a major component of leukotoxin regulation is at the level of RNA transcription or stability. Interestingly, the lkt RNAs in JP2 are regulated during growth phase, being greatly reduced in cells approaching stationary phase. Thus, the regulation of lkt RNA can be affected by both genotype and environment. Images PMID:2004819

  19. Identification and characterization of genetic cluster groups of Actinobacillus actinomycetemcomitans isolated from the human oral cavity.

    PubMed Central

    DiRienzo, J M; McKay, T L

    1994-01-01

    Actinobacillus actinomycetemcomitans is recognized as a primary pathogen in localized juvenile periodontitis (LJP). Restriction fragment length polymorphisms (RFLP) within a collection of subgingival plaque isolates of this bacterium were identified and characterized as the first step in understanding the pathogenesis of LJP. Over 800 isolates, from members of 18 families (LJP families) with at least one member with active LJP or a documented history of the disease and one or more siblings, less than 13 years of age, having no clinical evidence of LJP and 32 healthy control subjects, were assigned to one of 13 distinct RFLP groups (II to XIV) by using a previously characterized 4.7-kb DNA probe cloned from the reference strain FDC Y4. Isolates belonging to RFLP groups II, IV, V, and XIII predominated subgingival sites in the subjects. Members of RFLP groups II, IV, VII, VIII, X, and XI were recovered only from LJP family subjects, while group XIII and XIV variants were found exclusively in healthy controls. A synthetic oligonucleotide, homologous to the 5' end of the leukotoxin gene (lktA), and the A. actinomycetemcomitans plasmid, pVT745, were tested for their abilities to subdivide the 13 RFLP groups. The leukotoxin probe specifically identified all RFLP group II variants because of the absence of a HindIII site in the upstream noncoding region of the lkt gene complex. The plasmid probe was not as selective but may be useful for identifying clinical isolates belonging to RFLP group I. The use of these probes for the identification of genetic variants of A. actinomycetemcomitans that may be preferentially colonize diseased and healthy subjects will facilitate the study of the role of this important pathogen in periodontal diseases. Images PMID:7907346

  20. Effects of Aggregatibacter actinomycetemcomitans leukotoxin on neutrophil migration and extracellular trap formation

    PubMed Central

    Hirschfeld, Josefine; Roberts, Helen M.; Chapple, Iain L. C.; Parčina, Marijo; Jepsen, Søren; Johansson, Anders; Claesson, Rolf

    2016-01-01

    Background Aggressive periodontitis is associated with the presence of Aggregatibacter actinomycetemcomitans, a leukotoxin (Ltx)-producing periodontal pathogen. Ltx has the ability to lyse white blood cells including neutrophils. Objectives This study was aimed at investigating the interactions between neutrophils and Ltx with regard to the chemotactic properties of Ltx and the release of neutrophil extracellular traps (NETs). Methods Neutrophils from healthy blood donors were isolated and incubated for 30 min and 3 h with increasing concentrations of Ltx (1, 10, and 100 ng/mL) as well as with A. actinomycetemcomitans strains (NCTC 9710 and HK 1651) producing different levels of Ltx. Formation of NETs and cell lysis were assessed by microscopy, fluorescence-based assays, and measurement of released lactate dehydrogenase. Neutrophil migration in response to different Ltx gradients was monitored by real-time video microscopy, and image analysis was performed using ImageJ software. Results Although Ltx (10 and 100 ng/mL) and the leukotoxic A. actinomycetemcomitans strain HK 1651 lysed some neutrophils, other cells were still capable of performing NETosis in a concentration-dependent manner. Low doses of Ltx and the weakly leukotoxic strain NCTC 9710 did not lead to neutrophil lysis, but did induce some NETosis. Furthermore, all three concentrations of Ltx enhanced random neutrophil movement; however, low directional accuracy was observed compared with the positive control (fMLP). Conclusions The results indicate that Ltx acts both as a neutrophil activator and also causes cell death. In addition, Ltx directly induces NETosis in neutrophils prior to cell lysis. In future studies, the underlying pathways involved in Ltx-meditated neutrophil activation and NETosis need to be investigated further. PMID:27834173

  1. In vitro degradation of lysine by ruminal fluid-based fermentations and by Fusobacterium necrophorum.

    PubMed

    Elwakeel, E A; Amachawadi, R G; Nour, A M; Nasser, M E A; Nagaraja, T G; Titgemeyer, E C

    2013-01-01

    The objective of these studies was to characterize some factors affecting lysine degradation by mixed ruminal bacteria and by ruminal Fusobacterium necrophorum. Mixed ruminal bacteria degraded lysine, and addition of pure cultures of F. necrophorum did not increase lysine degradation. Addition of acetic or propionic acid strikingly reduced NH(3) production from lysine by mixed ruminal bacteria at pH 6, but not at pH 7. Although typical ruminal environments with acidic pH and normal concentrations of volatile fatty acids might inhibit lysine degradation by F. necrophorum, ruminal fluid contained enough bacteria with a lysine-degrading capacity to ferment 50 mM lysine in vitro. Of 7 strains of ruminal F. necrophorum tested, all grew on both lactate and lysine as the primary energy source. Both subspecies of ruminal F. necrophorum (necrophorum and funduliforme) used lysine as a primary C and energy source. Lysine and glutamic acid were effectively fermented by F. necrophorum, but alanine and tryptophan were not, and histidine and methionine were fermented only to a minor extent. The end products of lactate fermentation by F. necrophorum were propionate and acetate, and those of lysine degradation were butyrate and acetate. Fermentation of glutamic acid by F. necrophorum yielded acetate and butyrate in a ratio near to 2:1. The minimum inhibitory concentration of tylosin for F. necrophorum was not dependent on whether bacteria were grown with lactate or lysine, but F. necrophorum was more susceptible to monensin when grown on lysine than on lactate. Although F. necrophorum is generally resistant to monensin, the ionophore may reduce lysine degradation by F. necrophorum in the rumen. The essential oil components limonene, at 20 or 100 μg/mL, and thymol, at 100 μg/mL, inhibited F. necrophorum growth, whereas eugenol, guaiacol, and vanillin had no effect. Our findings may lead to ways to minimize ruminal lysine degradation and thus increase its availability to the animal.

  2. 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

  3. Killing of human myelomonocytic leukemia and lymphocytic cell lines by Actinobacillus actinomycetemcomitans leukotoxin.

    PubMed Central

    Simpson, D L; Berthold, P; Taichman, N S

    1988-01-01

    The purified leukotoxin of Actinobacillus actinomycetemcomitans kills human leukemic cell lines (e.g., HL-60, U937, and KG-1) and human T- and B-cell lines (e.g., JURKAT, MOLT-4, Daudi, and Raji) in a dose- and time-dependent manner. The 50% effective doses for these cell lines are similar to those established for human polymorphonuclear leukocytes and monocytes. In contrast, other human and nonhuman tumor cell lines are not susceptible to the leukotoxin. These human leukemia and lymphoid cell lines will serve as useful model systems with which to study the molecular specificity and mechanism(s) of action of the actinobacillus leukotoxin. Images PMID:3258584

  4. 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

  5. Cloning and sequencing of part of the S10 operon from Actinobacillus actinomycetemcomitans FDC Y4.

    PubMed

    Hayashida, H; Hotokezaka, H; Ohara, N; Kimura, M; Takagi, O; Yamada, T

    1997-06-01

    We have cloned and sequenced the 5.2 kb EcoRI fragment that contained part of the S10 operon from Actinobacillus actinomycetemcomitans FDC Y4. The order of the ribosomal protein genes was identical to that of the S10 operon of Haemophilus influenzae and Escherichia coli. The deduced amino acid sequences of ribosomal proteins in this operon displayed significant homologies (65.3%-100%) to those of H. influenzae, E. coli, Yersinia enterocolitica and Yersinia pseudotuberculosis. Phylogenetic trees obtained for these ribosomal proteins were similar to that obtained for 16S rRNA.

  6. Gene cloning of an Actinobacillus actinomycetemcomitans Y4 antigen which reacts with peripheral blood sera in patients with advanced destructive periodontitis.

    PubMed

    Arakawa, S; Hata, S; Ishikawa, I; Tsuchida, N

    1990-01-01

    Actinobacillus actinomycetemcomitans has been implicated in the aetiology of juvenile periodontitis and advanced destructive periodontitis. Levels of IgG antibody against A. actinomycetemcomitans in peripheral blood sera of patients with advanced destructive periodontitis are high, as are those against Bacteroides gingivalis. To clone the genes of antigens reactive with sera of such patients, a library of the A. actinomycetemcomitans strain Y4 DNA in lambda L47 was constructed and then screened, using an immunochemical detection method, with serum from a patient with the advanced disease. Six clones from among nearly 1000 reacted with the serum and also with that of another patient. They were designated 3, 4, 6, 7, 8 and 9. Restriction enzyme and Southern blot analyses indicated that clones 8 and 9 were identical and that all the clones were overlapping because they shared in common the 4 and 5 kbp HincII DNA fragments of A. actinomycetemcomitans. The cloned DNA fragment hybridized to the DNA of two other strains of A. actinomycetemcomitans but not to those of six periodontopathic bacteria examined. These findings suggest that a DNA sequence encoding an A. actinomycetemcomitans strain Y4 antigen strongly reactive with sera of patients with advanced destructive periodontitis was cloned. This sequence is present specifically in A. actinomycetemcomitans but not in other bacteria isolated from patients with periodontal diseases. Thus, the cloned DNA could serve as a probe for the diagnosis of periodontitis.

  7. Distinctive characteristics of transcriptional profiles from two epithelial cell lines upon interaction with Actinobacillus actinomycetemcomitans.

    PubMed

    Mans, J J; Baker, H V; Oda, D; Lamont, R J; Handfield, M

    2006-08-01

    Transcriptional profiling and gene ontology analyses were performed to investigate the unique responses of two different epithelial cell lines to an Actinobacillus actinomycetemcomitans challenge. A total of 2867 genes were differentially regulated among all experimental conditions. The analysis of these 2867 genes revealed that the predominant specific response to infection in HeLa cells was associated with the regulation of enzyme activity, RNA metabolism, nucleoside and nucleic acid transport and protein modification. The predominant specific response in immortalized human gingival keratinocytes (IHGK) was associated with the regulation of angiogenesis, chemotaxis, transmembrane receptor protein tyrosine kinase signaling, cell differentiation, apoptosis and response to stress. Of particular interest, stress response genes were significantly - yet differently - affected in both cell lines. In HeLa cells, only three regulated genes impacted the response to stress, and the response to unfolded protein was the only term that passed the ontology filters. This strikingly contrasted with the profiles obtained for IHGK, in which 61 regulated genes impacted the response to stress and constituted an extensive network of cell responses to A. actinomycetemcomitans interaction (response to pathogens, oxidative stress, unfolded proteins, DNA damage, starvation and wounding). Hence, while extensive similarities were found in the transcriptional profiles of these two epithelial cell lines, significant differences were highlighted. These differences were predominantly found in pathways that are associated with host-pathogen interactions.

  8. 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.

  9. 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.

  10. Integration host factor is required for replication of pYGK-derived plasmids in Aggregatibacter actinomycetemcomitans.

    PubMed

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

    2014-08-01

    In this study, we show that integration host factor protein (IHF) is required for replication of pYGK plasmids in Aggregatibacter actinomycetemcomitans. YGK plasmids were not replicated in A. actinomycetemcomitans strains lacking either the α- or β- subunit of IHF. However, the deletion mutants were complemented, and plasmid replication was restored when the promoter region and gene for either ihfA or ihfB was cloned into pYGK. We also identified two motifs that resemble the consensus IHF-binding site in a 813-bp fragment containing the pYGK origin of replication. Using electrophoretic mobility shift assays, purified IHFα-IHFβ protein complex was shown to bind to probes containing either of these motifs. To our knowledge, this is the first report showing that plasmid replication is IHF-dependent in the family Pasteurellaceae. In addition, using site-direct mutagenesis, the XbaI and KpnI restriction sites in the suicide vector pJT1 were modified to generate plasmid pJT10. The introduction of these new unique sites in pJT10 facilitates the transfer of transcriptional or translational lacZ fusion constructs for the generation of single-copy chromosomal insertion of the reporter construct. Plasmid pJT10 and its derivatives will be useful for genetic studies in Aggregatibacter (Actinobacillus) and probably other genera of Pasteurellaceae, including Haemophilus, Pasteurella, and Mannheimia.

  11. Aggregatibacter actinomycetemcomitans: Virulence of its leukotoxin and association with aggressive periodontitis

    PubMed Central

    Åberg, Carola Höglund; Kelk, Peyman; Johansson, Anders

    2015-01-01

    Periodontitis is an infection-induced inflammatory disease that causes loss of the tooth supporting tissues. Much focus has been put on comparison of the microbial biofilm in the healthy periodontium with the diseased one. The information arising from such studies is limited due to difficulties to compare the microbial composition in these two completely different ecological niches. A few longitudinal studies have contributed with information that makes it possible to predict which individuals who might have an increased risk of developing aggressive forms of periodontitis, and the predictors are either microbial or/and host-derived factors. The most conspicuous condition that is associated with disease risk is the presence of Aggregatibacter actinomycetemcomitans at the individual level. This Gram-negative bacterium has a great genetic variation with a number of virulence factors. In this review we focus in particular on the leukotoxin that, based on resent knowledge, might be one of the most important virulence factors of A. actinomycetemcomitans. PMID:25494963

  12. 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

  13. 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.

  14. Occurrence of temperate bacteriophages in different Actinobacillus actinomycetemcomitans serotypes isolated from periodontally healthy individuals.

    PubMed

    Willi, K; Sandmeier, H; Asikainen, S; Saarela, M; Meyer, J

    1997-02-01

    The occurrence of temperate bacteriophages was studied in 34 isolates of Actinobacillus actinomycetemcomitans derived from 27 periodontally healthy Finnish individuals both by lysis/plaque assays and by DNA hybridizations. In addition the serotype, the ribotype and the arbitrarily primed polymerase chain reaction (AP-PCR) profile were determined for each A. actinomycetemcomitans strain. Fourteen isolates showed hybridization patterns very similar to that of a known lysogen when probed with the genome of the previously characterized temperate phage Aa phi 23. Only 6 of these 14 strains had produced lysis or single plaques on suitable indicator strains. Phage Aa phi 247 derived from one of these lysogens was indistinguishable from Aa phi 23 by electron microscopy, and the genomes showed highly related DNA hybridization patterns. The remaining 20 isolates exhibited hybridization patterns very different from that of Aa phi 23 DNA. Seven of these strains also gave lysis or single plaques, suggesting that 21 of the 34 strains were lysogenic. These data indicate that the prophages per se do not represent a virulence factor exclusively associated with periodontal disease. Presence of an Aa phi 23-related prophage correlated with serotype a and AP-PCR type 1 of the bacterial host. This may indicate that Aa phi 23 and related phages have a limited host range.

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

    PubMed

    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.

  16. Resistance of Actinobacillus actinomycetemcomitans and differential susceptibility of oral Haemophilus species to the bactericidal effects of hydrogen peroxide.

    PubMed Central

    Miyasaki, K T; Wilson, M E; Reynolds, H S; Genco, R J

    1984-01-01

    We compared the sensitivities of oral and nonoral isolates of Actinobacillus actinomycetemcomitans, Haemophilus segnis, H. aphrophilus, and H. paraphrophilus to the bactericidal action of reagent hydrogen peroxide (H2O2). Susceptibility to a range of H2O2 concentrations (10(-6) to 10(-3) M) was assessed by incubating bacterial suspensions for 1 h at 37 degrees C in the presence of H2O2 and plating on chocolate agar to determine the concentration of H2O2 that would produce a 50% reduction in CFU (50% lethal dose). As a group, A. actinomycetemcomitans was more resistant to H2O2 than the oral haemophili, and H. aphrophilus was much more sensitive than all other organisms tested. The range of 50% lethal dose values for A. actinomycetemcomitans was between 8.5 X 10(-5) and 10(-3) M H2O2 or above. In contrast, H. aphrophilus exhibited 50% lethal dose values from below 1 X 10(-6) to 3.4 X 10(-4) M H2O2. The resistance of A. actinomycetemcomitans to H2O2 may be sufficient to protect these organisms from direct H2O2-mediated killing by host phagocytes. PMID:6500706

  17. Interleukin-1β is internalised by viable Aggregatibacter actinomycetemcomitans biofilm and locates to the outer edges of nucleoids.

    PubMed

    Paino, Annamari; Lohermaa, Elina; Sormunen, Raija; Tuominen, Heidi; Korhonen, Jari; Pöllänen, Marja T; Ihalin, Riikka

    2012-11-01

    The opportunistic pathogen Aggregatibacter actinomycetemcomitans causes periodontitis, which is a biofilm infection that destroys tooth-supportive tissues. Interleukin (IL)-1β, a central proinflammatory cytokine of periodontitis, is an essential first line cytokine for local inflammation that modulates the cell proliferation and anti-pathogen response of human gingival keratinocytes. Previously, we demonstrated that A. actinomycetemcomitans biofilms bind IL-1β; however, whether this binding is an active process is not known. In this study, we showed for the first time with immuno-electron microscopy that viable bacterial biofilm cells internalised IL-1β when co-cultured with an organotypic mucosa. Decreased biofilm viability hindered the ability of biofilm to sequester IL-1β and caused IL-1β leakage into the culture medium. In some A. actinomycetemcomitans cells, intracellular IL-1β localized to the outer edges of the nucleoids. We identified the DNA-binding protein HU as an IL-1β interacting protein with mass spectroscopy and showed the interaction of recombinant HU and IL-1βin vitro using enzyme-linked immunosorbent assay (ELISA). Close contact with a viable A. actinomycetemcomitans biofilm decreased the proliferation and apoptosis of human gingival keratinocytes as demonstrated using Ki-67 and the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining, respectively. Our results suggest that viable A. actinomycetemcomitans biofilms may disturb the critical first steps of local inflammation in periodontitis by binding and internalising IL-1β. The interaction of IL-1β with conserved HU provides a potential mechanism for shaping bacterial gene expression.

  18. 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

  19. 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.

  20. In Vitro Effect of Porphyromonas gingivalis Methionine Gamma Lyase on Biofilm Composition and Oral Inflammatory Response

    PubMed Central

    Stephen, Abish S.; Millhouse, Emma; Sherry, Leighann; Aduse-Opoku, Joseph; Culshaw, Shauna; Ramage, Gordon; Bradshaw, David J.; Burnett, Gary R.; Allaker, Robert P.

    2016-01-01

    Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (p<0.001), whilst A. naeslundii (p<0.01) and Streptococcus spp. (p<0.05) decreased in mutant biofilms. Keratinocytes produced less IL-8, IL-6 and IL-1α when stimulated with the mutant biofilms compared to wild type. Lack of mgl in P. gingivalis has been shown to affect microbial ecology in vitro, giving rise to a markedly different biofilm composition, with a more pro-inflammatory cytokine response from the keratinocytes observed. A possible role for methanethiol in biofilm formation

  1. Association of Actinobacillus actinomycetemcomitans leukotoxin with nucleic acids on the bacterial cell surface.

    PubMed Central

    Ohta, H; Hara, H; Fukui, K; Kurihara, H; Murayama, Y; Kato, K

    1993-01-01

    Actinobacillus actinomycetemcomitans, a periodontopathic gram-negative bacterium, produces a leukotoxin that is a member of the RTX cytotoxin family. Although genes may function in toxin secretion, the leukotoxin is not secreted extracellularly but remains associated with the bacterial cell surface. We report here that this toxin-cell surface association is mediated by nucleic acids and directly demonstrate that the extracellular secretion of toxin occurs in growing cultures with increased ionic strength of medium. All examinations were performed with freshly harvested A. actinomycetemcomitans 301-b from anaerobic fructose-limited chemostat cultures. The occurrence of cell surface-localized DNA was shown by directly digesting whole cells with the restriction endonuclease EcoRI or HindIII, which yielded many DNA fragments. The cell surface DNA constituted about 20% of the total cellular DNA. The leukotoxin was released from the whole cells by digestion with DNase I as well as restriction endonucleases. Because the leukotoxin binds ionically to DNA, it is dependent on the ionic strength of buffers or media. Accordingly, the toxin was released from cells suspended in saline at pH 7.5 in the presence of increasing amounts of MgCl2 (0 to 10 mM) or NaCl (0 to 50 mM). Moreover, a considerable quantity of leukotoxin was detected in the culture supernatant of fructose-limited chemostat cultures when sodium succinate solution was pumped into the steady state as an additional salt (30 and then 50 mM). This toxin-DNA association was also found in well-characterized strains including not only the leukotoxin-producing ATCC 29522 but also the toxin production-variable ATCC 29523 and the non-leukotoxin-producing ATCC 33384 when these strains were grown in the chemostat culture. Images PMID:8406888

  2. Setup of an In Vitro Test System for Basic Studies on Biofilm Behavior of Mixed-Species Cultures with Dental and Periodontal Pathogens

    PubMed Central

    Standar, Kerstin; Kreikemeyer, Bernd; Redanz, Sylvio; Münter, Wanja L.; Laue, Michael; Podbielski, Andreas

    2010-01-01

    Background Caries and periodontitis are important human diseases associated with formation of multi-species biofilms. The involved bacteria are intensively studied to understand the molecular basis of the interactions in such biofilms. This study established a basic in vitro single and mixed-species culture model for oral bacteria combining three complimentary methods. The setup allows a rapid screening for effects in the mutual species interaction. Furthermore, it is easy to handle, inexpensive, and reproducible. Methods Streptococcus mitis, S. salivarius and S. sanguinis, typical inhabitants of the healthy oral cavity, S. mutans as main carriogenic species, and Porphyromonas gingivalis, Fusobacterium nucleatum, Parvimonas micra, S. intermedius and Aggregatibacter actinomycetemcomitans as periodontitis-associated bacteria, were investigated for their biofilm forming ability. Different liquid growth media were evaluated. Safranin-staining allowed monitoring of biofilm formation under the chosen conditions. Viable counts and microscopy permitted investigation of biofilm behavior in mixed-species and transwell setups. Findings S. mitis, F. nucleatum, P. gingivalis and P. micra failed to form biofilm structures. S. mutans, S. sanguinis, S. intermedius and S. salivarius established abundant biofilm masses in CDM/sucrose. A. actinomycetemcomitans formed patchy monolayers. For in depth analysis S. mitis, S. mutans and A. actinomycetemcomitans were chosen, because i) they are representatives of the physiological-, cariogenic and periodontitis-associated bacterial flora, respectively and ii) their difference in their biofilm forming ability. Microscopic analysis confirmed the results of safranin staining. Investigation of two species combinations of S. mitis with either S. mutans or A. actinomycetemcomitans revealed bacterial interactions influencing biofilm mass, biofilm structure and cell viability. Conclusions This setup shows safranin staining, microscopic analysis and

  3. Despite large-scale T cell activation, only a minor subset of T cells responding in vitro to Actinobacillus actinomycetemcomitans differentiate into effector T cells.

    PubMed

    Zadeh, H H; Tanavoli, S; Haines, D D; Kreutzer, D L

    2000-06-01

    Recent studies in our laboratory have demonstrated that Actinobacillus actinomycetemcomitans has a potent T cell stimulatory effect, activating more than half of all T cells. However, since the fate of these activated T cells was not known, the present study sought to determine whether all of these T cells differentiate into effector cells. To that end, the intracellular expression of T cell cytokines (IL-2, IFN-gamma, IL-4 and IL-10) in response to A. actinomycetemcomitans was determined by flow cytometry. Results demonstrated a time-dependent increase in the expression of the cytokines, most reaching peak levels at 24-48 h. At 48 h, the proportion of T cells expressing each of the cytokines were as follows: IL-2 (1.7%+/-0.3), IFN-gamma (1.8%+/-0.5), IL-4 (1.0%+/-0.2) and IL-10 (1.5%+/-0.5). These data indicated that only 2-5% of all T cells stimulated with A. actinomycetemcomitans expressed any T cell cytokines. The finding of large-scale T cell activation in the absence of cytokine expression suggests that the activation of T cells in response to A. actinomycetemcomitans is incomplete. To investigate this phenomenon, peripheral blood mononuclear cells (PBMC) were cultured with A. actinomycetemcomitans for 24 h followed by sorting of the activated (CD69+) cells by immunomagnetic separation and restimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Results demonstrated that nearly 90% of the T cells were unresponsive to further restimulation. A possible explanation for this unresponsiveness is the induction of clonal anergy among the responding T cells. To determine possible preferential effects of the stimulation on specific cytokines, the expression of each cytokine among T cells responding to A. actinomycetemcomitans was compared to the maximum levels achieved by PMA + ionomycin stimulation. Results showed that number of IL-2+ and IFN-gamma+ T cells observed in response to A. actinomycetemcomitans were between 2% and 7% of those seen in

  4. The in Vitro Antimicrobial Efficacy of PDT against Periodontopathogenic Bacteria.

    PubMed

    Haag, Philippe A; Steiger-Ronay, Valerie; Schmidlin, Patrick R

    2015-11-13

    Periodontitis, an inflammatory disease, is caused by biofilms with a mixed microbial etiology and involves the progressive destruction of the tooth-supporting tissues. A rising number of studies investigate the clinical potential of photodynamic therapy (PDT) as an adjunct during active therapy. The aim of the present review was to evaluate the available literature for the in vitro antimicrobial efficacy of photodynamic therapy focusing on the periodontopathogenic bacteria Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum. The focused question was: "Is it possible to decrease (at least 3 log steps or 99.9%) or even eliminate bacterial growth by photodynamic therapy in vitro when compared to untreated control groups or control groups treated by placebo?" In general, PDT resulted in a substantial reduction of surviving bacteria. However, not all studies showed the desired reduction or elimination. The ranges of log10-reduction were 0.38 (58%) to a complete eradication (100%) for P. gingivalis, 0.21 (39%) to 100% for A. actinomycetemcomitans and 0.3 (50%) to 100% for F. nucleatum. In conclusion, further and particularly more comparable studies are needed to evaluate if PDT can be clinically successful as an adjuvant in periodontal therapy.

  5. Systemic Immune Responses in Pregnancy and Periodontitis: Relationship to Pregnancy Outcomes in the Obstetrics and Periodontal Therapy (OPT) Study

    PubMed Central

    Ebersole, Jeffrey L.; Novak, M. John; Michalowicz, Bryan S.; Hodges, James S.; Steffen, Michelle J.; Ferguson, James E.; DiAngelis, Anthony; Buchanan, William; Mitchell, Dennis A.; Papapanou, Panos N.

    2014-01-01

    Background Our previous studies reported on the obstetric, periodontal, and microbiologic outcomes of women participating in the Obstetrics and Periodontal Therapy (OPT) Study. This article describes the systemic antibody responses to selected periodontal bacteria in the same patients. Methods Serum samples, obtained from pregnant women at baseline (13 to 16 weeks; 6 days of gestation) and 29 to 32 weeks, were analyzed by enzyme-linked immunosorbent assay for serum immunoglobulin G (IgG) antibody to Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Campylobacter rectus, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia (previously T. forsythensis), and Treponema denticola. Results At baseline, women who delivered live preterm infants had significantly lower total serum levels of IgG antibody to the panel of periodontal pathogens (P = 0.0018), to P. gingivalis (P = 0.0013), and to F. nucleatum (P = 0.0200) than women who delivered at term. These differences were not significant at 29 to 32 weeks. Changes in IgG levels between baseline and 29 to 32 weeks were not associated with preterm birth when adjusted for treatment group, clinical center, race, or age. In addition, delivery of low birth weight infants was not associated with levels of antibody at baseline or with antibody changes during pregnancy. Conclusions Live preterm birth is associated with decreased levels of IgG antibody to periodontal pathogens in women with periodontitis when assessed during the second trimester. Changes in IgG antibody during pregnancy are not associated with birth outcomes. PMID:19485826

  6. The in Vitro Antimicrobial Efficacy of PDT against Periodontopathogenic Bacteria

    PubMed Central

    Haag, Philippe A.; Steiger-Ronay, Valerie; Schmidlin, Patrick R.

    2015-01-01

    Periodontitis, an inflammatory disease, is caused by biofilms with a mixed microbial etiology and involves the progressive destruction of the tooth-supporting tissues. A rising number of studies investigate the clinical potential of photodynamic therapy (PDT) as an adjunct during active therapy. The aim of the present review was to evaluate the available literature for the in vitro antimicrobial efficacy of photodynamic therapy focusing on the periodontopathogenic bacteria Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum. The focused question was: “Is it possible to decrease (at least 3 log steps or 99.9%) or even eliminate bacterial growth by photodynamic therapy in vitro when compared to untreated control groups or control groups treated by placebo?” In general, PDT resulted in a substantial reduction of surviving bacteria. However, not all studies showed the desired reduction or elimination. The ranges of log10-reduction were 0.38 (58%) to a complete eradication (100%) for P. gingivalis, 0.21 (39%) to 100% for A. actinomycetemcomitans and 0.3 (50%) to 100% for F. nucleatum. In conclusion, further and particularly more comparable studies are needed to evaluate if PDT can be clinically successful as an adjuvant in periodontal therapy. PMID:26580607

  7. Inflammatory Bone Loss in Experimental Periodontitis Induced by Aggregatibacter actinomycetemcomitans in Interleukin-1 Receptor Antagonist Knockout Mice

    PubMed Central

    Izawa, A.; 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.

    2014-01-01

    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

  8. cis Elements and trans factors are both important in strain-specific regulation of the leukotoxin gene in Actinobacillus actinomycetemcomitans.

    PubMed Central

    Kolodrubetz, D; Spitznagel, J; Wang, B; Phillips, L H; Jacobs, C; Kraig, E

    1996-01-01

    Actinobacillus actinomycetemcomitans, the etiologic agent of localized juvenile periodontitis, produces a potent leukotoxin that kills human neutrophils. The production of leukotoxin RNA can vary more than 50-fold among isolates of A. actinomycetemcomitans, and strains expressing high levels of leukotoxin RNA are most often found at sites of periodontal disease. To assess the relative contributions of transcription factors and promoter sequences in setting the disparate levels of leukotoxin RNA found, we have undertaken classical cis/trans analyses. First, the leukotoxin promoter regions from moderately leukotoxic (Y4) and minimally leukotoxic (ATCC 33384) strains of A. actinomycetemcomitans were cloned, sequenced, and compared with the previously sequences leukotoxin promoter region of the high-producer strain JP2. The Y4 and ATCC 33384 promoter regions each contain a 528-bp segment that is absent from JP2. Interestingly, the analysis of various deletion constructs in A. actinomycetemcomitans indicated that Y4, despite the large insertion, initiates leukotoxin RNA synthesis at the same promoter as JP2 does. To perform cis/trans analyses, these three leukotoxin promoter regions were cloned into a plasmid upstream of the reporter gene beta-galactosidase. Each plasmid was transformed into JP2, Y4, and ATCC 33384, and the beta-galactosidase levels were determined. The results indicated that the sequences responsible for down-regulating leukotoxin RNA levels in Y4 relative to JP2 are found within the transcribed region of the Y4 leukotoxin operon. Importantly, in ATCC 33384, strain-specific trans factors and promoter sequence differences are equally significant in determining the lower levels of leukotoxin RNA. We hypothesize that either strain ATCC 33384 has a negative regulatory protein (which is missing or mutated in JP2/Y4) or that JP2 and Y4 carry an activator that is missing or mutated in ATCC 33384. PMID:8751884

  9. The pga gene cluster in Aggregatibacter actinomycetemcomitans is necessary for the development of natural competence in Ca(2+) -promoted biofilms.

    PubMed

    Hisano, K; Fujise, O; Miura, M; Hamachi, T; Matsuzaki, E; Nishimura, F

    2014-04-01

    Natural competence is the ability of bacteria to incorporate extracellular DNA into their genomes. This competence is affected by a number of factors, including Ca(2+) utilization and biofilm formation. As bacteria can form thick biofilms in the presence of extracellular Ca(2+) , the additive effects of Ca(2+) -promoted biofilm formation on natural competence should be examined. We evaluated natural competence in Aggregatibacter actinomycetemcomitans, an important periodontal pathogen, in the context of Ca(2+) -promoted biofilms, and examined whether the pga gene cluster, required for bacterial cell aggregation, is necessary for competence development. The A. actinomycetemcomitans cells grown in the presence of 1 mm CaCl2 exhibited enhanced cell aggregation and increased levels of cell-associated Ca(2+) . Biofilm-derived cells grown in the presence of Ca(2+) exhibited the highest levels of natural transformation frequency and enhanced expression of the competence regulator gene, tfoX. Natural competence was enhanced by the additive effects of Ca(2+) -promoted biofilms, in which high levels of pga gene expression were also detected. Mutation of the pga gene cluster disrupted biofilm formation and competence development, suggesting that these genes play a critical role in the ability of A. actinomycetemcomitans to adapt to its natural environment. The Ca(2+) -promoted biofilms may enhance the ability of bacteria to acquire extracellular DNA.

  10. 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.

  11. Transcriptional regulation of Aggregatibacter actinomycetemcomitans lsrACDBFG and lsrRK operons and their role in biofilm formation.

    PubMed

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

    2013-01-01

    Autoinducer-2 (AI-2) is required for biofilm formation and virulence of the oral pathogen Aggregatibacter actinomycetemcomitans, and we previously showed that lsrB codes for a receptor for AI-2. The lsrB gene is expressed as part of the lsrACDBFG operon, which is divergently transcribed from an adjacent lsrRK operon. In Escherichia coli, lsrRK encodes a repressor and AI-2 kinase that function to regulate lsrACDBFG. To determine if lsrRK controls lsrACDBFG expression and influences biofilm growth of A. actinomycetemcomitans, we first defined the promoters for each operon. Transcriptional reporter plasmids containing the 255-bp lsrACDBFG-lsrRK intergenic region (IGR) fused to lacZ showed that essential elements of lsrR promoter reside 89 to 255 bp upstream from the lsrR start codon. Two inverted repeat sequences that represent potential binding sites for LsrR and two sequences resembling the consensus cyclic AMP receptor protein (CRP) binding site were identified in this region. Using electrophoretic mobility shift assay (EMSA), purified LsrR and CRP proteins were shown to bind probes containing these sequences. Surprisingly, the 255-bp IGR did not contain the lsrA promoter. Instead, a fragment encompassing nucleotides +1 to +159 of lsrA together with the 255-bp IGR was required to promote lsrA transcription. This suggests that a region within the lsrA coding sequence influences transcription, or alternatively that the start codon of A. actinomycetemcomitans lsrA has been incorrectly annotated. Transformation of ΔlsrR, ΔlsrK, ΔlsrRK, and Δcrp deletion mutants with lacZ reporters containing the lsrA or lsrR promoter showed that LsrR negatively regulates and CRP positively regulates both lsrACDBFG and lsrRK. However, in contrast to what occurs in E. coli, deletion of lsrK had no effect on the transcriptional activity of the lsrA or lsrR promoters, suggesting that another kinase may be capable of phosphorylating AI-2 in A. actinomycetemcomitans. Finally, biofilm

  12. Aggregatibacter actinomycetemcomitans outer membrane vesicles are internalized in human host cells and trigger NOD1- and NOD2-dependent NF-κB activation.

    PubMed

    Thay, Bernard; Damm, Anna; Kufer, Thomas A; Wai, Sun Nyunt; Oscarsson, Jan

    2014-10-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. We recently demonstrated that outer membrane vesicles (OMVs) disseminated by A. actinomycetemcomitans could deliver multiple proteins, including biologically active cytolethal distending toxin (CDT), into the cytosol of HeLa cells and human gingival fibroblasts (HGF). In the present work, we have used immunoelectron and confocal microscopy analysis and fluorescently labeled vesicles to further investigate mechanisms for A. actinomycetemcomitans OMV-mediated delivery of bacterial antigens to these host cells. Our results supported that OMVs were internalized into the perinuclear region of HeLa cells and HGF. Colocalization analysis revealed that internalized OMVs colocalized with the endoplasmic reticulum and carried antigens, detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells. Consistent with OMV internalization mediating intracellular antigen exposure, the vesicles acted as strong inducers of cytoplasmic peptidoglycan sensor NOD1- and NOD2-dependent NF-κB activation in human embryonic kidney cells. Moreover, NOD1 was the main sensor of OMV-delivered peptidoglycan in myeloid THP1 cells, contributing to the overall inflammatory responses induced by the vesicles. This work reveals a role of A. actinomycetemcomitans OMVs as a trigger of innate immunity via carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs).

  13. Amphotericin B down-regulates Aggregatibacter actinomycetemcomitans-induced production of IL-8 and IL-6 in human gingival epithelial cells.

    PubMed

    Imai, Haruka; Fujita, Tsuyoshi; Kajiya, Mikihito; Ouhara, Kazuhisa; Miyagawa, Tsuyoshi; Matsuda, Shinji; Shiba, Hideki; Kurihara, Hidemi

    2014-08-01

    Gingival epithelium is the primary barrier against microorganism invasion and produces inflammatory cytokines. Amphotericin B, a major antifungal drug, binds to cholesterol in the mammalian cell membrane in addition to fungal ergosterol. Amphotericin B has been shown to regulate inflammatory cytokines in host cells. To investigate the suppressive effect of amphotericin B on the gingival epithelium, we examined the expression of interleukin (IL)-8 and IL-6 and involvement of MAP kinase in human gingival epithelial cells (HGEC) stimulated by Aggregatibacter actinomycetemcomitans. Amphotericin B and the p38 MAP kinase inhibitor down-regulated the A. actinomycetemcomitans-induced increase in the expression of IL-8 and IL-6 at the mRNA. The ERK inhibitor suppressed the A. actinomycetemcomitans-induced IL-8 mRNA expression. Amphotericin B inhibited the A. actinomycetemcomitans-induced phosphorylation of ERK and p38 MAP kinase. Furthermore, amphotericin B inhibited the A. actinomycetemcomitans-induced production of prostaglandin E2. These results suggest that amphotericin B regulate inflammatory responses in HGEC.

  14. 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

  15. Specific genetic variants of Actinobacillus actinomycetemcomitans correlate with disease and health in a regional population of families with localized juvenile periodontitis.

    PubMed Central

    DiRienzo, J M; Slots, J; Sixou, M; Sol, M A; Harmon, R; McKay, T L

    1994-01-01

    A geographically homogeneous population of 83 subjects, from 21 families with localized juvenile periodontitis (LJP), and 35 healthy control subjects was monitored, over a 5-year period, for the presence of the periodontal pathogen Actinobacillus actinomycetemcomitans. Restriction fragment length polymorphism (RFLP) analysis was used to monitor the distribution of genetic variants of this bacterium in LJP-susceptible subjects that converted from a healthy to a diseased periodontal status. A. actinomycetemcomitans was cultured from 57% of the LJP family members accessioned into the study. Nine of 36 LJP-susceptible subjects, in seven families, developed signs of periodontal destruction. All but one of these conversion subjects harbored A. actinomycetemcomitans. Bacterial variants representative of a single RFLP group (II) showed the strongest correlation with conversion (P < 0.002). Six of nine conversion subjects were infected with A. actinomycetemcomitans from this group. RFLP group II variants also prevailed in 8 of 22 probands but were absent in the 35 healthy control subjects. In contrast to the selective distribution of group II variants is diseased individuals, variants belonging to RFLP groups XIII and XIV were found exclusively in the control subjects. Thus, the use of RFLP to type clinical isolates of A. actinomycetemcomitans has resulted in the identification of genetic variants that predominate in LJP and health. These results indicate that studies concerned with the pathogenicity of this bacterium in LJP should be focused on the group II variants. PMID:7913695

  16. Nuclease-sensitive binding of an Actinobacillus actinomycetemcomitans leukotoxin to the bacterial cell surface.

    PubMed Central

    Ohta, H; Kato, K; Kokeguchi, S; Hara, H; Fukui, K; Murayama, Y

    1991-01-01

    A leukotoxin of Actinobacillus actinomycetemcomitans 301-b was solubilized from cell-associated membrane vesicles by treatment with externally added DNase and RNase and was further purified by a procedure which included ammonium sulfate fractionation, gel filtration chromatography, and ion-exchange chromatography. The purified toxin had a molecular mass of 113,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a high isoelectric point (approximately 8.8). From these characteristics, it was to be expected that the membrane vesicle toxin was almost identical to the leukotoxin extracted with polymyxin B in an earlier study (C.-C. Tsai, B. J. Shenker, J. M. DiRienzo, D. Malamud, and N. S. Taichman, Infect, Immun. 43:700-705, 1984). The treatment with DNase and RNase was also highly effective for solubilizing the leukotoxin directly from whole cells, suggesting that the toxin is secreted extracellularly but retained in nucleic acids on the outermost surface of bacterial cells. Images PMID:1937819

  17. Murine macrophage interleukin-1 release by capsularlike serotype-specific polysaccharide antigens of Actinobacillus actinomycetemcomitans.

    PubMed Central

    Takahashi, T; Nishihara, T; Ishihara, Y; Amano, K; Shibuya, N; Moro, I; Koga, T

    1991-01-01

    Serotype-specific polysaccharide antigens (SPAs) were extracted from whole cells of Actinobacillus actinomycetemcomitans ATCC 29523 (serotype a), Y4 (serotype b), and NCTC 9710 (serotype c) by autoclaving and purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300 columns. Y4 SPA induced interleukin-1 (IL-1) release by P388D1 murine macrophages. Polymyxin B had virtually no effect on the release of IL-1. Rabbit anti-murine IL-1 serum strongly suppressed the proliferation of C3H/HeJ mouse thymocytes induced with the culture supernatants of Y4 SPA-stimulated P388D1 cells and a submitogenic dose of concanavalin A. Gel filtration of the culture supernatants of Y4 SPA-stimulated macrophages on Sephacryl S-200 showed that an IL-1 peak at a point corresponding to approximately 16.5 kDa was eluted. The ability of SPAs from strains ATCC 29523 and NCTC 9710 to induce the release of IL-1 was lower than that of Y4 SPA. The IL-1-releasing ability of serotype a and c antigens was enhanced by deacetylation of both polysaccharides, suggesting that acetyl groups of these antigens might hinder the interaction between the antigens and macrophages. PMID:1987032

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

    PubMed Central

    Wilson, M E; Schifferle, R E

    1991-01-01

    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. Images PMID:1706323

  19. Differential transcriptional regulation of Aggregatibacter actinomycetemcomitans lsrACDBFG and lsrRK operons by integration host factor protein.

    PubMed

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

    2014-04-01

    We previously showed that the Aggregatibacter actinomycetemcomitans lsrACDBFG and lsrRK operons are regulated by LsrR and cyclic AMP receptor protein (CRP) and that proper regulation of the lsr locus is required for optimal biofilm growth by A. actinomycetemcomitans. Here, we identified sequences that reside immediately upstream from both the lsrA and lsrR start codons that closely resemble the consensus recognition sequence of Escherichia coli integration host factor (IHF) protein. A. actinomycetemcomitans IHFα and IHFβ were expressed and purified as hexahistidine fusion proteins, and using electrophoretic mobility shift assays (EMSAs), the IHFα-IHFβ protein complex was shown to bind to probes containing the putative IHF recognition sequences. In addition, single-copy chromosomal insertions of lsrR promoter-lacZ and lsrA promoter-lacZ transcriptional fusions in wild-type A. actinomycetemcomitans and ΔihfA and ΔihfB mutant strains showed that IHF differentially regulates the lsr locus and functions as a negative regulator of lsrRK and a positive regulator of lsrACDBFG. Deletion of ihfA or ihfB also reduced biofilm formation and altered biofilm architecture relative to the wild-type strain, and these phenotypes were partially complemented by a plasmid-borne copy of ihfA or ihfB. Finally, using 5' rapid amplification of cDNA ends (RACE), two transcriptional start sites (TSSs) and two putative promoters were identified for lsrRK and three TSSs and putative promoters were identified for lsrACDBFG. The function of the two lsrRK promoters and the positive regulatory role of IHF in regulating lsrACDBFG expression were confirmed with a series of lacZ transcriptional fusion constructs. Together, our results highlight the complex transcriptional regulation of the lsrACDBFG and lsrRK operons and suggest that multiple promoters and the architecture of the lsrACDBFG-lsrRK intergenic region may control the expression of these operons.

  20. Identification of the Exported Proteins of the Oral Opportunistic Pathogen Actinobacillus actinomycetemcomitans by Using Alkaline Phosphatase Fusions

    PubMed Central

    Ward, John; Fletcher, Julie; Nair, Sean P.; Wilson, Michael; Williams, Rachel J.; Poole, Stephen; Henderson, Brian

    2001-01-01

    A phoA fusion library of Actinobacillus actinomycetemcomitans genomic DNA has been screened to identify genes encoding exported and secreted proteins. A total of 8,000 colonies were screened, and 80 positive colonies were detected. From these, 48 genes were identified with (i) more than half having homology to known or hypothetical Haemophilus influenzae genes, (ii) 14 having no ascribed function, and (iii) 4 having very limited or no homology to known genes. The proteins encoded by these genes may, by virtue of their presence on the cell surface, be novel virulence determinants. PMID:11254647

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

    PubMed

    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.

  2. Bioactive glass combined with bisphosphonates provides protection against biofilms formed by the periodontal pathogen Aggregatibacter actinomycetemcomitans.

    PubMed

    Hiltunen, Anna K; Skogman, Malena E; Rosenqvist, Kirsi; Juvonen, Helka; Ihalainen, Petri; Peltonen, Jouko; Juppo, Anne; Fallarero, Adyary

    2016-03-30

    Biofilms play a pivotal role in the progression of periodontitis and they can be treated with antiseptics (i.e. chlorhexidine) or antibiotics, but these therapeutic alternatives are unable of ameliorating periodontal alveolar bone loss, which has been, on the other hand, successfully treated with bone-preserving agents. The improved bone formation achieved in animal models by the combination of two such agents: bioactive glass (BAG) and bisphosphonates has attracted the interest for further exploring dental applications. However, the antimicrobial effects that may result from combining them have not been yet investigated. Here, our aim was to explore the anti-biofilm effects that could result from combining BAG with bisphosphonates, particularly in a dental biofilm model. The experiments were performed with an oral cavity single-specie (Aggregatibacter actinomycetemcomitans) biofilm assay, which was optimized in this contribution. Risedronate displayed an intrinsic anti-biofilm effect, and all bisphosphonates, except clodronate, reduced biofilm formation when combined with BAG. In particular, the anti-biofilm activity of risedronate was significantly increased by the combination with BAG. Since it has been proposed that some of the antimicrobial effects of BAG are caused by local pH changes, studies of pH variations were performed to gain a mechanistic understanding. However, the observed anti-biofilm effects could not be explained with lowered pHs. Overall, these results do provide further support for the promising use of bisphosphonate-BAG combinations in dental applications. These findings are particularly relevant for patients undergoing cancer chemotherapy, or osteoporotic patients, which are known to be more vulnerable to periodontitis. In such cases, bisphosphonate treatment could play a double positive effect: local treatment of periodontitis (in combination with BAG) and systemic treatment of osteoporosis, prevention of hypercalcemia and metastases.

  3. Aggregatibacter actinomycetemcomitans leukotoxin induces cytosol acidification in LFA-1 expressing immune cells.

    PubMed

    Balashova, N; Dhingra, A; Boesze-Battaglia, K; Lally, E T

    2016-02-01

    Studies have suggested that Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) kills human lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18)-bearing immune cells through a lysosomal-mediated mechanism. Lysosomes are membrane-bound cellular organelles that contain an array of acid hydrolases that are capable of breaking down biomolecules. The lysosomal membrane bilayer confines the pH-sensitive enzymes within an optimal acidic (pH 4.8) environment thereby protecting the slightly basic cytosol (pH 6.8-7.5). In the current study, we have probed the effect of LtxA-induced cytolysis on lysosomal integrity in two different K562 erythroleukemia cell lines. K562-puro/LFA-1 cells were stably transfected with CD11a and CD18 cDNA to express LFA-1 on the cell surface while K562-puro, which does not express LFA-1, served as a control. Following treatment with 100 ng ml(-1) LtxA cells were analyzed by live cell imaging in conjunction with time-lapse confocal microscopy and by flow cytometry. Using a pH-sensitive indicator (pHrodo(®)) we demonstrated that the toxin causes a decrease in the intracellular pH in K562-puro/LFA-1 cells that is noticeable within the first 15 min of treatment. This process correlated with the disappearance of lysosomes in the cytosol as determined by both acridine orange and LysoTracker(®) Red DND-99 staining. These changes were not observed in K562-puro cells or when heat inactivated toxin was added to K562-puro/LFA-1. Our results suggest that LtxA induces lysosomal damage, cytosol acidification, which is followed by cell death in K562-puro/LFA-1 cells.

  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. Occurrence of Aggregatibacter actinomycetemcomitans in Indian chronic periodontitis patients and periodontally healthy adults

    PubMed Central

    Joshi, Vinayak Mahableshwar; Bhat, Kishore Gajanan; Kugaji, Manohar Suresh; Ingalgi, Preeti Shivaji

    2016-01-01

    Background: Aggregatibacter actinomycetemcomitans (Aa), an important primary periodontal pathogen, is known for its strong virulence characteristics that cause periodontal disease. We investigated Aa occurrence in Indian individuals using culture and 16 s rDNA polymerase chain reaction (PCR). Materials and Methods: A cross-sectional study with 100 participants each in the healthy and chronic periodontitis (CP) groups was conducted. The subgingival plaque was collected and immediately plated on selective media for Aa. The remaining plaque samples were used for DNA extraction. PCR was performed using specific primers for Aa. Statistical Analysis Used: The detection of bacteria and the clinical parameters between the groups were compared using the Mann–Whitney U-test. For assessing the agreement between the results of anaerobic culture and PCR, Kappa analyses were performed. Results: Aa levels using culture and PCR was 51% and 69% in the CP group and 12% and 30% in the healthy group, respectively. The two groups showed significant differences (P < 0.00001). The detection accuracy of culture and PCR was assessed, and the coefficient of accuracy (k) was highly significant in the healthy (0.3103; P < 0.0001) and CP groups (0.1536; P < 0.0497). Conclusions: Aa was predominantly found in the CP group compared with the healthy group, which is consistent with previous findings. Our results showed that both techniques can be used for detecting Aa. An ideal technique for detecting subgingival microorganisms should be carefully selected depending on the scope of the intended future work. PMID:27143824

  6. Aggregatibacter actinomycetemcomitans leukotoxin induces cytosol acidification in LFA-1 expressing immune cells

    PubMed Central

    Balashova, Nataliya; Dhingra, Anuradha; Boesze-Battaglia, Kathleen; Lally, Edward T.

    2015-01-01

    Summary Studies have suggested that Aggregatibacter actinomycetemcomitans leukotoxin (LtxA) kills human LFA-1(CD11a/CD18)-bearing immune cells through a lysosomal-mediated mechanism. Lysosomes are membrane-bound cellular organelles that contain an array of acid hydrolases that are capable of breaking down biomolecules. The lysosomal membrane bilayer confines the pH-sensitive enzymes within an optimal acidic (pH 4.8) environment thereby protecting the slightly basic cytosol (pH 6.8–7.5). In the current study, we have probed the effect of LtxA-induced cytolysis on lysosomal integrity in two different K562 erythroleukemia cell lines. K562-puro/LFA-1 cells were stably transfected with CD11a and CD18 cDNA to express LFA-1 on the cell surface while K562-puro, which does not express LFA-1, served as a control. Following treatment with 100 ng/ml LtxA cells were analyzed by live cell imaging in conjunction with time-lapse confocal microscopy and by flow cytometry. Using a pH sensitive indicator (pHrodo®, Life Technologies) we demonstrated that the toxin causes a decrease in the intracellular pH in K562-puro/LFA-1 cells noticeable within the first 15 min of treatment. This process correlated with the disappearance of lysosomes in the cytosol as determined by both acridine orange and LysoTracker® Red DND-99 (Life Technologies) staining. These changes were not observed in K562-puro cells or when heat inactivated toxin was added to K562-puro/LFA-1. Our results suggest that LtxA induces lysosomal damage, cytosol acidification, which is followed by cell death in K562-puro/LFA-1 cells. PMID:26361372

  7. 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.

  8. 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

  9. Aggregatibacter actinomycetemcomitans cytolethal distending toxin activates the NLRP3 inflammasome in human macrophages, leading to the release of proinflammatory cytokines.

    PubMed

    Shenker, Bruce J; Ojcius, David M; Walker, Lisa P; Zekavat, Ali; Scuron, Monika Damek; Boesze-Battaglia, Kathleen

    2015-04-01

    The cytolethal distending toxin (Cdt) is produced from a number of bacteria capable of causing infection and inflammatory disease. Our previous studies with Actinobacillus actinomycetemcomitans Cdt demonstrate not only that the active toxin subunit functions as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase but also that macrophages exposed to the toxin were stimulated to produce proinflammatory cytokines. We now demonstrate that the Cdt-induced proinflammatory response involves the activation of the NLRP3 inflammasome. Specific inhibitors and short hairpin RNA (shRNA) were employed to demonstrate requirements for NLRP3 and ASC as well as caspase-1. Furthermore, Cdt-mediated inflammasome activation is dependent upon upstream signals, including reactive oxygen species (ROS) generation and Cdt-induced increases in extracellular ATP levels. Increases in extracellular ATP levels contribute to the activation of the P2X7 purinergic receptor, leading to K+ efflux. The relationship between the abilities of the active toxin subunit CdtB to function as a lipid phosphatase, activate the NLRP3 inflammasome, and induce a proinflammatory cytokine response is discussed. These studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as Aggregatibacter actinomycetemcomitans.

  10. Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Activates the NLRP3 Inflammasome in Human Macrophages, Leading to the Release of Proinflammatory Cytokines

    PubMed Central

    Ojcius, David M.; Walker, Lisa P.; Zekavat, Ali; Scuron, Monika Damek; Boesze-Battaglia, Kathleen

    2015-01-01

    The cytolethal distending toxin (Cdt) is produced from a number of bacteria capable of causing infection and inflammatory disease. Our previous studies with Actinobacillus actinomycetemcomitans Cdt demonstrate not only that the active toxin subunit functions as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase but also that macrophages exposed to the toxin were stimulated to produce proinflammatory cytokines. We now demonstrate that the Cdt-induced proinflammatory response involves the activation of the NLRP3 inflammasome. Specific inhibitors and short hairpin RNA (shRNA) were employed to demonstrate requirements for NLRP3 and ASC as well as caspase-1. Furthermore, Cdt-mediated inflammasome activation is dependent upon upstream signals, including reactive oxygen species (ROS) generation and Cdt-induced increases in extracellular ATP levels. Increases in extracellular ATP levels contribute to the activation of the P2X7 purinergic receptor, leading to K+ efflux. The relationship between the abilities of the active toxin subunit CdtB to function as a lipid phosphatase, activate the NLRP3 inflammasome, and induce a proinflammatory cytokine response is discussed. These studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as Aggregatibacter actinomycetemcomitans. PMID:25644004

  11. 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...

  12. Comparative genomic hybridization and transcriptome analysis with a pan-genome microarray reveal distinctions between JP2 and non-JP2 genotypes of Aggregatibacter actinomycetemcomitans.

    PubMed

    Huang, Y; Kittichotirat, W; Mayer, M P A; Hall, R; Bumgarner, R; Chen, C

    2013-02-01

    It was postulated that the highly virulent JP2 genotype of Aggregatibacter actinomycetemcomitans may possess a constellation of distinct virulence determinants not found in non-JP2 genotypes. This study compared the genome content and the transcriptome of the serotype b JP2 genotype and the closely related serotype b non-JP2 genotype of A. actinomycetemcomitans. A custom-designed pan-genomic microarray of A. actinomycetemcomitans was constructed and validated against a panel of 11 sequenced reference strains. The microarray was subsequently used for comparative genomic hybridization of serotype b strains of JP2 (six strains) and non-JP2 (six strains) genotypes, and for transcriptome analysis of strains of JP2 (three strains) and non-JP2 (two strains). Two JP2-specific and two non-JP2-specific genomic islands were identified. In one instance, distinct genomic islands were found to be inserted into the same locus among strains of different genotypes. Transcriptome analysis identified five operons, including the leukotoxin operon, to have at least two genes with an expression ratio of 2 or greater between genotypes. Two of the differentially expressed operons were members of the membrane-bound nitrate reductase system (nap operon) and the Tol-Pal system of gram-negative bacterial species. This study is the first to demonstrate the differences in the full genome content and gene expression between A. actinomycetemcomitans strains of JP2 and non-JP2 genotypes. The information is essential for designing hypothesis-driven experiments to examine the pathogenic mechanisms of A. actinomycetemcomitans.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. REHABILITATION AND FUNCTIONAL OUTCOMES AFTER EXTENSIVE SURGICAL DEBRIDEMENT OF A KNEE INFECTED BY FUSOBACTERIUM NECROPHORUM: A CASE REPORT

    PubMed Central

    Briggs, Matthew S.; Kegelmeyer, Deborah K.; Kloos, Anne D.

    2013-01-01

    Background and Purpose: Joint infection is a rare but serious complication after knee injury that should be part of a physical therapist's differential diagnosis. This case report presents the care of a 17 year‐old female athlete with septic arthritis from a Fusobacterium infection after sustaining a right lateral meniscus tear. Joint pathology combined with the aggressive infectious agent led to arthrofibrosis of her knee joint and resultant activity limitations and participation restrictions. The purpose of this case report is to highlight a rare and unique pathology, the serious effects that a joint infection can have on musculoskeletal function, and the challenges encountered during the rehabilitation process. Case Description: The subject was a 17 year‐old volleyball player who injured her right knee while playing volleyball. Within 7 days, the subject developed a severe joint infection that spread into surrounding gluteal, quadriceps, and gastrocnemius musculature. The infection was surgically debrided eight times during a 10‐week inpatient hospital stay. A manipulation under anesthesia was performed to restore range of motion in her knee joint. Outpatient physical therapy was initiated 4 days later in order to restore musculoskeletal function. Outcome: Over eight months of physical therapy services were utilized to address the impairments and activity limitations caused by her joint dysfunction. She met her physical therapy goals and made significant improvements on the Knee Outcome Survey and the Lower Extremity Functional Scale. Success in physical therapy and completion of additional strength training exercise allowed this subject to return to competitive softball at the club level during her freshman year of college. Discussion: Though rare after musculoskeletal injury, joint infection can lead to soft tissue damage, partial or complete degradation of articular cartilage, and arthrofibrosis causing significant disability. Physical therapists must

  19. 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

  20. 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.

  1. Inner-membrane protein MorC is involved in fimbriae production and biofilm formation in Aggregatibacter actinomycetemcomitans.

    PubMed

    Smith, Kenneth P; Ruiz, Teresa; Mintz, Keith P

    2016-03-01

    Fimbrial subunit synthesis, secretion and assembly on the surface of the periodontal pathogen Aggregatibacter actinomycetemcomitans are essential for biofilm formation. A recent quantitative proteomics study employing an afimbriated strain and a developed mutant isogenic for the inner-membrane protein morphogenesis protein C (MorC) revealed that the abundance of the proteins of the fimbrial secretion apparatus in the membrane is dependent on MorC. To investigate further the relationship between MorC and fimbriation, we identified and complemented the defect in fimbriae production in the afimbriated laboratory strain. The transformed strain expressing a plasmid containing genes encoding the WT fimbrial subunit and the prepilin peptidase displayed all of the hallmarks of a fimbriated bacterium including the distinct star-like colony morphology, robust biofilm formation, biofilm architecture composed of discrete microcolonies and the presence of fimbriae. When the identical plasmid was transformed into a morC mutant strain, the bacterium did not display any of the phenotypes of fimbriated strains. Extension of these studies to a naturally fimbriated clinical strain showed that the resulting morC mutant maintained the characteristic colony morphology of fimbriated strains. There was, however, a reduction in the secretion of fimbrial subunits, and fewer fimbriae were observed on the surface of the mutant strain. Furthermore, the morC mutant of the fimbriated strain displayed a significantly altered biofilm microcolony architecture, while maintaining a similar biofilm mass to the parent strain. These results suggest that MorC influences fimbrial secretion and microcolony formation in A. actinomycetemcomitans.

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

    PubMed Central

    Kaur, Manpreet

    2014-01-01

    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. PMID:25169107

  3. Prevalence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in Japanese patients with generalized chronic and aggressive periodontitis.

    PubMed

    Tomita, Sachiyo; Komiya-Ito, Akiyo; Imamura, Kentaro; Kita, Daichi; Ota, Koki; Takayama, Saori; Makino-Oi, Asako; Kinumatsu, Takashi; Ota, Mikio; Saito, Atsushi

    2013-01-01

    This study aimed to investigate the prevalence and levels of major periodontal pathogens, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in subgingival plaque samples of a group of Japanese patients with aggressive periodontitis (AgP) and chronic periodontitis (CP). A total of 40 patients with clinical diagnosis of AgP or CP and 10 periodontally healthy volunteers were subjected to clinical and microbiological analysis. Subgingival plaque samples were analyzed for A. actinomycetemcomitans, P. gingivalis and T. forsythia with a real-time polymerase chain reaction (PCR) technique. The prevalence of P. gingivalis and T. forsythia was relatively high in patients with periodontitis: over 60% of AgP or CP patients harbored these pathogens whereas they were not detected in the subgingival plaque samples from periodontally healthy individuals. P. gingivalis and T. forsythia were relatively frequently detected together in AgP and CP patients. No significant differences in the prevalence or level of the 3 pathogens were found between periodontitis groups. The proportion of T. forsythia was approximately 4-fold higher in CP group than in AgP group (P = 0.02). In periodontitis patients, a significant positive correlation was found between periodontal parameters (probing depth and clinical attachment level) and the numbers of total bacteria, P. gingivalis and T. forsythia. No distinct pattern of the subgingival profile of these pathogens was discerned between the two disease entities, except for the difference in the proportion of T. forsythia. The red complex bacteria, P. gingivalis and T. forsythia were highly prevalent in this population of Japanese AgP and CP patients, collaborating their roles in periodontitis.

  4. Disease severity associated with presence in subgingival plaque of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Tannerella forsythia, singly or in combination, as detected by nested multiplex PCR.

    PubMed

    Ready, D; D'Aiuto, F; Spratt, D A; Suvan, J; Tonetti, M S; Wilson, M

    2008-10-01

    This study used a nested multiplex PCR method to detect three periodontal pathogens in subgingival plaque collected before treatment and at 2 and 6 months posttreatment from 107 patients with severe, generalized periodontitis. The proportions of the patients who harbored these bacteria before periodontal treatment were as follows: Tannerella forsythia, 81%; Porphyromonas gingivalis, 78%; and Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans, 47%. At 2 months posttreatment there was a significant reduction in the numbers of patients harboring P. gingivalis (46%; P < 0.001) or T. forsythia (63%; P = 0.043) but not A. actinomycetemcomitans (50%) compared to pretreatment data. At 6 months posttreatment, significantly fewer patients harbored P. gingivalis (43%; P < 0.001); A. actinomycetemcomitans, (31%; P = 0.025), or T. forsythia (63%; P = 0.030). Interestingly, at baseline and at 2 months posttherapy, subjects who harbored only a single pathogen had a greater level of periodontal disease than subjects who harbored two, or all three, of these periodontal pathogens. These data suggest that a reduction in the number of species present may be associated with an increase in the severity of periodontal diseases.

  5. [Susceptibility of potential periodontopathic bacteria to metronidazole, spiramycin and their combination].

    PubMed

    Mouton, C; Dextraze, L; Mayrand, D

    1984-03-01

    A total of 65 bacterial strains originating mostly from subgingival plaque were tested for their susceptibilities to metronidazole, spiramycin, and their combination, ornidazole, erythromycin and tetracycline by means of an agar dilution technique. All agents were active against all anaerobic Gram-negative rods. Bacteroides gingivalis and Fusobacterium nucleatum showed marked susceptibility to metronidazole (MIC less than or equal to 0.06 microgram/ml) whereas 4-64 micrograms/ml were required to inhibit the capnophilic Actinobacillus actinomycetemcomitans and Capnocytophaga. Gram-positive facultatives were resistant to nitro-imidazoles but were inhibited at macrolide concentrations less than or equal to 0.5 microgram/ml. Except for F. nucleatum and Veillonella strains (2 less than or equal to MIC less than or equal to 128 micrograms/ml) macrolides were active against all other anaerobic bacteria tested. At concentrations less than or equal to 2 micrograms/ml the combination of spiramycin and metronidazole (2 : 1) was active against virtually all bacteria tested but our results failed to show a synergistic effect.

  6. 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

  7. MyD88 is essential for alveolar bone loss induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide in mice.

    PubMed

    Madeira, M F M; Queiroz-Junior, C M; Cisalpino, D; Werneck, S M C; Kikuchi, H; Fujise, O; Ryffel, B; Silva, T A; Teixeira, M M; Souza, D G

    2013-12-01

    Aggregatibacter actinomycetemcomitans is a Gram-negative bacteria highly associated with localized aggressive periodontitis. The recognition of microbial factors, such as lipopolysaccharide from A. actinomycetemcomitans ((Aa)LPS), in the oral environment is made mainly by surface receptors known as Toll-like receptors (TLR). TLR4 is the major LPS receptor. This interaction leads to the production of inflammatory cytokines by myeloid differentiation primary-response protein 88 (MyD88) -dependent and -independent pathways, which may involve the adaptor Toll/interleukin-1 receptor-domain-containing adaptor inducing interferon-β (TRIF). The aim of this study was to assess the involvement of MyD88 in alveolar bone loss induced by (Aa)LPS in mice. C57BL6/J wild-type (WT) mice, MyD88, TRIF or TRIF/MyD88 knockout mice received 10 injections of Aa LPS strain FDC Y4 (5 μg in 3 μl), in the palatal gingival tissue of the right first molar, every 48 h. Phosphate-buffered saline was injected in the opposite side and used as control. Animals were sacrificed 24 h after the 10th injection and the maxillae were removed for macroscopic and biochemical analyses. The injections of Aa LPS induced significant alveolar bone loss in WT mice. In the absence of MyD88 or TRIF/MyD88 no bone loss induced by (Aa)LPS was observed. In contrast, responses in TRIF(-/-) mice were similar to those in WT mice. Diminished bone loss in the absence of MyD88 was associated with fewer TRAP-positive cells and increased expression of osteoblast markers, RUNX2 and osteopontin. There was also reduced tumor necrosis factor-α production in MyD88(-/-) mice. There was less osteoclast differentiation of hematopoietic bone marrow cells from MyD88(-/-) mice after (Aa)LPS stimulation. Hence, the signaling through MyD88 is pivotal for (Aa)LPS-induced osteoclast formation and alveolar bone loss.

  8. 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

  9. Synergistic Antibacterial Effects of Nanoparticles Encapsulated with Scutellaria baicalensis and Pure Chlorhexidine on Oral Bacterial Biofilms

    PubMed Central

    Leung, Ken Cham-Fai; Seneviratne, Chaminda Jayampath; Li, Xuan; Leung, Ping Chung; Lau, Clara Bik San; Wong, Chi-Hin; Pang, Ka Yan; Wong, Chun Wai; Wat, Elaine; Jin, Lijian

    2016-01-01

    Scutellaria baicalensis (SB) is a traditional Chinese medicine for treating infectious and inflammatory diseases. Our recent study shows potent antibacterial effects of nanoparticle-encapsulated chlorhexidine (Nano-CHX). Herein, we explored the synergistic effects of the nanoparticle-encapsulated SB (Nano-SB) and Nano-CHX on oral bacterial biofilms. Loading efficiency of Nano-SB was determined by thermogravimetric analysis, and its releasing profile was assessed by high-performance liquid chromatographyusing baicalin (a flavonoid compound of SB) as the marker. The mucosal diffusion assay on Nano-SB was undertaken in a porcine model. The antibacterial effects of the mixed nanoparticles (Nano-MIX) of Nano-SB and Nano-CHX at 9:1 (w/w) ratio were analyzed in both planktonic and biofilm modes of representative oral bacteria. The Nano-MIX was effective on the mono-species biofilms of Streptococcus (S.) mutans, S. sobrinus, Fusobacterium (F.) nucleatum, and Aggregatibacter (A.) actinomycetemcomitans (MIC 50 μg/mL) at 24 h, and exhibited an enhanced effect against the multi-species biofilms such as S. mutans, F. nucleatum, A. actinomycetemcomitans, and Porphyromonas (P.) gingivalis (MIC 12.5 μg/mL) at 24 h that was supported by the findings of both scanning electron microscopy (SEM) and confocal scanning laser microscopy (CLSM). This study shows enhanced synergistic antibacterial effects of the Nano-MIX on common oral bacterial biofilms, which could be potentially developed as a novel antimicrobial agent for clinical oral/periodontal care. PMID:28335189

  10. Antibacterial Activity of Myristica fragrans against Oral Pathogens

    PubMed Central

    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. PMID:23049613

  11. 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.

  12. Biochemical and morphological characterization of the killing of human monocytes by a leukotoxin derived from Actinobacillus actinomycetemcomitans.

    PubMed Central

    Taichman, N S; Dean, R T; Sanderson, C J

    1980-01-01

    A potent, heat-labile leukotoxic material was extracted from Actinobacillus actinomycetemcomitans (strain Y4), an anaerobic gram-negative microorganism originally isolated from subgingival plaque in a patient with juvenile periodontitis. The cytopathic effects of Y4 toxin on purified monocytes were studied by the extracellular release of radioactive cytoplasmic markers and cell enzymes and by time-lapse microcinematography. Y4 toxin rapidly bound to the cells, producing dose- and time-dependent alterations culminating in cell death and release of intracellular constituents into the culture medium. The evidence to be presented suggests that the cell membrane of the monocyte may be the primary target in the development of these phenomena. Previous studies have shown that Y4 toxin also kills human polymorphonuclear leukocytes but not other cell types. It is conceivable that disruption of polymorphonuclear leukocytes and monocytes by Y4 toxin in the gingival crevice area may be relevant in the pathogenesis of juvenile periodontitis. Images Fig. 1 PMID:6155347

  13. Breaking the Gingival Epithelial Barrier: Role of the Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin in Oral Infectious Disease

    PubMed Central

    DiRienzo, Joseph M.

    2014-01-01

    The Gram-negative bacterium Aggregatibacter actinomycetemcomitans is part of the HACEK group that causes infective endocarditis, a constituent of the oral flora that promotes some forms of periodontal disease and a member of the family of species that secrete a cytolethal distending toxin (Cdt). The family of bacteria that express the cdt genes participate in diseases that involve the disruption of a mucosal or epithelial layer. In vitro studies have shown that human gingival epithelial cells (HGEC) are native targets of the Cdt that typically induces DNA damage that signals growth arrest at the G2/M interphase of the cell cycle. The gingival epithelium is an early line of defense in the oral cavity against microbial assault. When damaged, bacteria collectively gain entry into the underlying connective tissue where microbial products can affect processes and pathways in infiltrating inflammatory cells culminating in the destruction of the attachment apparatus of the tooth. One approach has been the use of an ex vivo gingival explant model to assess the effects of the Cdt on the morphology and integrity of the tissue. The goal of this review is to provide an overview of these studies and to critically examine the potential contribution of the Cdt to the breakdown of the protective gingival barrier. PMID:24861975

  14. The immunodominant outer membrane antigen of Actinobacillus actinomycetemcomitans is located in the serotype-specific high-molecular-mass carbohydrate moiety of lipopolysaccharide.

    PubMed Central

    Page, R C; Sims, T J; Engel, L D; Moncla, B J; Bainbridge, B; Stray, J; Darveau, R P

    1991-01-01

    Most patients with juvenile periodontitis manifest serum antibodies, sometimes at very high titers, to antigens of Actinobacillus actinomycetemcomitans, but the antigens inducing the immune response have been only partly characterized. We separated A. actinomycetemcomitans serotype b cells into protein, lipopolysaccharide (LPS), and soluble polysaccharide fractions and characterized them. Coomassie blue- and silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels were used to detect protein and LPS components, and gas-liquid chromatography was used to determine their carbohydrate and fatty acid composition. Western blots, dot blots, and enzyme-linked immunosorbent assay inhibition with high-titer sera from juvenile periodontitis patients revealed which components were highest in antibody binding activity. These results showed that the major portion of the immunoglobulin G binding activity resides in the purified mannan-free LPS, with lesser amounts in the total protein fraction. Using Sephacryl S-300 chromatography, we separated LPS into high-molecular-mass components with high carbohydrate contents by gas-liquid chromatography and a low-molecular-mass component consisting mainly of lipid A and the inner core sugar heptulose. The results of quantitative dot blot assays and enzyme-linked immunosorbent assay inhibition show that the serotype-specific antibody binding activity is highly concentrated in the high-molecular-mass carbohydrate-rich LPS fraction and is almost completely absent in the low-molecular-weight lipid-rich fraction. Our observations contrast with previous reports that the predominant serotype antigen of A. actinomycetemcomitans resides in a mannan-rich polysaccharide isolated from spent culture medium. These observations support the conclusion that the immunodominant antigen of the outer membrane is the O antigen of the LPS. Images PMID:1716610

  15. Preliminary Extraction and Identification of the 44.5 kDa Outer Membrane Proteins Isolated from Bovine Fusobacterium necrophorum (AB).

    PubMed

    Xu, Jing; Chen, Li-Zhi; Liu, Xiao-Ying; Feng, Er-Kai; Wang, Sun-Jie; Cao, Yue

    2013-12-01

    Fusobacterium necrophorum (AB) in the pharynx, respiratory tract, female reproductive tract or urinary system is the causative agent of footrot and hepatic abscesses in animals and acute Lemierre's syndrome in humans. Current methods do not effectively protect animals and humans against F. necrophorum (AB). The outer membrane proteins (OMP) of F. necrophorum (AB) can be used as new material to protect against the diseases induced by F. necrophorum (AB). The aim of this study was to extract OMP and examine the immunogenic response of OMP. The preliminary extraction of OMP of F. necrophorum (AB) was identified by SDS-PAGE and stained by Coomassie Brilliant Blue R-250 (CB B R-250) and silver staining methods. The results showed that only a major band of 44.5 kDa was observed when staining the gel using CB B R-250. This band represented the target protein. In contrast, many small bands were observed by the silver staining method. The OMP also exhibited immune biological activities according to western blot analysis. The brightest band among the multi-banding observed was the OMP. Thus, the OMP was obtained and had immunogenic activity. The results provide a new direction to protect animals and humans against F. necrophorum (AB) in the clinical setting.

  16. A cost-effectiveness analysis of identifying Fusobacterium necrophorum in throat swabs followed by antibiotic treatment to reduce the incidence of Lemierre's syndrome and peritonsillar abscesses.

    PubMed

    Bank, S; Christensen, K; Kristensen, L H; Prag, J

    2013-01-01

    The main purpose of this paper was to estimate the cost per quality-adjusted life year (QALY) saved by identifying Fusobacterium necrophorum in throat swabs followed by proper antibiotic treatment, to reduce the incidence of Lemierre's syndrome and peritonsillar abscesses (PTA) originating from a pharyngitis. The second purpose was to estimate the population size required to indicate that antibiotic treatment has an effect. Data from publications and our laboratory were collected. Monte Carlo simulation and one-way sensitivity analysis were used to analyse cost-effectiveness. The cost-effectiveness analysis shows that examining throat swabs from 15- to 24-year-olds for F. necrophorum followed by antibiotic treatment will probably be less costly than most other life-saving medical interventions, with a median cost of US$8,795 per QALY saved. To indicate a reduced incidence of Lemierre's syndrome and PTA in Denmark, the intervention probably has to be followed for up to 5 years. Identifying F. necrophorum in throat swabs from 15- to 24-year-olds followed by proper antibiotic treatment only requires a reduction of 20-25 % in the incidence of Lemierre's syndrome and PTA to be cost-effective. This study warrants further examination of the effect of antibiotic treatment on the outcome of F. necrophorum acute and recurrent pharyngitis, as well as the effect on Lemierre's syndrome and PTA.

  17. 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

  18. 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

  19. A systematic review of Fusobacterium necrophorum-positive acute tonsillitis: prevalence, methods of detection, patient characteristics, and the usefulness of the Centor score.

    PubMed

    Klug, T E; Rusan, M; Fuursted, K; Ovesen, T; Jorgensen, A W

    2016-12-01

    The purpose of this study was to review the current evidence for an association between Fusobacterium necrophorum (FN) and acute tonsillitis (AT), to assess the prevalence of FN in AT, to identify the better FN detection method, and to characterize the clinical characteristics of FN-positive patients with uncomplicated AT. A systematic literature search was conducted in the PubMed, EMBASE, and SweMed+ databases for studies reporting on the recovery rates of FN in patients with AT. A total of 498 unique records were identified. Eleven studies were included in a qualitative synthesis and six studies were included in a meta-analysis. Considerable heterogeneity (I(2) = 60 %) and risk of bias and confounders was found among the studies, though a subset of studies (prospective) had lower heterogeneity and higher study quality. FN was recovered significantly more frequently from patients (21.2 %) compared to healthy controls (7.6 %) (p < 0.001). FN recovery rates were similar between culture-based studies (20.3 %) and studies using polymerase chain reaction (PCR) methodology (22.2 %) (p = 0.462). The typical FN-positive AT patient was a smoking young (15-25 years) male presenting with tonsillar exudates and a Centor score of 2 or higher. A clear association between FN and AT was found. FN is likely to be a significant and prevalent pathogen in AT, especially in teenagers and young adults. However, no evidence for causality between FN and AT has been established and it is unexplored if timely antibiotic therapy directed against FN accelerates the resolution of symptoms and decreases the risk of complications.

  20. Immunoglobulin class and subclass distribution of antibodies reactive with the immunodominant antigen of Actinobacillus actinomycetemcomitans serotype b.

    PubMed Central

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

    1993-01-01

    The aims of this study were to determine the immunodominant antigens of Actinobacillus actinomycetemcomitans serotype b (Aab) for the different immunoglobulin (Ig) classes and subclasses and to determine the relative levels of these different Igs in serum. Seropositive early-onset periodontitis patients were sampled, and the Ig classes IgG, IgA, and IgM and subclasses IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 were studied. Reactivity with Aab antigens was assessed by using the Western blot (immunoblot) in limiting dilution analysis and radioimmunoassay with sera from 13 early-onset periodontitis subjects. A smeared antigen in the upper portion of the immunoblots, typical of high-molecular-weight LPS, was immunodominant for IgG, IgA, IgM, IgG1, IgG2, IgG3, IgA1, and IgA2. This smeared antigen was present in every patient for all of these Igs at the endpoint. A few additional antigens were also present at the endpoint in some patients, but none were present in more than half of the subjects. The distribution of antibody titers by Ig classes reactive with the Aab immunodominant antigen was IgG > IgA > IgM. The distribution of antibody titers by IgG subclass was IgG2 > IgG1 approximately IgG3. Further quantitation by radioimmunoassay revealed that the mean concentration of IgG2 (65.7 micrograms/ml) was significantly greater than that of IgG1 (8.8 micrograms/ml). The IgA subclass distribution was IgA1 >> IgA2, with IgA1 apparently being second only to IgG2. Therefore, the Aab antigen eliciting the highest antibody level in virtually all Ig classes and subclasses appeared to be lipopolysaccharide, and IgG2 was markedly elevated over all other serum Ig classes or subclasses reactive with Aab. Images PMID:8500879

  1. Transcriptional regulation of the Aggregatibacter actinomycetemcomitans ygiW-qseBC operon by QseB and integration host factor proteins.

    PubMed

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

    2014-12-01

    The QseBC two-component system plays a pivotal role in regulating virulence and biofilm growth of the oral pathogen Aggregatibacter actinomycetemcomitans. We previously showed that QseBC autoregulates the ygiW-qseBC operon. In this study, we characterized the promoter that drives ygiW-qseBC expression. Using lacZ transcriptional fusion constructs and 5'-rapid amplification of cDNA ends, we showed that ygiW-qseBC expression is driven by a promoter that initiates transcription 53 bases upstream of ygiW and identified putative cis-acting promoter elements, whose function was confirmed using site-specific mutagenesis. Using electrophoretic mobility shift assays, two trans-acting proteins were shown to interact with the ygiW-qseBC promoter. The QseB response regulator bound to probes containing the direct repeat sequence CTTAA-N6-CTTAA, where the CTTAA repeats flank the -35 element of the promoter. The ygiW-qseBC expression could not be detected in A. actinomycetemcomitans ΔqseB or ΔqseBC strains, but was restored to WT levels in the ΔqseBC mutant when complemented by single copy chromosomal insertion of qseBC. Interestingly, qseB partially complemented the ΔqseBC strain, suggesting that QseB could be activated in the absence of QseC. QseB activation required its phosphorylation since complementation did not occur using qseB(pho-), encoding a protein with the active site aspartate substituted with alanine. These results suggest that QseB is a strong positive regulator of ygiW-qseBC expression. In addition, integration host factor (IHF) bound to two sites in the promoter region and an additional site near the 5' end of the ygiW ORF. The expression of ygiW-qseBC was increased by twofold in ΔihfA and ΔihfB strains of A. actinomycetemcomitans, suggesting that IHF is a negative regulator of the ygiW-qseBC operon.

  2. Mouse interleukin-1 receptor antagonist induced by Actinobacillus actinomycetemcomitans lipopolysaccharide blocks the effects of interleukin-1 on bone resorption and osteoclast-like cell formation.

    PubMed Central

    Nishihara, T; Ohsaki, Y; Ueda, N; Saito, N; Mundy, G R

    1994-01-01

    We have reported that P388D1 cell line murine macrophages stimulated with lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans release interleukin-1 (IL-1) inhibitor. The IL-1 inhibitor was purified from conditioned media of P388D1 cells stimulated with A. actinomycetemcomitans LPS for 72 h to homogeneity by a four-step procedure: acetic acid extraction from conditioned media; Bio-Gel P-60 gel filtration chromatography; DEAE-Sepharose CL-6B column chromatography; and reverse-phase high-performance liquid chromatography on a C18 hydrophobic support. The purified IL-1 inhibitor gave a single band of protein with a molecular mass of 26 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified IL-1 inhibitor was a heat- and acid-stable protein that was inactivated by digestion with trypsin and reduction with dithiothreitol. This inhibitory factor suppressed the proliferation of C3H/HeJ mouse thymocytes and the proliferation of IL-1-dependent cell lines, D10.G4.1 and RPMI 1788, induced by IL-1. However, this inhibitor did not affect the proliferation of IL-2-dependent CTLL-2 cells induced by IL-2, the proliferation of C3H/HeJ mouse thymocytes stimulated with a mitogenic dose of concanavalin A, and the proliferation of IL-6-dependent B9 cells induced by IL-6. Furthermore, the IL-1 inhibitor significantly blocked stimulation of bone resorption in organ cultures of newborn mouse calvaria and inhibited the osteoclast-like cell formation in mouse marrow cultures. A monoclonal antibody prepared against the purified IL-1 inhibitor reacted with mouse recombinant IL-1 receptor antagonist (rIL-1ra), and a polyclonal antibody to mouse rIL-1ra reacted with the IL-1 inhibitor by Western blot (immunoblot) analysis. These results indicate that the IL-1 inhibitor is an identical molecule to rIL-1ra, suggesting that the IL-1 inhibitor (IL-1ra) released by macrophages stimulated with LPS from A. actinomycetemcomitans may play an important mediative role

  3. 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

  4. 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.

  5. A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

    PubMed Central

    Ahlstrand, Tuuli; Tuominen, Heidi; Beklen, Arzu; Torittu, Annamari; Oscarsson, Jan; Sormunen, Raija; Pöllänen, Marja T.; Permi, Perttu; Ihalin, Riikka

    2017-01-01

    ABSTRACT Intrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1β. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1β and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI− mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1β in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1β internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations. PMID:27459270

  6. A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8.

    PubMed

    Ahlstrand, Tuuli; Tuominen, Heidi; Beklen, Arzu; Torittu, Annamari; Oscarsson, Jan; Sormunen, Raija; Pöllänen, Marja T; Permi, Perttu; Ihalin, Riikka

    2017-02-17

    Intrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1β. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1β and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI(-) mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1β in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1β internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations.

  7. 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

  8. Antimicrobial activity of nanoparticulate metal oxides against peri-implantitis pathogens.

    PubMed

    Vargas-Reus, Miguel A; Memarzadeh, Kaveh; Huang, Jie; Ren, Guogang G; Allaker, Robert P

    2012-08-01

    Dental plaque accumulation may result in peri-implantitis, an inflammatory process causing loss of supporting bone that may lead to dental implant failure. The antimicrobial activities of six metal and metal oxide nanoparticles and two of their composites against bacterial pathogens associated with peri-implantitis were examined under anaerobic conditions. The activities of nanoparticles of silver (Ag), cuprous oxide (Cu(2)O), cupric oxide (CuO), zinc oxide (ZnO), titanium dioxide (TiO(2)), tungsten oxide (WO(3)), Ag+CuO composite and Ag+ZnO composite were assessed by minimum inhibitory (bacteriostatic) concentration (MIC) and minimum bactericidal concentration (MBC) determination against Prevotella intermedia, Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans. Time-kill assays were carried out to examine the dynamics of the antimicrobial activity with ZnO nanoparticles. MIC and MBC values were in the range of <100 μg/mL to 2500 μg/mL and <100 μg/mL to >2500 μg/mL, respectively. The activity of the nanoparticles tested in descending order was Ag>Ag+CuO>Cu(2)O>CuO>Ag+ZnO>ZnO>TiO(2)>WO(3). Time-kill assays with ZnO demonstrated a significant decrease in growth of all species tested within 4h, reaching 100% within 2h for P. gingivalis and within 3h for F. nucleatum and P. intermedia. Coating titanium surfaces of dental and orthopaedic implants with antimicrobial nanoparticles should lead to an increased rate of implant success.

  9. 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

  10. Antimicrobial effect of adjunctive use of chlorhexidine mouthrinse in untreated gingivitis: a randomized, placebo-controlled study.

    PubMed

    Becerik, Sema; Türkoğlu, Oya; Emingil, Gülnur; Vural, Caner; Ozdemir, Güven; Atilla, Gül

    2011-06-01

    The aim of this study was to examine the effectiveness of chlorhexidine mouthrinse (CHX) in addition to daily plaque control on subgingival microbiota in patients with untreated gingivitis. Fifty gingivitis patients were randomized to CHX or placebo groups. CHX group rinsed with 0.2% CHX, while placebo group rinsed with placebo mouthrinse for 4 weeks. Subgingival plaque samples were collected and plaque index (PI), papilla bleeding index (PBI), calculus index, and probing pocket depth (PPD) were recorded at baseline and at 4 weeks. The amounts of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum, and total bacteria were detected by quantitative real-time PCR method. In the CHX group the total bacteria count was significantly reduced in posterior teeth at 4 weeks (p < 0.05), while no significant decrease was observed in the placebo group (p > 0.05). CHX mouthrinse as an adjunct to daily plaque control could be useful in the management of plaque-associated gingivitis and in reducing the subgingival total bacteria count especially in posterior teeth.

  11. Polyclonal B-cell activation induced by extracts of Gram-negative bacteria isolated from periodontally diseased sites.

    PubMed Central

    Bick, P H; Carpenter, A B; Holdeman, L V; Miller, G A; Ranney, R R; Palcanis, K G; Tew, J G

    1981-01-01

    The objective of this research was to determine whether gram-negative bacteria frequently isolated from periodontally diseased sites contained polyclonal B-cell activators. Polyclonal B-cell activation, which results in nonspecific activation of multiple B-cell clones was analyzed by a hemolysis-in-gel assay designed to detect a broad range of antibody specificities. Extracts from numerous bacterial strains, including Bacteroides gingivalis, Bacteroides melaninogenicus subsp. melaninogenicus, B. melaninogenicus subsp. intermedius, Fusobacterium nucleatum, Selenomonas sputigena, Capnocytophaga ochracea, and Actinobacillus actinomycetemcomitans, were tested. Extracts of the above organisms were found to stimulate polyclonal antibody responses in cultures of normal human peripheral blood lymphocytes, although the magnitude of stimulation varied among the extracts. Optimal antibody-forming cell responses were found at stimulator doses between 5 and 1,000 micrograms/ml. We conclude that the resident gram-negative subgingival flora associated with periodontal lesions possesses potent polyclonal B-cell activators. These activators may contribute to disease pathogenesis by inducing B lymphocytes to produce antibody, osteolytic factors, or both and possibly other mediators of inflammation. PMID:6975240

  12. [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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps.

    PubMed

    Doke, M; Fukamachi, H; Morisaki, H; Arimoto, T; Kataoka, H; Kuwata, H

    2016-08-01

    Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps (NETs). These are extracellular net-like fibers comprising DNA and antimicrobial components such as histones, LL-37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NETs to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram-negative periodontopathogenic bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD, putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA- and nucD-encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA, which required both Mg(2+) and Ca(2+) for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NETs.

  19. 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.

  20. Laser Supported Reduction of Specific Microorganisms in the Periodontal Pocket with the Aid of an Er,Cr:YSGG Laser: A Pilot Study

    PubMed Central

    Gutknecht, N.; Van Betteray, C.; Ozturan, S.; Vanweersch, L.; Franzen, R.

    2015-01-01

    Objective. The aim of this study was to evaluate the effectiveness of a radial firing tip of an Er,Cr:YSGG laser as an adjunct to a nonsurgical periodontal treatment. Methods. Twelve patients with chronic or aggressive periodontitis were treated by conventional periodontal treatment using ultrasonic devices and hand instruments and, additionally, in two quadrants with an Er,Cr:YSGG laser. A new radial firing tip (RFPT 14-5, Biolase) was used with 1.5 W, 30 Hz, 11% air, 20% water, and pulse duration 140 μs. Microbiological smears were taken before treatment, one day after lasing, and three and six months after lasing. Pocket depths of all periodontal sites were measured before and six months after treatment. Results. The total bacterial load of Prevotella intermedia, Tannerella forsythia, Treponema denticola, Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans inside the pocket was reduced significantly throughout the whole examination time. Greater pocket depth reductions were observed in all groups. There was a slight higher reduction of pocket depth in the lased group after six months. Conclusions. These results support the thesis that Er,Cr:YSGG laser supported periodontal treatment leads to a significant reduction of periopathogenes and thereby helps the maintenance of periodontal health. PMID:25879057

  1. [Antimicrobial effects of tea tree oil (Melaleuca alternifolia) on oral microorganisms].

    PubMed

    Kulik, E; Lenkeit, K; Meyer, J

    2000-01-01

    The essential oil of Melaleuca alternifolia (tea tree oil) exhibits antimicrobial activity against a wide range of Gram-positive and Gram-negative bacteria, yeasts and fungi. In this study the bacteriostatic and bacteriocidal/fungicidal activity of a tea tree oil solution, of a new tea tree oil (Tebodont) and the respective placebo-gel, of a chlorhexidindigluconate-solution and of PlakOut was tested in vitro against ten different oral microorganisms. Minimum inhibitory concentrations were in the range from 0.0293% to 1.25% for the tea tree oil solution and from 0.0082% to 1.25% for the tea tree oil gel. The values for minimum bacteriocidal/fungicidal concentrations were in the range from 0.0521% to 2.5% for the tea tree oil solution and from <0.0098% to 3.33% for the tea tree oil gel. The most susceptible microorganisms were Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis, whereas Streptococcus mutans and Prevotella intermedia were the least susceptible ones. Both for the chlorhexidindigluconate solution and for PlakOut the values for the minimal inhibitory concentration and for the minimal cidal concentration were between <0.0002% and 0.0125%.

  2. 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.

  3. Antimicrobial photodynamic efficiency of novel cationic porphyrins towards periodontal Gram-positive and Gram-negative pathogenic bacteria.

    PubMed

    Prasanth, Chandra Sekhar; Karunakaran, Suneesh C; Paul, Albish K; Kussovski, Vesselin; Mantareva, Vanya; Ramaiah, Danaboyina; Selvaraj, Leslie; Angelov, Ivan; Avramov, Latchezar; Nandakumar, Krishnankutty; Subhash, Narayanan

    2014-01-01

    The Gram-negative Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum are major causative agents of aggressive periodontal disease. Due to increase in the number of antibiotic-resistant bacteria, antimicrobial Photodynamic therapy (aPDT) seems to be a plausible alternative. In this work, photosensitization was performed on Gram-positive and Gram-negative bacteria in pure culture using new-age cationic porphyrins, namely mesoimidazolium-substituted porphyrin derivative (ImP) and pyridinium-substituted porphyrin derivative (PyP). The photophysical properties of both the sensitizers including absorption, fluorescence emission, quantum yields of the triplet excited states and singlet oxygen generation efficiencies were evaluated in the context of aPDT application. The studied porphyrins exhibited high ability to accumulate into bacterial cells with complete penetration into early stage biofilms. As compared with ImP, PyP was found to be more effective for photoinactivation of bacterial strains associated with periodontitis, without any signs of dark toxicity, owing to its high photocytotoxicity.

  4. Novel bioactive tetracycline-containing electrospun polymer fibers as a potential antibacterial dental implant coating.

    PubMed

    Shahi, R G; Albuquerque, M T P; Münchow, E A; Blanchard, S B; Gregory, R L; Bottino, M C

    2016-09-01

    The purpose of this investigation was to determine the ability of tetracycline-containing fibers to inhibit biofilm formation of peri-implantitis-associated pathogens [i.e., Porphyromonas gingivalis (Pg), Fusobacterium nucleatum (Fn), Prevotella intermedia (Pi), and Aggregatibacter actinomycetemcomitans (Aa)]. Tetracycline hydrochloride (TCH) was added to a poly(DL-lactide) [PLA], poly(ε-caprolactone) [PCL], and gelatin [GEL] polymer blend solution at distinct concentrations to obtain the following fibers: PLA:PCL/GEL (TCH-free, control), PLA:PCL/GEL + 5 % TCH, PLA:PCL/GEL + 10 % TCH, and PLA:PCL/GEL + 25 % TCH. The inhibitory effect of TCH-containing fibers on biofilm formation was assessed by colony-forming units (CFU/mL). Qualitative analysis of biofilm inhibition was done via scanning electron microscopy (SEM). Statistical significance was reported at p < 0.05. Complete inhibition of biofilm formation on the fibers was observed in groups containing TCH at 10 and 25 wt%. Fibers containing TCH at 5 wt% demonstrated complete inhibition of Aa biofilm. Even though a marked reduction in CFU/mL was observed with an increase in TCH concentration, Pi proved to be the most resilient microorganism. SEM images revealed the absence of or a notable decrease in bacterial biofilm on the TCH-containing nanofibers. Collectively, our data suggest that tetracycline-containing fibers hold great potential as an antibacterial dental implant coating.

  5. Subgingival bacteria in Ghanaian adolescents with or without progression of attachment loss

    PubMed Central

    Dahlén, Gunnar; Claesson, Rolf; Åberg, Carola Höglund; Haubek, Dorte; Johansson, Anders; Kwamin, Francis

    2014-01-01

    Objective This study describes subgingival bacterial profiles associated with clinical periodontal status in Ghanaian adolescents with or without progression of attachment loss. Materials and methods Among 500 adolescents included in a cohort study, 397 returned 2 years later for a periodontal re-examination, including full-mouth CAL measurements. At follow-up, a subgroup of 98 adolescents was also subjected to bacterial sampling with paper points at four periodontal sites (mesial aspect of 11, 26, 31, and 46) and analyzed with the checkerboard DNA–DNA hybridization technique against DNA-probes from nine periodontitis-associated bacterial species. Results The 98 Ghanaian adolescents examined in the present study were similar to the entire group examined at the 2-year follow-up with respect to age, gender, and CAL ≥3 mm. A high detection frequency of Fusobacterium nucleatum and Prevotella intermedia (>99%) using checkerboard analysis was found, while for Aggregatibacter actinomycetemcomitans the detection frequency was <50%. A strong correlation was found at the individual level between the presence of P. intermedia and the total CAL change, and P. intermedia and Porphyromonas gingivalis were strongly correlated with a change in CAL and probing pocket depth (PPD) at the sampled sites. In a linear regression model, a significant discriminating factor for the total CAL change in the dentition during the 2-year follow-up period was obtained for P. intermedia and public school. Conclusion This study indicates that subgingival bacterial species other than A. actinomycetemcomitans, for example, P. intermedia, have a significant association with periodontal breakdown (change in CAL) in Ghanaian adolescents with progression of periodontal attachment loss. PMID:24834145

  6. Effect of ozone on periodontopathogenic species--an in vitro study.

    PubMed

    Eick, Sigrun; Tigan, Marius; Sculean, Anton

    2012-04-01

    The in vitro study was aimed to determine the effect of ozone on periodontopathogenic microorganisms. Ozone was generated for 6 s-2 × 24 s (corresponding to 0.56 mg-2 × 2.24 mg of ozone) against 23 mainly anaerobic periodontopathogenic species. Agar diffusion test was used as a screening method. Then, the killing activity was tested in a serum-free environment and with 25% v/v inactivated serum. Further, the effect of ozone on bactericidal activity of native serum was analyzed against Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans. Agar diffusion test showed a high efficacy of ozone against microorganisms, especially against Porphyromonas gingivalis. This result was confirmed by the killing tests; most of the strains in a concentration of 10(5) were completely eliminated after twofold 18-s application of ozone. Only four of the six potentially "superinfecting" species (Staphylococcus aureus, Enterococcus faecalis, Enterobacter cloacae, Candida albicans) survived in part. Addition of heat-inactivated serum reduced the killing rate of ozone by 78% after 6-s and by 47% after twofold 18-s exposures; no strain was completely eradicated after any application of ozone. The bactericidal effect of native serum was enhanced after application of ozone; no effect was visible on the included A. actinomycetemcomitans strain which was found to be completely resistant to the bactericidal action of serum. In conclusion, (a) ozone has a strong antibacterial activity against putative periodontopathogenic microorganisms, and (b) the bactericidal effect is reduced in the presence of serum. Ozone may have potential as an adjunctive application to mechanical treatment in periodontitis patients.

  7. 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

  8. The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Active Subunit CdtB Contains a Cholesterol Recognition Sequence Required for Toxin Binding and Subunit Internalization.

    PubMed

    Boesze-Battaglia, Kathleen; Walker, Lisa P; Zekavat, Ali; Dlakić, Mensur; Scuron, Monika Damek; Nygren, Patrik; Shenker, Bruce J

    2015-10-01

    Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted, indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the proinflammatory response in macrophages (interleukin 1β [IL-1β] and tumor necrosis factor alpha [TNF-α] release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and, further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells.

  9. Human immune responses to oral micro-organisms. I. Association of localized juvenile periodontitis (LJP) with serum antibody responses to Actinobacillus actinomycetemcomitans.

    PubMed Central

    Ebersole, J L; Taubman, M A; Smith, D J; Genco, R J; Frey, D E

    1982-01-01

    The association between periodontal disease in humans and serum and salivary antibody to Actinobacillus actinomycetemcomitans strain Y4 was determined. An Elisa was used to examine anti-Y4 antibody of the IgM, IgG, IgA and IgE isotypes in serum from 127 individuals and IgA in parotid saliva. Patients diagnosed as having localized juvenile periodontitis (n = 37) had significantly higher levels and frequency of serum IgG antibodies to Y4 than all other groups. Serum and salivary IgA and serum IgE antibody levels were significantly increased in patients with both localized and generalized types of juvenile periodontitis (n = 48) when compared to all other patient groups. Specificity studies suggested that the antigenic determinants that were differentiating the group responses were unique to the Y4 organism. These results indicate that serum antibodies to Y4 may reflect an infectious process with this micro-organism and that these responses may provide some diagnostic value in delineating different types of periodontal diseases. PMID:7094425

  10. MicroRNAs responsive to Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis LPS modulate expression of genes regulating innate immunity in human macrophages.

    PubMed

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

    2014-07-01

    MicroRNAs (miRNAs) are a class of small, noncoding RNAs that regulate post-transcriptional expression of their respective target genes and are responsive to various stimuli, including LPS. Here we examined the early (4 h) miRNA responses of THP1-differentiated macrophages challenged with LPS derived from the periodontal pathogens, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis or environmentally-modified LPS obtained from P. gingivalis 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 interleukin-6 receptorα (IL-6Rα) and IFN-γ inducible protein 30 and let-7f targeting of suppressor of cytokine signaling 4 and thrombospondin-1. Transfection experiments confirmed miR-29b and let-7f modulation of IL-6Rα and SOCS4 protein expression levels, respectively. Thus, we have demonstrated convergent/divergent miRNA responses to wild type LPS 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.

  11. Actinobacillus actinomycetemcomitans lipopolysaccharide stimulates the phosphorylation of p44 and p42 MAP kinases through CD14 and TLR-4 receptor activation in human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Kawasaki-Cárdenas, Perla; Cruz-Arroyo, Santa Rita; Pérez-Garzón, Miguel; Maldonado-Frías, Silvia

    2006-04-25

    Tyrosine phosphorylation is an early step in lipopolysaccharide (LPS) stimulated monocytes and macrophages that appears to play a key role in signal transduction. We have demonstrated that LPS purified from Actinobacillus actinomycetemcomitans also increases protein tyrosine phosphorylation in human gingival fibroblasts (HGF). This effect was elicited rapidly after LPS stimulation at concentrations that stimulate anti-bacterial responses in human gingival fibroblasts. Two main proteins, with an apparent molecular weight of 44 and 42 kDa, were phosphorylated after LPS stimulation of the human gingival fibroblasts. The phosphorylation was detected after 5 to 15 min and reached the maximum at 30 min of treatment. The increase in tyrosine phosphorylation was apparent following stimulation with LPS at 10 ng/ml and the response was dose dependent up to 10 microg/ml. Pretreatment with the tyrosine kinase inhibitors, herbimycin A and genistein inhibited the LPS-stimulated phosphorylation of p44 and p42 MAP kinases in a dose dependent manner. Pretreatment of human gingival fibroblasts with antibodies anti-CD14 or anti-TLR-4 but not anti-TLR-2 inhibited the LPS-induced tyrosine phosphorylation of p44 and p42. Additionally, LPS-induced p44 and p42 phosphorylation was inhibited by polymyxin treatment. These findings demonstrate that LPS from A. actinomycetemcomintans increases rapidly p44 and p42 phosphorylation (ERK 1 and ERK 2, respectively) in human gingival fibroblasts. Our data also suggest that CD14 and TLR-4 receptors are involved in the LPS effects in human gingival fibroblasts.

  12. Involvement of Ganglioside GM3 in G2/M Cell Cycle Arrest of Human Monocytic Cells Induced by Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin

    PubMed Central

    Mise, Koji; Akifusa, Sumio; Watarai, Shinobu; Ansai, Toshihiro; Nishihara, Tatsuji; Takehara, Tadamichi

    2005-01-01

    Actinobacillus actinomycetemcomitans produces a toxin called cytolethal distending toxin (CDT), which causes host cell DNA damage leading to the induction of DNA damage checkpoint pathways. CDT consists of three subunits, CdtA, CdtB, and CdtC. CdtB is the active subunit of CDT and exerts its effect as a nuclease that damages nuclear DNA, triggering cell cycle arrest. In the present study, we confirmed that the only combination of toxin proteins causing cell cycle arrest was that of all three recombinant CDT (rCDT) protein subunits. Furthermore, in order for rCDT to demonstrate toxicity, it was necessary for CdtA and CdtC to access the cell before CdtB. The coexistence of CdtA and CdtC was necessary for these subunits to bind to the cell. Cells treated with the glucosylceramide synthesis inhibitor 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol showed resistance to the cytotoxicity induced by rCDT. Furthermore, LY-B cells, which are deficient in the biosynthesis of sphingolipid, also showed resistance to the cytotoxicity induced by rCDT. To evaluate the binding of each subunit for glucosylceramides, we performed thin-layer chromatography immunostaining. The results indicated that each subunit reacted with the glycosphingolipids GM1, GM2, GM3, Gb3, and Gb4. The rCDT mixture incubated with liposomes containing GM3 displayed partially reduced toxicity. These results indicate that GM3 can act as a CDT receptor. PMID:16040998

  13. 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

  14. Correlation of Aggregatibacter actinomycetemcomitans Detection with Clinical/Immunoinflammatory Profile of Localized Aggressive Periodontitis Using a 16S rRNA Microarray Method: A Cross-Sectional Study

    PubMed Central

    Gonçalves, Patricia F.; Klepac-Ceraj, Vanja; Huang, Hong; Paster, Bruce J.; Aukhil, Ikramuddin; Wallet, Shannon M.; Shaddox, Luciana M.

    2013-01-01

    Objective The objective of this study was to determine whether the detection of Aggregatibacter actinomycetemcomitans (Aa) correlates with the clinical and immunoinflammatory profile of Localized Aggressive Periodontitis (LAP), as determined by by 16S rRNA gene-based microarray. Subjects and Methods Subgingival plaque samples from the deepest diseased site of 30 LAP patients [PD ≥ 5 mm, BoP and bone loss] were analyzed by 16S rRNA gene-based microarrays. Gingival crevicular fluid (GCF) samples were analyzed for 14 cyto/chemokines. Peripheral blood was obtained and stimulated in vitro with P.gingivalis and E.coli to evaluate inflammatory response profiles. Plasma lipopolysaccharide (LPS) levels were also measured. Results Aa was detected in 56% of LAP patients and was shown to be an indicator for different bacterial community structures (p<0.01). Elevated levels of pro-inflammatory cyto/chemokines were detected in LPS-stimulated blood samples in both Aa-detected and Aa-non-detected groups (p>0.05). Clinical parameters and serum LPS levels were similar between groups. However, Aa-non-detected GCF contained higher concentration of IL-8 than Aa-detected sites (p<0.05). TNFα and IL1β were elevated upon E.coli LPS stimulation of peripheral blood cells derived from patients with Aa-detected sites. Conclusions Our findings demonstrate that the detection of Aa in LAP affected sites, did not correlate with clinical severity of the disease at the time of sampling in this cross-sectional study, although it did associate with lower local levels of IL-8, a different subgingival bacterial profile and elevated LPS-induced levels of TNFα and IL1β. PMID:24376864

  15. 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

  16. 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

  17. The TadV Protein of Actinobacillus actinomycetemcomitans Is a Novel Aspartic Acid Prepilin Peptidase Required for Maturation of the Flp1 Pilin and TadE and TadF Pseudopilins†

    PubMed Central

    Tomich, Mladen; Fine, Daniel H.; Figurski, David H.

    2006-01-01

    The tad locus of Actinobacillus actinomycetemcomitans encodes genes for the biogenesis of Flp pili, which allow the bacterium to adhere tenaciously to surfaces and form strong biofilms. Although tad (tight adherence) loci are widespread among bacterial and archaeal species, very little is known about the functions of the individual components of the Tad secretion apparatus. Here we characterize the mechanism by which the pre-Flp1 prepilin is processed to the mature pilus subunit. We demonstrate that the tadV gene encodes a prepilin peptidase that is both necessary and sufficient for proteolytic maturation of Flp1. TadV was also found to be required for maturation of the TadE and TadF pilin-like proteins, which we term pseudopilins. Using site-directed mutagenesis, we show that processing of pre-Flp1, pre-TadE, and pre-TadF is required for biofilm formation. Mutation of a highly conserved glutamic acid residue at position +5 of Flp1, relative to the cleavage site, resulted in a processed pilin that was blocked in assembly. In contrast, identical mutations in TadE or TadF had no effect on biofilm formation, indicating that the mechanisms by which Flp1 pilin and the pseudopilins function are distinct. We also determined that two conserved aspartic acid residues in TadV are critical for function of the prepilin peptidase. Together, our results indicate that the A. actinomycetemcomitans TadV protein is a member of a novel subclass of nonmethylating aspartic acid prepilin peptidases. PMID:16980493

  18. Prelude to Oral Microbes and Chronic Diseases: Past, Present and Future

    PubMed Central

    Atanasova, Kalina R; Yilmaz, Özlem

    2015-01-01

    Associations between oral and systemic health are ancient. Oral opportunistic bacteria, particularly, Porphyromonas gingivalis and Fusobacterium nucleatum, have recently been deviated from their traditional roles and arguably ascended to central players based on their participations in complex co-dependent mechanisms of diverse systemic chronic diseases risk and pathogenesis, including cancers, rheumatoid-arthritis, and diabetes. PMID:25813714

  19. Prelude to oral microbes and chronic diseases: past, present and future.

    PubMed

    Atanasova, Kalina R; Yilmaz, Özlem

    2015-07-01

    Associations between oral and systemic health are ancient. Oral opportunistic bacteria, particularly, Porphyromonas gingivalis and Fusobacterium nucleatum, have recently been deviated from their traditional roles as periodontal pathogens and arguably ascended to central players based on their participations in complex co-dependent mechanisms of diverse systemic chronic diseases risk and pathogenesis, including cancers, rheumatoid-arthritis, and diabetes.

  20. Subgingival microbiota levels and their associations with periodontal status at the sampled sites in an adult Sudanese population using miswak or toothbrush regularly.

    PubMed

    Darout, Ismail A; Skaug, Nils; Albandar, Jasim M

    2003-04-01

    Little information is available on the effect of miswak use on gingival microbiota. We assessed levels of 28 oral bacteria in subgingival plaque of adult Sudanese miswak (n = 38) and toothbrush users (n = 36) age range 20-53 years (mean 34.6 years) to study associations between these bacteria, oral hygiene method, and periodontal status at the sampled sites. A pooled subgingival plaque sample from 6 probing sites of 1 selected tooth in each jaw was obtained from each subject. Whole genomic DNA probes and the checkerboard DNA-DNA hybridization were used in assessing 74 pooled samples. Using 10(5) bacterial cells threshold, between 2.6% and 47.4% of miswak users and between 2.8% and 36.1% of toothbrush users harbored the investigated species. The percentages of subjects with the investigated species at 10(6) bacterial cells varied between 2.6% and 39.5% in miswak and between 2.8% and 36.1% in toothbrush users. Miswak users harbored significantly higher Streptococcus intermedius, Actinobacillus actinomycetemcomitans, Veillonella parvula, Actinomyces israelii, and Capnocytophaga gingivalis, and significantly lower Selenomonas sputigena, Streptococcus salivarius, Actinomyces naeslundii, and Streptococcus oralis than did toothbrush users. Probing pocket depth > or = 6 mm showed significantly (P < 0.05) higher levels of Porphyromonas gingivalis, Treponema denticola, Bacteroides forsythus, Fusobacterium nucleatum, and V. parvula than those 4-5 mm. Our results indicate that the type of oral hygiene had a significant effect on levels of 11 out of 28 bacterial species, and that the type of effect was also dependent on type of bacteria and probing pocket depth.

  1. 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

  2. Effect of bioactive dental adhesive on periodontal and endodontic pathogens.

    PubMed

    Wang, Lin; Xie, Xianju; Weir, Michael D; Fouad, Ashraf F; Zhao, Liang; Xu, Hockin H K

    2016-11-01

    The objectives of this study were to: (1) develop a new bioactive dental bonding agent with nanoparticles of amorphous calcium phosphate and dimethylaminohexadecyl methacrylate for tooth root caries restorations and endodontic applications, and (2) investigate biofilm inhibition by the bioactive bonding agent against eight species of periodontal and endodontic pathogens for the first time. Bonding agent was formulated with 5 % of dimethylaminohexadecyl methacrylate. Nanoparticles of amorphous calcium phosphate at 30 wt% was mixed into adhesive. Eight species of biofilms were grown on resins: Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Parvimonas micra, Enterococcus faecalis, Enterococcus faecium. Colony-forming units, live/dead assay, biomass, metabolic activity and polysaccharide of biofilms were determined. The results showed that adding dimethylaminohexadecyl methacrylate and nanoparticles of amorphous calcium phosphate into bonding agent did not decrease dentin bond strength (P > 0.1). Adding dimethylaminohexadecyl methacrylate reduced the colony-forming units of all eight species of biofilms by nearly three orders of magnitude. The killing efficacy of dimethylaminohexadecyl methacrylate resin was: P. gingivalis > A. actinomycetemcomitans > P. intermedia > P. nigrescens > F. nucleatum > P. micra > E. faecalis > E. faecium. Dimethylaminohexadecyl methacrylate resin had much less biomass, metabolic activity and polysaccharide of biofilms than those without dimethylaminohexadecyl methacrylate (P < 0.05). In conclusion, a novel dental adhesive was developed for root caries and endodontic applications, showing potent inhibition of biofilms of eight species of periodontal and endodontic pathogens, and reducing colony-forming units by three orders of magnitude. The bioactive adhesive is promising for tooth root restorations to provide

  3. 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.

  4. Effect of Helicobacter pylori infection on chronic periodontitis by the change of microecology and inflammation

    PubMed Central

    Hu, Zhekai; Zhang, Yu; Li, Zhiyu; Yu, Yuedi; Kang, Wenyan; Han, Yingnan; Geng, Xiwen; Ge, Shaohua; Sun, Yundong

    2016-01-01

    Helicobacter pylori (H. pylori), a pathogen inducing peptic disease, is recently found to be binding to the progress of periodontitis. Most previous studies are case-controlled, and they investigate the risk of H. pylori infection in disease the development of while few studies evaluate the correlation between H. pylori and periodontal pathogens. Therefore, we investigated the correlation between H. pylori infection with periodontal parameters, periodontal pathogens and inflammation. The results indicated that patients with H. pylori showed significantly higher probing depth and attachment loss than those without (p < 0.05). Among 28 subgingival plaque samples from 14 patients, the frequencies of Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Treponema denticola were significantly higher with H. pylori infection than those without H. pylori infection (p < 0.05). However, the frequency of Aggregatibacter actinomycetemcomitans was lower (p < 0.05). Furthermore, after human acute monocytic leukemia cell line (THP-1) was stimulated with cagA-positive standard strains (cagA+ H. pylori 26695), the expression of periodontitis-related molecules Wnt5a, interleukin 8 (IL-8), interleukin 6 (IL-6) and interferon gamma (IFN-γ) significantly increased (p < 0.05). Conversely, the expression of tumor necrosis factor alpha (TNF-α) was almost stable. Meanwhile, cagA+ H. pylori promoted significantly higher expression of IL-8 and Wnt5a than isogenic cagA mutants strains (cagA− H. pylori 26695) did. Taken together, our data suggested that H. pylori might promote the growth of some periodontal pathogens and aggravate the progress of chronic periodontitis. PMID:27602578

  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. Microbiological Efficacy of Photodynamic Therapy as an Adjunct to Non-surgical Periodontal Treatment: A Clinical Trial

    PubMed Central

    Talebi, Mohammadreza; Taliee, Rojin; Mojahedi, Masoud; Meymandi, Mansour; Torshabi, Maryam

    2016-01-01

    Introduction: The efficiency of routine scaling and root planning is negatively influenced by the tooth anatomy and residual bacteria all possibly affecting the treatment outcomes in future. The present study compared the microbiologic effectiveness of the photodynamic therapy (PDT) as an adjunctive treatment modality for nonsurgical treatment in chronic periodontitis. Methods: In this randomized controlled clinical trial, 18 chronic periodontitis patients were selected. Four quadrants were randomly treated by scaling and root planning (SRP), diode laser (810n m wavelength, 1.5 W and 320 μm fiber, contact and sweeping technique), SRP + PDT (with diode laser 808 nm, 0.5 W) and laser + SRP (with diode laser 808 nm, 1 W) in each patient. Presence of periodontal pathogen species in the treated areas were measured before the treatment, at 1 and 3 months afterwards. The identification and reproduction of the specific genes of pathogen bacteria were done by means of polymerase chain reaction (PCR) technique. Presence of oral pathogen bacteria in the treatment groups were analyzed by chi-square test. A semi quantitative analysis was used to measure the intensity of white light in each band. This was calculated by number of pixels in each band. Results: In the qualitative analysis, Fusobacterium nucleatum (Fn) and Treponema denticola (Td) species were killed after 1 month in all treatment modalities. PDT had more effects to decrease Prevotella intermedia (Pi) species than SRP while Tannerella forsythensis count (Tf) species increased in all treatments. Furthermore, Actinobacillus actinomycetemcomitans (Aa) species decreased in all treatments and Porphyromonas gingivalis (P.g) species increased in all treatments after 1 and 3 months. Conclusion: It can be concluded that PDT was more effective as an adjunctive treatment to SRP than SRP alone; however, no distinct differences were found between both treatment modalities regarding reduction of certain pathogen bacteria. PMID

  7. 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.

  8. Necrotizing pneumonia in children: report of 41 cases between 2006 and 2011 in a French tertiary care center.

    PubMed

    Lemaître, Chloé; Angoulvant, François; Gabor, Flaviu; Makhoul, Juliette; Bonacorsi, Stéphane; Naudin, Jérôme; Alison, Marianne; Faye, Albert; Bingen, Edouard; Lorrot, Mathie

    2013-10-01

    Forty-one children hospitalized for necrotizing pneumonia were retrospectively analyzed. Necrotizing pneumonia represented 0.8% of community-acquired pneumonia and 6% of hospitalized community-acquired pneumonia. The chest radiograph revealed necrosis on admission in onethird of cases. Twenty-one cases (51%) were documented, including 13 Staphylococcus aureus, all Panton-Valentine leukocidin positive, 7 Streptococcus pneumoniae and 1 Fusobacterium nucleatum.

  9. Red wine and oenological extracts display antimicrobial effects in an oral bacteria biofilm model.

    PubMed

    Muñoz-González, Irene; Thurnheer, Thomas; Bartolomé, Begoña; Moreno-Arribas, M Victoria

    2014-05-21

    The antimicrobial effects of red wine and its inherent components on oral microbiota were studied by using a 5-species biofilm model of the supragingival plaque that includes Actinomyces oris, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans and Veillonella dispar. Microbiological analysis (CFU counting and confocal laser scanning microscopy) of the biofilms after the application of red wine, dealcoholized red wine, and red wine extract solutions spiked or not with grape seed and inactive dry yeast extracts showed that the solutions spiked with seed extract were effective against F. nucleatum, S. oralis and A. oris. Also, red wine and dealcoholized wine had an antimicrobial effect against F. nucleatum and S. oralis. Additional experiments showed almost complete and early degradation of flavan-3-ol precursors [(+)-catechin and procyanidin B2] when incubating biofilms with the red wine extract. To our knowledge, this is the first study of antimicrobial properties of wine in an oral biofilm model.

  10. The T Cell Response to Actinobacillus actinomycetemcomitans

    DTIC Science & Technology

    2006-05-01

    disease (11). Other clinical features of the disease include deep pain on mastication, first molar mobility, distolabial migration of maxillary incisors...transformed into one shot chemically competent E. coli TOP -10 (InVitrogen). 40 .tl from each transformation was grown at 37°C overnight on LB plates...5. Peripheral Blood Mononuclear Cells (PBMCs) isolation The 13 ml vacutainer with SST Gel and Clot Activator, or "Tiger Top ," was centrifuged at 3800

  11. Photoinactivation Using Visible Light Plus Water-Filtered Infrared-A (vis+wIRA) and Chlorine e6 (Ce6) Eradicates Planktonic Periodontal Pathogens and Subgingival Biofilms

    PubMed Central

    Al-Ahmad, Ali; Walankiewicz, Aleksander; Hellwig, Elmar; Follo, Marie; Tennert, Christian; Wittmer, Annette; Karygianni, Lamprini

    2016-01-01

    Alternative treatment methods for pathogens and microbial biofilms are required due to the widespread rise in antibiotic resistance. Antimicrobial photodynamic therapy (aPDT) has recently gained attention as a novel method to eradicate pathogens. The aim of this study was to evaluate the antimicrobial effects of a novel aPDT method using visible light (vis) and water infiltrated infrared A (wIRA) in combination with chlorine e6 (Ce6) against different periodontal pathogens in planktonic form and within in situ subgingival oral biofilms. Eight different periodontal pathogens were exposed to aPDT using vis+wIRA and 100 μg/ml Ce6 in planktonic culture. Additionally, pooled subgingival dental biofilm was also treated by aPDT and the number of viable cells determined as colony forming units (CFU). Live/dead staining was used in combination with confocal laser scanning microscopy to visualize and quantify antimicrobial effects within the biofilm samples. Untreated negative controls as well as 0.2% chlorhexidine-treated positive controls were used. All eight tested periodontal pathogens including Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Eikenella corrodens, Actinomyces odontolyticus, Fusobacterium nucleatum, Parvimonas micra, Slackia exigua, and Atopobium rimae and the aPDT-treated subgingival biofilm were eliminated over the ranges of 3.43–8.34 and 3.91–4.28 log10 CFU in the log10 scale, respectively. Thus, aPDT showed bactericidal effects on the representative pathogens as well as on the in situ subgingival biofilm. The live/dead staining also revealed a significant reduction (33.45%) of active cells within the aPDT-treated subgingival biofilm. Taking the favorable tissue healing effects of vis+wIRA into consideration, the significant antimicrobial effects revealed in this study highlight the potential of aPDT using this light source in combination with Ce6 as an adjunctive method to treat periodontitis as well as periimplantitis. The

  12. Photoinactivation Using Visible Light Plus Water-Filtered Infrared-A (vis+wIRA) and Chlorine e6 (Ce6) Eradicates Planktonic Periodontal Pathogens and Subgingival Biofilms.

    PubMed

    Al-Ahmad, Ali; Walankiewicz, Aleksander; Hellwig, Elmar; Follo, Marie; Tennert, Christian; Wittmer, Annette; Karygianni, Lamprini

    2016-01-01

    Alternative treatment methods for pathogens and microbial biofilms are required due to the widespread rise in antibiotic resistance. Antimicrobial photodynamic therapy (aPDT) has recently gained attention as a novel method to eradicate pathogens. The aim of this study was to evaluate the antimicrobial effects of a novel aPDT method using visible light (vis) and water infiltrated infrared A (wIRA) in combination with chlorine e6 (Ce6) against different periodontal pathogens in planktonic form and within in situ subgingival oral biofilms. Eight different periodontal pathogens were exposed to aPDT using vis+wIRA and 100 μg/ml Ce6 in planktonic culture. Additionally, pooled subgingival dental biofilm was also treated by aPDT and the number of viable cells determined as colony forming units (CFU). Live/dead staining was used in combination with confocal laser scanning microscopy to visualize and quantify antimicrobial effects within the biofilm samples. Untreated negative controls as well as 0.2% chlorhexidine-treated positive controls were used. All eight tested periodontal pathogens including Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Eikenella corrodens, Actinomyces odontolyticus, Fusobacterium nucleatum, Parvimonas micra, Slackia exigua, and Atopobium rimae and the aPDT-treated subgingival biofilm were eliminated over the ranges of 3.43-8.34 and 3.91-4.28 log10 CFU in the log10 scale, respectively. Thus, aPDT showed bactericidal effects on the representative pathogens as well as on the in situ subgingival biofilm. The live/dead staining also revealed a significant reduction (33.45%) of active cells within the aPDT-treated subgingival biofilm. Taking the favorable tissue healing effects of vis+wIRA into consideration, the significant antimicrobial effects revealed in this study highlight the potential of aPDT using this light source in combination with Ce6 as an adjunctive method to treat periodontitis as well as periimplantitis. The present

  13. Could gut microbiota serve as prognostic biomarker associated with colorectal cancer patients' survival? A pilot study on relevant mechanism

    PubMed Central

    Wei, Zhiliang; Cao, Shougen; Liu, Shanglong; Yao, Zengwu; Sun, Teng; Li, Yi; Li, Jiante; Zhang, Dongfeng; Zhou, Yanbing

    2016-01-01

    Evidences have shown that dysbiosis could promote the progression of colorectal cancer (CRC). However, the association of dysbiosis and prognosis of CRC is barely investigated. Therefore, we used 16S rRNA gene sequencing approach to determine differences in microbiota among tumor tissues of different prognosis and found that Fusobacterium nucleatum and Bacteroides fragilis were more abundant in worse prognosis groups, while Faecalibacterium prausnitzii displayed higher abundance in survival group. To further explore the prognostic value of the found bacteria, Kaplan–Meier and Cox proportional regression analyses were used and the results exhibited that high abundance of F. nucleatum and B. fragilis were independent indicators of poor patient's survival. Besides, the expression of major inflammatory mediator were analyzed using PCR and western blot methods, and it turned out that high abundance of F. nucleatum was associated with increased expression of TNF-α, β-catenin and NF-κB, while COX-2, MMP-9 and NF-κB were positively related with high B. fragilis level, and high level of F. prausnitzii showed lower expression of β-catenin, MMP-9 and NF-κB. Moreover, immunohistochemical analysis indicated that KRAS and BRAF expression were prominent in F. nucleatum and B. fragilis high abundance group, while MLH1 showed lower expression. In conclusion, F. nucleatum, B. fragilis and F. prausnitzii can be identified as useful prognostic biomarkers for CRC, and dysbiosis might worsen the patients' prognosis by up-regulating gut inflammation level. PMID:27323816

  14. Changes in salivary microbiota increase volatile sulfur compounds production in healthy male subjects with academic-related chronic stress

    PubMed Central

    Marcondes, Fernanda Klein; Groppo, Francisco Carlos; Rolim, Gustavo Sattolo; de Moraes, Antonio Bento Alves; Cogo-Müller, Karina; Franz-Montan, Michelle

    2017-01-01

    Objective To investigate the associations among salivary bacteria, oral emanations of volatile sulfur compounds, and academic-related chronic stress in healthy male subjects. Materials and methods Seventy-eight healthy male undergraduate dental students were classified as stressed or not by evaluation of burnout, a syndrome attributed to academic-related chronic stress. This evaluation was carried out using the Maslach Burnout Inventory—Student Survey questionnaire. Oral emanations of hydrogen sulfide, methyl mercaptan, and dimethyl sulfide were measured using an Oral Chroma™ portable gas chromatograph. The amounts in saliva of total bacteria and seven bacteria associated with halitosis were quantified by qPCR. The in vitro production of H2S by S. moorei and/or F. nucleatum was also measured with the Oral Chroma™ instrument. Results The stressed students group showed increased oral emanations of hydrogen sulfide and dimethyl sulfide, together with higher salivary Solobacterium moorei levels (p < 0.05, Mann Whitney test). There were moderate positive correlations between the following pairs of variables: Fusobacterium nucleatum and S. moorei; F. nucleatum and hydrogen sulfide; Tannerella forsythia and F. nucleatum; T. forsythia and S. moorei. These correlations only occurred for the stressed group (p < 0.05, Spearman correlation). The in vitro experiment demonstrated that S. moorei increased H2S production by F. nucleatum (p < 0.05, ANOVA and Tukey’s test). Conclusion The increased amount of S. moorei in saliva, and its coexistence with F. nucleatum and T. forsythia, seemed to be responsible for increased oral hydrogen sulfide in the healthy male stressed subjects. PMID:28319129

  15. Myrsinoic acid B inhibits the production of hydrogen sulfide by periodontal pathogens in vitro.

    PubMed

    Ito, Satomi; Shimura, Susumu; Tanaka, Tomoko; Yaegaki, Ken

    2010-06-01

    Recently, we reported that myrsinoic acid B purified from Myrsine seguinii inhibited methyl mercaptan (CH(3)SH) production by Fusobacterium nucleatum JCM8532. Since hydrogen sulfide (H(2)S) is the main component of physiological halitosis, while CH(3)SH is involved in pathological oral halitosis, the objective of this study is to determine whether myrsinoic acid B inhibits H(2)S production by oral microorganisms. F. nucleatum, Porphyromonas gingivalis and Treponema denticola were incubated with myrsinoic acid B and a substrate such as l-cysteine or l-methionine. H(2)S or CH(3)SH concentration in the headspace air, was determined using a gas chromatograph. The concentration of myrsinoic acid B inhibiting 50% (IC(50)) of H(2)S production by F. nucleatum was 0.142 µg ml(-1), and the IC(50) of P. gingivalis and T. denticola were 2.71 µg ml(-1) and 28.9 µg ml(-1), respectively. The presence of pyruvate, a by-product of H(2)S production, was determined. The IC(50) values of myrsinoic acid B for pyruvate production were 22.9 µg ml(-1) for F. nucleatum, 87.7 µg ml(-1) for P. gingivalis and 165 µg ml(-1) for T. denticola. We concluded that myrsinoic acid B inhibited the production of both H(2)S and pyruvate by periodontal pathogens.

  16. The protective effect of recombinant FomA-expressing Lactobacillus acidophilus against periodontal infection.

    PubMed

    Ma, Li; Ding, Qinfeng; Feng, Xiping; Li, Fei

    2013-10-01

    A number of studies have shown that the outer membrane protein FomA found in Fusobacterium nucleatum demonstrates great potential as an immune target for combating periodontitis. Lactobacillus acidophilus is a useful antigen delivery vehicle for mucosal immunisation, and previous studies by our group have shown that L. acidophilus acts as a protective factor in periodontal health. In this study, making use of the immunogenicity of FomA and the probiotic properties of L. acidophilus, we constructed a recombinant form of L. acidophilus expressing the FomA protein and detected the FomA-specific IgG in the serum and sIgA in the saliva of mice through oral administration with the recombinant strains. When serum containing FomA-specific antibodies was incubated with the F. nucleatum in vitro, the number of Porphyromonas gingivalis cells that coaggregated with the F. nucleatum cells was significantly reduced. Furthermore, a mouse gum abscess model was successfully generated, and the range of gingival abscesses in the immune mice was relatively limited compared with the control group. The level of IL-1β in the serum and local gum tissues of the immune mice was consistently lower than in the control group. Our findings indicated that oral administration of the recombinant L. acidophilus reduced the risk of periodontal infection with P. gingivalis and F. nucleatum.

  17. Effect of S-PRG Eluate on Biofilm Formation and Enzyme Activity of Oral Bacteria

    PubMed Central

    Yoneda, Masahiro; Suzuki, Nao; Masuo, Yosuke; Fujimoto, Akie; Iha, Kosaku; Yamada, Kazuhiko; Iwamoto, Tomoyuki; Hirofuji, Takao

    2012-01-01

    Recently, the antibacterial activity of a composite resin containing prereacted glass ionomer (S-PRG) filler was revealed. We examined the effect of an S-PRG eluate on various biologic activities of Streptococcus mutans and Porphyromonas gingivalis. Adherence ability of S. mutans was evaluated by microtiter plate assay; protease and gelatinase activities of P. gingivalis were examined by synthetic substrate hydrolysis and gelatin film spot assay, respectively. Coaggregation of P. gingivalis with Fusobacterium nucleatum was also examined. S-PRG eluate was found to suppress streptococcal adherence. S-PRG eluate inhibited the protease and gelatinase activities of P. gingivalis and the coaggregation between P. gingivalis and F. nucleatum. These results indicate that S-PRG eluate suppresses streptococcal adherence and inhibits the protease and coaggregation activities of P. gingivalis. These findings may prompt research into novel strategies for preventing caries and periodontitis. PMID:22693508

  18. Antimicrobial efficacy of denture adhesives on some oral malodor-related microbes.

    PubMed

    Polyzois, Gregory; Stefaniotis, Theodoros; Papaparaskevas, Joseph; Donta, Catherine

    2013-01-01

    The objective of the study was to determine the antimicrobial efficacy of three denture adhesives toward Streptococcus oralis, mutans, Prevotella oralis and Fusobacterium nucleatum. Adhesives used were Corega Ultra(®), Fixodent Pro Original(®) and Biotene(®) Denture Grip. For Streptococcus oralis and Streptococcus mutans, four tubes of Trypticase Soy Broth 10 mL and 1 g denture of adhesive were used. In addition four tubes of Trypticase Soy Broth 10 mL without any denture adhesive was employed as control. For Prevotella oralis and Fusobacterium nucleatum, four tubes of thioglycolate 10 mL and 1 g denture adhesive were used for each one, while four tubes of thioglycolate 10 mL without adhesive served as control. All samples were incubated for 48 h at 37°C. After 48 h, the number of colonies was counted and the mean was extracted as cfu/mL. The results were evaluated with ANOVA on ranked data and Tukey's post hoc test at α = 0.05. Streptococcus oralis, mutans, Prevotella oralis and Fusobacterium nucleatum showed decreased number of colonies for each denture adhesive compared to the control. Under the conditions of this in vitro study, all the tested denture adhesives showed antimicrobial efficacy. However, in contrast to the hypothesis, there were differences among them. Corega Ultra(®) and Biotene(®) Denture Grip were more effective for all the tested oral malodor-related microbes than Fixodent Pro Original(®).

  19. Structural Basis of Cooperative Ligand Binding by the Glycine Riboswitch

    SciTech Connect

    E Butler; J Wang; Y Xiong; S Strobel

    2011-12-31

    The glycine riboswitch regulates gene expression through the cooperative recognition of its amino acid ligand by a tandem pair of aptamers. A 3.6 {angstrom} crystal structure of the tandem riboswitch from the glycine permease operon of Fusobacterium nucleatum reveals the glycine binding sites and an extensive network of interactions, largely mediated by asymmetric A-minor contacts, that serve to communicate ligand binding status between the aptamers. These interactions provide a structural basis for how the glycine riboswitch cooperatively regulates gene expression.

  20. Antibacterial and antibiofilm activities of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) against periodontopathic bacteria.

    PubMed

    Sun, Mengjun; Zhou, Zichao; Dong, Jiachen; Zhang, Jichun; Xia, Yiru; Shu, Rong

    2016-10-01

    Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are two major omega-3 polyunsaturated fatty acids (n-3 PUFAs) with antimicrobial properties. In this study, we evaluated the potential antibacterial and antibiofilm activities of DHA and EPA against two periodontal pathogens, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum). MTT assay showed that DHA and EPA still exhibited no cytotoxicity to human oral tissue cells when the concentration came to 100 μM and 200 μM, respectively. Against P. gingivalis, DHA and EPA showed the same minimum inhibitory concentration (MIC) of 12.5 μM, and a respective minimum bactericidal concentration (MBC) of 12.5 μM and 25 μM. However, the MIC and MBC values of DHA or EPA against F. nucleatum were both greater than 100 μM. For early-stage bacteria, DHA or EPA displayed complete inhibition on the planktonic growth and biofilm formation of P. gingivalis from the lowest concentration of 12.5 μM. And the planktonic growth of F. nucleatum was slightly but not completely inhibited by DHA or EPA even at the concentration of 100 μM, however, the biofilm formation of F. nucleatum at 24 h was significantly restrained by 100 μM EPA. For exponential-phase bacteria, 100 μM DHA or EPA completely killed P. gingivalis and significantly decreased the viable counts of F. nucleatum. Meanwhile, the morphology of P. gingivalis was apparently damaged, and the virulence factor gene expression of P. gingivalis and F. nucleatum was strongly downregulated. Besides, the viability and the thickness of mature P. gingivalis biofilm, together with the viability of mature F. nucleatum biofilm were both significantly decreased in the presence of 100 μM DHA or EPA. In conclusion, DHA and EPA possessed antibacterial activities against planktonic and biofilm forms of periodontal pathogens, which suggested that DHA and EPA might be potentially supplementary therapeutic agents for prevention

  1. 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

  2. Identification and localization of extraradicular biofilm-forming bacteria associated with refractory endodontic pathogens.

    PubMed

    Noguchi, Nobuo; Noiri, Yuichiro; Narimatsu, Masahiro; Ebisu, Shigeyuki

    2005-12-01

    Bacterial biofilms have been found to develop on root surfaces outside the apical foramen and be associated with refractory periapical periodontitis. However, it is unknown which bacterial species form extraradicular biofilms. The present study aimed to investigate the identity and localization of bacteria in human extraradicular biofilms. Twenty extraradicular biofilms, used to identify bacteria using a PCR-based 16S rRNA gene assay, and seven root-tips, used to observe immunohistochemical localization of three selected bacterial species, were taken from 27 patients with refractory periapical periodontitis. Bacterial DNA was detected from 14 of the 20 samples, and 113 bacterial species were isolated. Fusobacterium nucleatum (14 of 14), Porphyromonas gingivalis (12 of 14), and Tannellera forsythensis (8 of 14) were frequently detected. Unidentified and uncultured bacterial DNA was also detected in 11 of the 14 samples in which DNA was detected. In the biofilms, P. gingivalis was immunohistochemically detected in all parts of the extraradicular biofilms. Positive reactions to anti-F. nucleatum and anti-T. forsythensis sera were found at specific portions of the biofilm. These findings suggested that P. gingivalis, T. forsythensis, and F. nucleatum were associated with extraradicular biofilm formation and refractory periapical periodontitis.

  3. In vitro evaluation of the effect of nicotine, cotinine, and caffeine on oral microorganisms.

    PubMed

    Cogo, Karina; Montan, Michelle Franz; Bergamaschi, Cristiane de Cássia; D Andrade, Eduardo; Rosalen, Pedro Luiz; Groppo, Francisco Carlos

    2008-06-01

    The aim of this in vitro study was to evaluate the effects of nicotine, cotinine, and caffeine on the viability of some oral bacterial species. It also evaluated the ability of these bacteria to metabolize those substances. Single-species biofilms of Streptococcus gordonii, Porphyromonas gingivalis, or Fusobacterium nucleatum and dual-species biofilms of S. gordonii -- F. nucleatum and F. nucleatum -- P. gingivalis were grown on hydroxyapatite discs. Seven species were studied as planktonic cells, including Streptococcus oralis, Streptococcus mitis, Propionibacterium acnes, Actinomyces naeslundii, and the species mentioned above. The viability of planktonic cells and biofilms was analyzed by susceptibility tests and time-kill assays, respectively, against different concentrations of nicotine, cotinine, and caffeine. High-performance liquid chromatography was performed to quantify nicotine, cotinine, and caffeine concentrations in the culture media after the assays. Susceptibility tests and viability assays showed that nicotine, cotinine, and caffeine cannot reduce or stimulate bacterial growth. High-performance liquid chromatography results showed that nicotine, cotinine, and caffeine concentrations were not altered after bacteria exposure. These findings indicate that nicotine, cotinine, and caffeine, in the concentrations used, cannot affect significantly the growth of these oral bacterial strains. Moreover, these species do not seem to metabolize these substances.

  4. Comparative Study on the Characteristics of Weissella cibaria CMU and Probiotic Strains for Oral Care.

    PubMed

    Jang, Hye-Jin; Kang, Mi-Sun; Yi, Sung-Hun; Hong, Ji-Young; Hong, Sang-Pil

    2016-12-20

    Probiotics have been demonstrated as a new paradigm to substitute antibiotic treatment for dental caries, gingivitis, and chronic periodontitis. The present work was conducted to compare the characteristics of oral care probiotics: Weissella cibaria CMU (Chonnam Medical University) and four commercial probiotic strains. Survival rates under poor oral conditions, acid production, hydrogen peroxide production, as well as inhibition of biofilm formation, coaggregation, antibacterial activity, and inhibition of volatile sulfur compounds were evaluated. The viability of W. cibaria CMU was not affected by treatment of 100 mg/L lysozyme for 90 min and 1 mM hydrogen peroxide for 6 h. Interestingly, W. cibaria produced less acid and more hydrogen peroxide than the other four probiotics. W. cibaria inhibited biofilm formation by Streptococcus mutans at lower concentrations (S. mutans/CMU = 8) and efficiently coaggregated with Fusobacterium nucleatum. W. cibaria CMU and two commercial probiotics, including Lactobacillus salivarius and Lactobacillus reuteri, showed high antibacterial activities (>97%) against cariogens (S. mutans and Streptococcus sobrinus), and against periodontopathogens (F. nucleatum and Porphyromonas gingivalis). All of the lactic acid bacterial strains in this study significantly reduced levels of hydrogen sulfide and methyl mercaptan produced by F. nucleatum and P. gingivalis (p < 0.05). These results suggest that W. cibaria CMU is applicable as an oral care probiotic.

  5. Epigenetic regulation of human β-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria.

    PubMed

    Yin, L; Chung, W O

    2011-07-01

    Gingival epithelia utilize multiple signaling pathways to regulate innate immune responses to various oral bacteria, but little is understood about how these bacteria alter epithelial epigenetic status. In this study we report that DNA methyltransferase (DNMT1) and histone deacetylase expression were decreased in gingival epithelial cells treated with oral pathogen Porphyromonas gingivalis and nonpathogen Fusobacterium nucleatum. Pretreatment with trichostatin A and sodium butyrate, which increase acetylation of chromatin histones, significantly enhanced the gene expression of antimicrobial proteins human β-defensin 2 (hBD2) and CC chemokine ligand 20 (CCL20) in response to both bacterial challenges. Pretreatment with DNMT inhibitor 5'-azacytidine increased hBD2 and CCL20 expression in response to F. nucleatum, but not to P. gingivalis. Furthermore, we observed a differential pattern of protein levels of H3K4me3, which has been associated with chromatin remodeling and activation of gene transcription, in response to P. gingivalis vs. F. nucleatum. This study provides a new insight into the bacteria-specific innate immune responses via epigenetic regulation.

  6. The effects of stress hormones on growth of selected periodontitis related bacteria.

    PubMed

    Jentsch, H F R; März, Diana; Krüger, Monika

    2013-12-01

    The focus of this study was to examine in vitro the effects of stress hormones (catecholamines: epinephrine, norepinephrine, dopamine and hydrocortisone: cortisol) on the growth of four anaerobic species of periodontitis-related bacteria (Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia and Tannerella forsythia) and one facultative anaerobic species (Eikenella corrodens). Bacterial growth was determined by two different methods: fluorescence in situ hybridization (FISH), and the viable count by culture method. To simulate stress, each single strain was grown in a special growth medium with three different concentrations of each hormone, using an anaerobic chamber at 37 °C. Growth of F. nucleatum increased in the presence of all stress hormones. Growth of P. gingivalis was not significantly influenced by any hormone. Growth of P. intermedia and E. corrodens was inhibited by almost all stress hormones tested. Both methods of analysis revealed that the highest concentrations of norepinephrine and cortisol increased the growth of T. forsythia. Different hormones have a different effect on the growth of periodontitis-related bacteria in vitro. It appears that bacterial viability is more strongly influenced than is bacterial metabolic activity. The growth of F. nucleatum particularly and partially of T. forsythia is increased by several stress hormones and may have an additional negative impact on periodontal disease.

  7. Arginine-Ornithine Antiporter ArcD Controls Arginine Metabolism and Interspecies Biofilm Development of Streptococcus gordonii*♦

    PubMed Central

    Sakanaka, Akito; Kuboniwa, Masae; Takeuchi, Hiroki; Hashino, Ei; Amano, Atsuo

    2015-01-01

    Arginine is utilized by the oral inhabitant Streptococcus gordonii as a substrate of the arginine deiminase system (ADS), eventually producing ATP and NH3, the latter of which is responsible for microbial resistance to pH stress. S. gordonii expresses a putative arginine-ornithine antiporter (ArcD) whose function has not been investigated despite relevance to the ADS and potential influence on inter-bacterial communication with periodontal pathogens that utilize amino acids as a main energy source. Here, we generated an S. gordonii ΔarcD mutant to explore the role of ArcD in physiological homeostasis and bacterial cross-feeding. First, we confirmed that S. gordonii ArcD plays crucial roles for mediating arginine uptake and promoting bacterial growth, particularly under arginine-limited conditions. Next, metabolomic profiling and transcriptional analysis of the ΔarcD mutant revealed that deletion of this gene caused intracellular accumulation of ornithine leading to malfunction of the ADS and suppression of de novo arginine biosynthesis. The mutant strain also showed increased susceptibility to low pH stress due to reduced production of ammonia. Finally, accumulation of Fusobacterium nucleatum was found to be significantly decreased in biofilm formed by the ΔarcD mutant as compared with the wild-type strain, although ornithine supplementation restored fusobacterium biovolume in dual-species biofilms with the ΔarcD mutant and also enhanced single species biofilm development by F. nucleatum. Our results are the first direct evidence showing that S. gordonii ArcD modulates not only alkali and energy production but also interspecies interaction with F. nucleatum, thus initiating a middle stage of periodontopathic biofilm formation, by metabolic cross-feeding. PMID:26085091

  8. Effects of tongue cleaning on bacterial flora in tongue coating and dental plaque: a crossover study

    PubMed Central

    2014-01-01

    Background The effects of tongue cleaning on reconstruction of bacterial flora in dental plaque and tongue coating itself are obscure. We assessed changes in the amounts of total bacteria as well as Fusobacterium nucleatum in tongue coating and dental plaque specimens obtained with and without tongue cleaning. Methods We conducted a randomized examiner-blind crossover study using 30 volunteers (average 23.7 ± 3.2 years old) without periodontitis. After dividing randomly into 2 groups, 1 group was instructed to clean the tongue, while the other did not. On days 1 (baseline), 3, and 10, tongue coating and dental plaque samples were collected after recording tongue coating score (Winkel tongue coating index: WTCI). After a washout period of 3 weeks, the same examinations were performed with the subjects allocated to the alternate group. Genomic DNA was purified from the samples and applied to SYBR® Green-based real-time PCR to quantify the amounts of total bacteria and F. nucleatum. Results After 3 days, the WTCI score recovered to baseline, though the amount of total bacteria in tongue coating was significantly lower as compared to the baseline. In plaque samples, the bacterial amounts on day 3 and 10 were significantly lower than the baseline with and without tongue cleaning. Principal component analysis showed that variations of bacterial amounts in the tongue coating and dental plaque samples were independent from each other. Furthermore, we found a strong association between amounts of total bacteria and F. nucleatum in specimens both. Conclusions Tongue cleaning reduced the amount of bacteria in tongue coating. However, the cleaning had no obvious contribution to inhibit dental plaque formation. Furthermore, recovery of the total bacterial amount induced an increase in F. nucleatum in both tongue coating and dental plaque. Thus, it is recommended that tongue cleaning and tooth brushing should both be performed for promoting oral health. PMID:24423407

  9. Reducing the bioactivity of Tannerella forsythia lipopolysaccharide by Porphyromonas gingivalis.

    PubMed

    Kim, Young-Jae; Lee, Sung-Hoon

    2014-08-01

    Tannerella forsythia is considered a pathogen of periodontitis and forms a biofilm with multi-species bacteria in oral cavity. Lipopolysaccharide is a powerful immunostimulator and induces inflammation and shock. The purpose of this study was to investigate the characteristics of T. forsythia LPS in its co-cultivation with Fusobacterium nucleatum or Porphyromonas gingivalis. T. forsythia was co-cultured in the presence and absence of F. nucleatum and P. gingivalis and then T. forsythia LPS was extracted. The extracts were analyzed by SDS-PAGE and NF-κB reporter CHO cell lines. THP-1 cells were treated with the LPS and evaluated induction of cytokine expression by real-time RT-PCR and ELISA. For analysis of the bioactivity of T. forsythia LPS, the binding assay on LPS-binding protein (LBP) and CD14 was processed. The extracts did not contaminate other molecules except LPS and showed TLR4 agonists. Co-cultured T. forsythia LPS with P. gingivalis exhibited a lower level of induction of TNF-α, IL-1β, and IL-6 expression than single- or co-cultured T. forsythia LPS with F. nucleatum in the conditions of human serum. However, the three T. forsythia LPS did not show difference of cytokine induction in the serum free conditions. Co-cultured T. forsythia LPS with P. gingivalis exhibited a lower affinity to LBP and CD14 as binding site of O-antigen and attached at a lower level to THP-1 cells compared to single- or co-cultured T. forsythia LPS with F. nucleatum. The virulence of T. forsythia LPS was decreased by co-culturing with P. gingivalis and their affinity to LBP and CD14 was reduced, which may due to modification of O-antigen chain by P. gingivalis.

  10. 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

  11. Clinical appearance of orofacial infections of odontogenic origin in relation to microbiological findings.

    PubMed Central

    Heimdahl, A; von Konow, L; Satoh, T; Nord, C E

    1985-01-01

    Fifty-eight patients with acute orofacial infections of odontogenic origin were classified into two groups with respect to the severity of infection. A total of 174 anaerobic and 22 aerobic bacterial strains were isolated. Anaerobic gram-negative rods were isolated more frequently from the patients with severe infections than from the patients with infections judged as mild (P less than 0.05). The occurrence of Fusobacterium nucleatum especially appeared to be associated with the severity of the infections (P less than 0.05). Penicillin resistance among the anaerobes was rarely found, while resistance to erythromycin was a common finding. All aerobic and anaerobic bacteria were susceptible to clindamycin, and all obligate anaerobic bacteria were susceptible to nitroimidazoles. PMID:4031041

  12. Antibacterial activity of commercially available plant-derived essential oils against oral pathogenic bacteria.

    PubMed

    Bardají, D K R; Reis, E B; Medeiros, T C T; Lucarini, R; Crotti, A E M; Martins, C H G

    2016-01-01

    This work investigated the antibacterial activity of 15 commercially available plant-derived essential oils (EOs) against a panel of oral pathogens. The broth microdilution method afforded the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the assayed EOs. The EO obtained from Cinnamomum zeylanicum (Lauraceae) (CZ-EO) displayed moderate activity against Fusobacterium nucleatum (MIC and MBC = 125 μg/mL), Actinomyces naeslundii (MIC and MBC = 125 μg/mL), Prevotella nigrescens (MIC and MBC = 125 μg/mL) and Streptococcus mutans (MIC = 200 μg/mL; MBC = 400 μg/mL). (Z)-isosafrole (85.3%) was the main chemical component of this oil. We did not detect cinnamaldehyde, previously described as the major constituent of CZ-EO, in specimens collected in other countries.

  13. Antibacterial efficacy of triple-layered poly(lactic-co-glycolic acid)/nanoapatite/lauric acid guided bone regeneration membrane on periodontal bacteria.

    PubMed

    Saarani, Nur Najiha; Jamuna-Thevi, Kalitheerta; Shahab, Neelam; Hermawan, Hendra; Saidin, Syafiqah

    2017-01-20

    A guided bone regeneration (GBR) membrane has been extensively used in the repair and regeneration of damaged periodontal tissues. One of the main challenges of GBR restoration is bacterial colonization on the membrane, constitutes to premature membrane degradation. Therefore, the purpose of this study was to investigate the antibacterial efficacy of triple-layered GBR membrane composed of poly(lactic-co-glycolic acid) (PLGA), nanoapatite (NAp) and lauric acid (LA) with two types of Gram-negative periodontal bacteria, Fusobacterium nucleatum and Porphyromonas gingivalis through a disc diffusion and bacterial count tests. The membranes exhibited a pattern of growth inhibition and killing effect against both bacteria. The increase in LA concentration tended to increase the bactericidal activities which indicated by higher diameter of inhibition zone and higher antibacterial percentage. It is shown that the incorporation of LA into the GBR membrane has retarded the growth and proliferation of Gram-negative periodontal bacteria for the treatment of periodontal disease.

  14. Oral Epithelial Cell Responses to Multispecies Microbial Biofilms

    PubMed Central

    Peyyala, R.; Kirakodu, S.S.; Novak, K.F.; Ebersole, J.L

    2013-01-01

    This report describes the use of a novel model of multispecies biofilms to stimulate profiles of cytokines/chemokines from oral epithelial cells that contribute to local inflammation in the periodontium. Streptococcus gordonii (Sg)/S. oralis (So)/S. sanguinis (Ss) and Sg/Fusobacterium nucleatum (Fn)/Porphyromonas gingivalis (Pg) biofilms elicited significantly elevated levels of IL-1α and showed synergistic stimulatory activity compared with an additive effect of the 3 individual bacteria. Only the Sg/Actinomyces naeslundii (An)/Fn multispecies biofilms elicited IL-6 levels above those of control. IL-8 was a primary response to the Sg/An/Fn biofilms, albeit the level was not enhanced compared with a predicted composite level from the monospecies challenges. These results represent some of the first data documenting alterations in profiles of oral epithelial cell responses to multispecies biofilms. PMID:23300185

  15. Term and preterm labour are associated with distinct microbial community structures in placental membranes which are independent of mode of delivery.

    PubMed

    Doyle, R M; Alber, D G; Jones, H E; Harris, K; Fitzgerald, F; Peebles, D; Klein, N

    2014-12-01

    Infection is considered a possible trigger for preterm labour, supported by evidence showing the presence of bacteria in the placenta and placental membranes from preterm births. In this study, 16S rDNA pyrosequencing was used to identify bacteria in placental membranes. Caesarean sections and vaginal deliveries at term were found to harbour common genera. Mycoplasma hominis, Aerococcus christensenii, Gardnerella vaginalis and Fusobacterium nucleatum were either only present in preterm membranes or in greater abundance than at term. These data support previous studies that used either targeted qPCR or broad-range 16S rDNA PCR and cloning but not a recent microbiome analysis of placental tissue using high-throughput sequencing.

  16. A SONOGRAPHIC SHORT CERVIX AS THE ONLY CLINICAL MANIFESTATION OF INTRA-AMNIOTIC INFECTION

    PubMed Central

    HASSAN, SONIA; ROMERO, ROBERTO; HENDLER, ISRAEL; GOMEZ, RICARDO; KHALEK, NAHLA; ESPINOZA, JIMMY; NIEN, JYH KAE; BERRY, STANLEY M.; BUJOLD, EMMANUEL; CAMACHO, NATALIA; SOROKIN, YORAM

    2006-01-01

    OBJECTIVE A sonographically short cervix is a powerful predictor of spontaneous preterm delivery. However, the etiology and optimal management of a patient with a short cervix in the mid-trimester of pregnancy remain uncertain. Microbial invasion of the amniotic cavity (MIAC) and intra-amniotic inflammation are frequently present in patients with spontaneous preterm labor or acute cervical insufficiency. This study was conducted to determine the rate of MIAC and intra-amniotic inflammation in patients with a cervical length <25 mm in the mid-trimester. STUDY DESIGN A retrospective cohort study was conducted of patients referred to our high risk clinic because of a sonographic short cervix or a history of a previous preterm birth. Amniocenteses were performed for the evaluation of MIAC and for karyotype analysis in patients with a short cervix. Fluid was cultured for aerobic and anaerobic bacteria, as well as genital mycoplasmas. Patients with MIAC were treated with antibiotics selected by their physician. RESULTS Of 152 patients with a short cervix at 14–24 weeks, 57 had amniotic fluid analysis. The prevalence of MIAC was 9% (5/57). Among these patients, the rate of preterm delivery (<32 weeks) was 40% (2/5). Microorganisms isolated from amniotic fluid included Ureaplasma urealyticum (n=4) and Fusobacterium nucleatum (n=1). Patients with a positive culture for Ureaplasma urealyticum received intravenous Azithromycin. Three patients with Ureaplasma urealyticum had a sterile amniotic fluid culture after treatment, and subsequently delivered at term. The patient with Fusobacterium nucleatum developed clinical chorioamnionitis and was induced. CONCLUSION 1) Sub-clinical MIAC was detected in 9% of patients with a sonographically short cervix (<25 mm); and 2) maternal parenteral treatment with antibiotics can eradicate MIAC caused by Ureaplasma urealyticum. This was associated with delivery at term in the three patients whose successful treatment was documented by

  17. Bacterial vaginosis and vaginal microorganisms in idiopathic premature labor and association with pregnancy outcome.

    PubMed Central

    Holst, E; Goffeng, A R; Andersch, B

    1994-01-01

    The vaginal microflora of 49 women in idiopathic preterm labor was compared with that of 38 term controls to determine whether the presence of bacterial vaginosis (BV) and/or specific microorganisms would influence the rate of preterm delivery. Demographic factors, pregnancy outcome, and reproductive history were also studied. BV, as defined by the presence of clue cells in a vaginal wet mount and characteristic microbial findings in a stained vaginal smear and vaginal culture, was more common in women with preterm labor and delivery than in controls (P < 0.01). The condition, diagnosed in 41% of women who had both preterm labor and delivery (n = 22) and in 11% each of women who had preterm labor but term delivery (n = 27) and controls, was associated with a 2.1-fold risk (95% confidence intervals, 1.2 to 3.7) for preterm birth prior to 37 weeks of gestation. BV was associated with low birth weight. Of 49 women with preterm labor, 67% (8 of 12) of women with BV were delivered of low-birth-weight neonates (< 2,500 g) compared with 22% (8 of 37) of women without the condition (P < 0.0005). The presence of hydrogen peroxide-producing facultative Lactobacillus spp. was strongly negatively associated with both preterm delivery and BV. BV-associated microorganisms, i.e., Mobiluncus, Prevotella, and Peptostreptococcus species, Porphyromonas asaccharolytica, Fusobacterium nucleatum, Mycoplasma hominis, and high numbers of Gardnerella vaginalis were significantly associated with preterm delivery; all species also strongly associated with BV (P = 0.0001 for each comparison). Mobiluncus curtisii and Fusobacterium nucleatum were recovered exclusively from women with preterm delivery. Our study clearly indicates that BV and its associated organisms are correlated with idiopathic premature delivery. PMID:8126176

  18. High in vitro antibacterial activity of Pac-525 against Porphyromonas gingivalis biofilms cultured on titanium.

    PubMed

    Li, Ji-yin; Wang, Xue-jin; Wang, Li-na; Ying, Xiao-xia; Ren, Xiang; Liu, Hui-ying; Xu, Li; Ma, Guo-wu

    2015-01-01

    In order to investigate the potential of short antimicrobial peptides (AMPs) as alternative antibacterial agents during the treatment of peri-implantitis, the cytotoxic activity of three short AMPs, that is, Pac-525, KSL-W, and KSL, was determined using the MTT assay. The antimicrobial activity of these AMPs, ranging in concentration from 0.0039 mg/mL to 0.5 mg/mL, against the predominant planktonic pathogens, including Streptococcus sanguis, Fusobacterium nucleatum, and Porphyromonas gingivalis, involved in peri-implantitis was investigated. Furthermore, 2-day-old P. gingivalis biofilms cultured on titanium surfaces were treated with Pac-525 and subsequently observed and analysed using confocal laser scanning microscopy (CLSM). The average cell proliferation curve indicated that there was no cytotoxicity due to the three short AMPs. The minimum inhibitory concentration and minimum bactericidal concentration values of Pac-525 were 0.0625 mg/mL and 0.125 mg/mL, respectively, for P. gingivalis and 0.0078 mg/mL and 0.0156 mg/mL, respectively, for F. nucleatum. Using CLSM, we confirmed that compared to 0.1% chlorhexidine, 0.5 mg/mL of Pac-525 caused a significant decrease in biofilm thickness and a decline in the percentage of live bacteria. These data indicate that Pac-525 has unique properties that might make it suitable for the inhibition the growth of pathogenic bacteria around dental implants.

  19. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    SciTech Connect

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  20. Novel model for multispecies biofilms that uses rigid gas-permeable lenses.

    PubMed

    Peyyala, Rebecca; Kirakodu, Sreenatha S; Ebersole, Jeffrey L; Novak, Karen F

    2011-05-01

    Oral biofilms comprise complex multispecies consortia aided by specific inter- and intraspecies interactions occurring among commensals and pathogenic bacterial species. Oral biofilms are primary initiating factors of periodontal disease, although complex multifactorial biological influences, including host cell responses, contribute to the individual outcome of the disease. To provide a system to study initial stages of interaction between oral biofilms and the host cells that contribute to the disease process, we developed a novel in vitro model system to grow biofilms on rigid gas-permeable contact lenses (RGPLs), which enable oxygen to permeate through the lens material. Bacterial species belonging to early- and late-colonizing groups were successfully established as single- or three-species biofilms, with each group comprising Streptococcus gordonii, Streptococcus oralis, and Streptococcus sanguinis; S. gordonii, Actinomyces naeslundii, and Fusobacterium nucleatum; or S. gordonii, F. nucleatum, and Porphyromonas gingivalis. Quantification of biofilm numbers by quantitative PCR (qPCR) revealed substantial differences in the magnitude of bacterial numbers in single-species and multispecies biofilms. We evaluated cell-permeable conventional nucleic acid stains acridine orange, hexidium iodide, and Hoechst 33258 and novel SYTO red, blue, and green fluorochromes for their effect on bacterial viability and fluorescence yield to allow visualization of the aggregates of individual bacterial species by confocal laser scanning microscopy (CLSM). Substantial differences in the quantity and distribution of the species in the multispecies biofilms were identified. The specific features of these biofilms may help us better understand the role of various bacteria in local challenge of oral tissues.

  1. Salivary Periodontopathic Bacteria in Children and Adolescents with Down Syndrome

    PubMed Central

    Lopes Devito, Karina; Ribeiro, Luiz Cláudio

    2016-01-01

    Objective To assess and compare salivary periodontopathic bacteria between groups of Down syndrome and non-Down syndrome children and adolescents. Materials and Methods This study included a sample of 30 Down syndrome children and adolescents (G-DS) and 30 age- and sex-matched non-Down syndrome subjects (G-ND). Clinical examination determined the gingival bleeding index (GBI) and plaque index. Unstimulated whole saliva samples were collected from all participants. The fluorescence in situ hybridization (FISH) technique identified the presence and density of eight periodontopathic bacteria in saliva. The statistical analysis included chi-square and Mann-Whitney U tests. Results In the G-DS group, bleeding on probing was more frequent (p = 0.037) and higher densities of Campylobacter rectus (p = 0.013), Porphyromonas gingivalis (p = 0.025), Treponema denticola (p = 0.026), Fusobacterium nucleatum (p = 0.013), Prevotella intermedia (p = 0.001) and Prevotella nigrescens (p = 0.008) were observed. Besides, in the G-DS, the densities of bacteria from the orange complex were significantly higher in the age group 3–7 years for F. nucleatum (p = 0.029), P. intermedia (p = 0.001) and P. nigrescens (p = 0.006). C. rectus was higher in the age group 8–12 years (p = 0.045). Conclusion The results showed that children and adolescents with Down syndrome have higher susceptibility to periodontal disease and number of periodontopathic bacteria. PMID:27727287

  2. Intracanal Antibiotic Medication for Sustained Root Surface Disinfection–A Laboratory Evaluation

    PubMed Central

    Zaruba, Markus Tobias Winfried; Filli, Tilla; Rechenberg, Dan-Krister; Thurnheer, Thomas; Attin, Thomas; Schmidlin, Patrick Roger

    2015-01-01

    Purpose: To measure the release of an antibiotic mixture of ciprofloxacin, cerfuroxim and metronidazole (TreVitaMix, TVM) through human dentine and to assess the growth inhibition of Fusobacterium nucleatum. Material and Methods: Twenty-four extracted human incisors were scaled and endodontically treated. Root canals were either filled with antibiotic tri-mixture (TVM) or with the carrier material alone (propylene glycol, PG) and were coronally and apically sealed with a flowable composite. Transradicular medicament release was spectrophotometrically measured at 277 nm in simulated body fluid for up to 21 days. In a second part, an agar diffusion assay (F. nucleatum) with representative TVM concentrations as determined in the first part was performed to study the growth inhibition. Samples were anaerobical incubated for 48 h and inhibition zones were measured. Results: TVM was spectrophotometrically detectable in the immersion solution and released in decreasing concentrations up to 21 days (222.5 ± 65.2 mg/ml at day 1 and 35.1 ± 15.6 mg/ml at day 21). In addition, inhibition zones were shown in the agar diffusion assay at representative TVM concentrations. The carrier material showed no antibacterial effect. Conlusion: TVM showed the potential to penetrate through dentine and to inhibit bacterial growth. Therefore, it might have the potential to disinfect the outer root surface in perio-endo lesions, but further research is needed to confirm these observations. PMID:26966464

  3. Alteration of Neutrophil Reactive Oxygen Species Production by Extracts of Devil's Claw (Harpagophytum)

    PubMed Central

    Muzila, Mbaki; Wright, Helen; Roberts, Helen; Grant, Melissa; Nybom, Hilde; Sehic, Jasna; Ekholm, Anders

    2016-01-01

    Harpagophytum, Devil's Claw, is a genus of tuberiferous xerophytic plants native to southern Africa. Some of the taxa are appreciated for their medicinal effects and have been traditionally used to relieve symptoms of inflammation. The objectives of this pilot study were to investigate the antioxidant capacity and the content of total phenols, verbascoside, isoverbascoside, and selected iridoids, as well as to investigate the capacity of various Harpagophytum taxa in suppressing respiratory burst in terms of reactive oxygen species produced by human neutrophils challenged with phorbol myristate acetate (PMA), opsonised Staphylococcus aureus, and Fusobacterium nucleatum. Harpagophytum plants were classified into different taxa according to morphology, and DNA analysis was used to confirm the classification. A putative new variety of H. procumbens showed the highest degree of antioxidative capacity. Using PMA, three Harpagophytum taxa showed anti-inflammatory effects with regard to the PBS control. A putative hybrid between H. procumbens and H. zeyheri in contrast showed proinflammatory effect on the response of neutrophils to F. nucleatum in comparison with treatment with vehicle control. Harpagophytum taxa were biochemically very variable and the response in suppressing respiratory burst differed. Further studies with larger number of subjects are needed to corroborate anti-inflammatory effects of different taxa of Harpagophytum. PMID:27429708

  4. The antibiofilm activity of lingonberry flavonoids against oral pathogens is a case connected to residual complexity.

    PubMed

    Riihinen, Kaisu R; Ou, Zhen M; Gödecke, Tanja; Lankin, David C; Pauli, Guido F; Wu, Christine D

    2014-09-01

    The antimicrobial activity of lingonberry (Vaccinium vitis-idaea L.) was evaluated against two oral pathogens, Streptococcus mutans and Fusobacterium nucleatum. Long-bed gel permeation chromatography (GPC; Sephadex LH-20) yielded purified flavonoids, with the most efficient minimum inhibitory concentrations (MICs) against planktonic cells in the anthocyanin and procyanidin primary fractions against F. nucleatum (63-125 μg/ml) and in the procyanidin rich fraction against S. mutans (16-31 μg/ml). The purified flavonol glycosides and procyanidins inhibited biofilm formation of S. mutans (MICs 16-31 μg/ml), while the corresponding reference compounds showed no activity. Secondary GPC purification yielded flavonol glycosides devoid of antibiofilm activity in the 50% MeOH fraction, while elution with 70% acetone recovered a brownish material with activity against S. mutans biofilm (MIC 8 μg/ml). Even after HPLC-PDA, NMR, and MALDI-TOF analyses, the structural identity of this material remained unknown, while its color and analytical characteristics appear to be consistent with flavonoid oxidation products.

  5. Peripheral blood neutrophil cytokine hyper-reactivity in chronic periodontitis.

    PubMed

    Ling, Martin R; Chapple, Iain L C; Matthews, John B

    2015-10-01

    Pro-inflammatory cytokine release (IL-8, IL-6, TNF-α, IL-1β) by peripheral blood neutrophils, isolated from periodontitis patients (before/after therapy) and matched controls, was determined after 18 h culture in the presence/absence of Escherichia coli LPS, opsonised Staphylococcus aureus, heat-killed Fusobacterium nucleatum and Porphyromonas gingivalis. All cultures demonstrated differences in the amounts of each cytokine detected (P < 0.0001), with a clear release pattern (IL-8 > IL-6 > TNF-α = IL-1β). Median cytokine release from unstimulated patient neutrophils was consistently, but non-significantly, higher than from control cells. Stimulated cytokine release from untreated patient neutrophils was also consistently higher than from control cells. This hyper-reactivity was significant for all tested cytokines when data for all stimuli were combined (P < 0.016). In terms of individual stimuli, significant hyper-reactivity was detected with LPS (IL-8), F. nucleatum (IL-8, TNF-α), opsonised S. aureus (IL-8, TNF-α, IL-1β) and P. gingivalis (IL-8, IL-1β). Cytokine production by patient neutrophils did not reduce following successful non-surgical periodontal therapy and, except for responses to F. nucleatum, the cytokine hyper-reactivity detected pre-therapy was retained. These data demonstrate that chronic periodontitis is characterised by neutrophils that constitutively exhibit cytokine hyper-reactivity, the effects of which could modulate local and systemic inflammatory-immune responses and influence the risk and severity of periodontitis-associated systemic inflammatory diseases.

  6. Hierarchical micro/nanostructured titanium with balanced actions to bacterial and mammalian cells for dental implants

    PubMed Central

    Zhu, Yu; Cao, Huiliang; Qiao, Shichong; Wang, Manle; Gu, Yingxin; Luo, Huiwen; Meng, Fanhao; Liu, Xuanyong; Lai, Hongchang

    2015-01-01

    A versatile strategy to endow dental implants with long-term antibacterial ability without compromising the cytocompatibility is highly desirable to combat implant-related infection. Silver nanoparticles (Ag NPs) have been utilized as a highly effective and broad-spectrum antibacterial agent for surface modification of biomedical devices. However, the high mobility and subsequent hazardous effects of the particles on mammalian cells may limit its practical applications. Thus, Ag NPs were immobilized on the surface of sand-blasted, large grit, and acid-etched (SLA) titanium by manipulating the atomic-scale heating effect of silver plasma immersion ion implantation. The silver plasma immersion ion implantation-treated SLA surface gave rise to both good antibacterial activity and excellent compatibility with mammalian cells. The antibacterial activity rendered by the immobilized Ag NPs was assessed using Fusobacterium nucleatum and Staphylococcus aureus, commonly suspected pathogens for peri-implant disease. The immobilized Ag NPs offered a good defense against multiple cycles of bacteria attack in both F. nucleatum and S. aureus, and the mechanism was independent of silver release. F. nucleatum showed a higher susceptibility to Ag NPs than S. aureus, which might be explained by the presence of different wall structures. Moreover, the immobilized Ag NPs had no apparent toxic influence on the viability, proliferation, and differentiation of rat bone marrow mesenchymal stem cells. These results demonstrated that good bactericidal activity could be obtained with very small quantities of immobilized Ag NPs, which were not detrimental to the mammalian cells involved in the osseointegration process, and promising for titanium-based dental implants with commercial SLA surfaces. PMID:26604743

  7. Influence of glycemic control on the levels of subgingival periodontal pathogens in patients with generalized chronic periodontitis and type 2 diabetes

    PubMed Central

    MIRANDA, Tamires Szeremeske; FERES, Magda; RETAMAL-VALDÉS, Belén; PEREZ-CHAPARRO, Paula Juliana; MACIEL, Suellen Silva; DUARTE, Poliana Mendes

    2017-01-01

    Abstract Objective This study evaluated the influence of glycemic control on the levels and frequency of subgingival periodontal pathogens in patients with type 2 diabetes mellitus (DM) and generalized chronic periodontitis (ChP). Material and Methods Fifty-six patients with generalized ChP and type 2 DM were assigned according to the levels of glycated hemoglobin (HbA1c) into one of the following groups: HbA1c<8% (n=28) or HbA1c≥8% (n=28). Three subgingival biofilm samples from sites with probing depth (PD)<5 mm and three samples from sites with PD≥5 mm were analyzed by quantitative Polymerase Chain Reaction (PCR) for the presence and levels of Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Eubacterium nodatum, Parvimona micra, Fusobacterium nucleatum ssp. and Prevotella intermedia. Results The mean counts of F. nucleatum ssp. were statistically significantly higher in the sites with PD≥5 mm of the HbA1c≥8% group (p<0.05). Frequencies of detection of T. forsythia, E. nodatum, P. micra and F. nucleatum ssp. were all higher in the sites with PD≥5 mm of the patients with HbA1c≥8%, compared with those of patients with HbA1c<8% (p<0.05). Frequency of detection of P. intermedia was higher in the sites with PD<5 mm of the patients with HbA1c≥8% than those of the patients with HbA1c<8% (p<0.05). Conclusions Poor glycemic control, as indicated by HbA1c≥8%, is associated with increased levels and frequencies of periodontal pathogens in the subgingival biofilm of subjects with type 2 DM and ChP. PMID:28198980

  8. Recovery of periodontopathogenic bacteria from embalmed human cadavers.

    PubMed

    Wood, Nelson; Johnson, Roger B

    2005-01-01

    There is recent interest in recovery of periodontopathogenic bacteria from arterial and bronchial tissues to identify a link between periodontal and cardiovascular or pulmonary diseases. This interest could provide a useful clinical correlation exercise for gross anatomy. Our objective was to perform a feasibility study to determine whether these bacteria could be recovered from two sites within eight (4 dentate, 4 edentulous) human embalmed cadavers from an anatomical dissection laboratory. Bacterial samples were collected from the right coronary artery and the right superior secondary bronchus and assayed for the presence and concentrations of the DNA of A. actinomycetemcomitans, E. corrodens, C. rectus, P. intermedia, P. gingivalis, B. forsythus, T. denticola, and F. nucleatum. Frequencies were compared using a Kruskal-Wallis H-test. Correlations between the presence of teeth, bacterial species, and site were determined by a Spearman's rho correlation test. A. actinomycetemcomitans and B. forsythus frequencies were different between the sites in edentulous subjects (P <0.05); the frequency of B. forsythus was different in dentate and edentulous subjects at the bronchus site (P <0.05). Numerous significant correlations were identified between strains of bacteria, site, and presence of teeth. Thus, it is possible for the DNA of periodontopathogenic bacteria to be recovered from human embalmed cadavers. Collection and identification of these bacteria from these cadavers could be a useful clinical correlation exercise for dental students in a gross anatomy class.

  9. Comprehensive Analysis of Bacterial Flora in Postoperative Maxillary Cyst Fluid by 16S rRNA Gene and Culture Methods

    PubMed Central

    Sano, Naoto; Yamashita, Yoshio; Fukuda, Kazumasa; Taniguchi, Hatsumi; Goto, Masaaki; Miyamoto, Hiroshi

    2012-01-01

    Intracystic fluid was aseptically collected from 11 patients with postoperative maxillary cyst (POMC), and DNA was extracted from the POMC fluid. Bacterial species were identified by sequencing after cloning of approximately 580 bp of the 16S rRNA gene. Identification of pathogenic bacteria was also performed by culture methods. The phylogenetic identity was determined by sequencing 517–596 bp in each of the 1139 16S rRNA gene clones. A total of 1114 clones were classified while the remaining 25 clones were unclassified. A total of 103 bacterial species belonging to 42 genera were identified in POMC fluid samples by 16S rRNA gene analysis. Species of Prevotella (91%), Neisseria (73%), Fusobacterium (73%), Porphyromonas (73%), and Propionibacterium (73%) were found to be highly prevalent in all patients. Streptococcus mitis (64%), Fusobacterium nucleatum (55%), Propionibacterium acnes (55%), Staphylococcus capitis (55%), and Streptococcus salivarius (55%) were detected in more than 6 of the 11 patients. The results obtained by the culture method were different from those obtained by 16S rRNA gene analysis, but both approaches may be necessary for the identification of pathogens, especially of bacteria that are difficult to detect by culture methods, and the development of rational treatments for patients with POMC. PMID:22685668

  10. Microbiological profile and antimicrobial susceptibility pattern of infected root canals associated with periapical abscesses.

    PubMed

    Sousa, E L R; Gomes, B P F A; Jacinto, R C; Zaia, A A; Ferraz, C C R

    2013-04-01

    The aim of this investigation was to identify microorganisms from root canals with periapical abscesses and assess the susceptibility of specific anaerobic bacteria to selected antimicrobials and their β-lactamase production. Sixty root canals were microbiologically investigated. The susceptibility of Anaerococcus prevotii, Fusobacterium necrophorum, F. nucleatum, Parvimonas micra, and Prevotella intermedia/nigrescens to antimicrobials was evaluated with the Etest, whereas β-lactamase production was assessed with nitrocefin. A total of 287 different bacterial strains were recovered, including 201 strict anaerobes. The most frequently strict isolated anaerobes were A. prevotii, P. micra, and F. necrophorum. The selected bacteria were susceptible to all the tested antibiotics, except A. prevotii and Fusobacterium species to azithromycin and erythromycin, as well as A. prevotii and F. necrophorum to metronidazole. None of the microorganisms produced β-lactamase. Gram-positive anaerobic bacteria predominated in the root canals with periapical abscesses. All microorganisms tested were susceptible to benzylpenicillin, amoxicillin, amoxicillin + clavulanate, cefaclor, and clindamycin, producing no β-lactamase.

  11. Oral Gram-negative anaerobic bacilli as a reservoir of β-lactam resistance genes facilitating infections with multiresistant bacteria.

    PubMed

    Dupin, Clarisse; Tamanai-Shacoori, Zohreh; Ehrmann, Elodie; Dupont, Anais; Barloy-Hubler, Frédérique; Bousarghin, Latifa; Bonnaure-Mallet, Martine; Jolivet-Gougeon, Anne

    2015-02-01

    Many β-lactamases have been described in various Gram-negative bacilli (Capnocytophaga, Prevotella, Fusobacterium, etc.) of the oral cavity, belonging to class A of the Ambler classification (CepA, CblA, CfxA, CSP-1 and TEM), class B (CfiA) or class D in Fusobacterium nucleatum (FUS-1). The minimum inhibitory concentrations of β-lactams are variable and this variation is often related to the presence of plasmids or other mobile genetic elements (MGEs) that modulate the expression of resistance genes. DNA persistence and bacterial promiscuity in oral biofilms also contribute to genetic transformation and conjugation in this particular microcosm. Overexpression of efflux pumps is facilitated because the encoding genes are located on MGEs, in some multidrug-resistant clinical isolates, similar to conjugative transposons harbouring genes encoding β-lactamases. All these facts lead us to consider the oral cavity as an important reservoir of β-lactam resistance genes and a privileged place for genetic exchange, especially in commensal strictly anaerobic Gram-negative bacilli.

  12. Dental plaque microbial profiles of children from Khartoum, Sudan, with congenital heart defects

    PubMed Central

    Mohamed Ali, Hiba; Berggreen, Ellen; Nguyen, Daniel; Wahab Ali, Raouf; Van Dyke, Thomas E.; Hasturk, Hatice; Mustafa, Manal

    2017-01-01

    ABSTRACT Few studies have focused on the bacterial species associated with the deterioration of the dental and gingival health of children with congenital heart defects (CHD). The aims of this study were (1) to examine the dental plaque of children with CHD in order to quantify bacterial load and altered bacterial composition compared with children without CHD; and (2) to investigate the correlation between the level of caries and gingivitis and dental biofilm bacteria among those children. In this cross-sectional study, participants were children (3–12 years) recruited in Khartoum State, Sudan. A total of 80 CHD cases from the Ahmed Gasim Cardiac Centre and 80 healthy controls from randomly selected schools and kindergartens were included. Participants underwent clinical oral examinations for caries (decayed, missing, and filled teeth indices [DMFT] for primary dentition, and DMFT for permanent dentition), and gingivitis (simplified gingival index [GI]). Pooled dental biofilm samples were obtained from four posterior teeth using paper points. Real-time quantitative polymerase chain reaction was used for the detection and quantification of Streptococcus mutans, Streptococcussanguinis, and Lactobacillus acidophilus. Checkerboard DNA–DNA hybridization was used for the detection of 40 additional bacterial species. CHD cases had a significantly higher caries experience (DMFT = 4.1 vs. 2.3, p < 0.05; DMFT = 1.4 vs. 0.7, p < 0.05) and a higher mean number of examined teeth with gingivitis (4.2 vs. 2.0; p < 0.05) compared with controls. S. mutans counts were significantly higher among the CHD cases (p < 0.05). Checkerboard results revealed that 18/40 bacterial species exhibited significantly higher mean counts among CHD cases (p < 0.01). Correlation analyses revealed that among CHD cases, the detection levels of Tannerella forsythia, Campylobacter rectus, Fusobacterium nucleatum subsp. vincentii, F. nucleatum subsp. nucleatum, and F. nucleatum subsp

  13. The Anticomplementary Activity of ’Fusobacterium polymorphum’ in Normal and C-4 Deficient Sources of Guinea Pig Complement.

    DTIC Science & Technology

    1977-01-12

    alternative activation pathway appears possible which involves the reaction of certain polymeric m i- tiators (inulin, endotoxin, or zyinosan) with non ...displayed the ability to consume C’3, and anti-C’2 did not interfere with this activity. These findings suggested that there was an alternate ( non ...included using 5 g dextrose per liter veronal buffered diluent (VBD) . VBD prepared with the stock buffer containing Mg~~ and Ca++ was designated VBD

  14. Oral microbiota species in acute apical endodontic abscesses

    PubMed Central

    George, Noelle; Flamiatos, Erin; Kawasaki, Kellie; Kim, Namgu; Carriere, Charles; Phan, Brian; Joseph, Raphael; Strauss, Shay; Kohli, Richie; Choi, Dongseok; Craig Baumgartner, J.; Sedgley, Christine; Maier, Tom; Machida, Curtis A.

    2016-01-01

    Background and objectives Acute apical abscesses are serious endodontic diseases resulting from pulpal infection with opportunistic oral microorganisms. The objective of this study was to identify and compare the oral microbiota in patients (N=18) exhibiting acute apical abscesses, originating from the demographic region in Portland, Oregon. The study hypothesis is that abscesses obtained from this demographic region may contain unique microorganisms not identified in specimens from other regions. Design Endodontic abscesses were sampled from patients at the Oregon Health & Science University (OHSU) School of Dentistry. DNA from abscess specimens was subjected to polymerase chain reaction amplification using 16S rRNA gene-specific primers and Cy3-dCTP labeling. Labeled DNA was then applied to microbial microarrays (280 species) generated by the Human Oral Microbial Identification Microarray Laboratory (Forsyth Institute, Cambridge, MA). Results The most prevalent microorganisms, found across multiple abscess specimens, include Fusobacterium nucleatum, Parvimonas micra, Megasphaera species clone CS025, Prevotella multisaccharivorax, Atopobium rimae, and Porphyromonas endodontalis. The most abundant microorganisms, found in highest numbers within individual abscesses, include F. nucleatum, P. micra, Streptococcus Cluster III, Solobacterium moorei, Streptococcus constellatus, and Porphyromonas endodontalis. Strong bacterial associations were identified between Prevotella multisaccharivorax, Acidaminococcaceae species clone DM071, Megasphaera species clone CS025, Actinomyces species clone EP053, and Streptococcus cristatus (all with Spearman coefficients >0.9). Conclusions Cultivable and uncultivable bacterial species have been identified in endodontic abscesses obtained from the Portland, Oregon demographic region, and taxa identifications correlated well with other published studies, with the exception of Treponema and Streptococcus cristae, which were not commonly

  15. Effect of gallium-arsenic laser on photosensitized periodontopathic anaerobic organisms: An in vitro study

    PubMed Central

    Mathur, Setu; Kothiwale, Shaila Veerappa; Nag, Buddhi Prakash; Mathur, Tanu; Bhansali, Ashoka; Khatri, Rohit Kumar

    2016-01-01

    Background: The mainstay of periodontal therapy is mechanical removal of subgingival plaque. There is considerable interest in supplementing it with the use of antibiotics and antiseptics. Many drawbacks are associated with these adjunctive pharmacological regimens such as development of resistance to antibiotics and disruption of microflora of the gastrointestinal tract. Hence, alternate means of killing subgingival bacteria are clearly desirable. One such method is the use of laser. Aim: This study aimed to investigate antibacterial capabilities of gallium-arsenic (Ga-As) laser on photosensitized periodontopathic organisms. The three bacteria selected for the study were Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella intermedia. Settings: The subjects for the study were selected from the patients visiting the Department of Periodontics, Karnataka Lingayat Education Society's Institute of Dental Sciences, Belgaum. Design: In vitro study design. Materials and Methods: Subgingival plaque samples collected from chronic periodontitis patients were cultured anaerobically for 72 h. Predetermined number of colonies of each bacterium was taken and was then divided into cases and control groups. Both groups were photosensitized using toluidine blue O (TBO) dye and the case groups were irradiated with Ga-As laser. Bacterial colonies were then serially diluted and were incubated for subculture. After incubation period, the number of viable bacterial count was performed. Statistical Analysis: Wilcoxon-signed rank test was carried out to determine significance of reduction on subsequent dilution within the bacterial group. Mann–Whitney U-test was performed to determine the significance of reduction between cases and control of particular bacterial group. Results: The results revealed substantial reduction in the viable bacterial count. F. nucleatum was found to be most sensitive to killing by laser irradiation followed by P. intermedia and then P

  16. Sialic acid transporter NanT participates in Tannerella forsythia biofilm formation and survival on epithelial cells.

    PubMed

    Honma, Kiyonobu; Ruscitto, Angela; Frey, Andrew M; Stafford, Graham P; Sharma, Ashu

    2016-05-01

    Tannerella forsythia is a periodontal pathogen implicated in periodontitis. This gram-negative pathogen depends on exogenous peptidoglycan amino sugar N-acetylmuramic acid (NAM) for growth. In the biofilm state the bacterium can utilize sialic acid (Neu5Ac) instead of NAM to sustain its growth. Thus, the sialic acid utilization system of the bacterium plays a critical role in the growth and survival of the organism in the absence of NAM. We sought the function of a T. forsythia gene annotated as nanT coding for an inner-membrane sugar transporter located on a sialic acid utilization genetic cluster. To determine the function of this putative sialic acid transporter, an isogenic nanT-deletion mutant generated by allelic replacement strategy was evaluated for biofilm formation on NAM or Neu5Ac, and survival on KB epithelial cells. Moreover, since T. forsythia forms synergistic biofilms with Fusobacterium nucleatum, co-biofilm formation activity in mixed culture and sialic acid uptake in culture were also assessed. The data showed that the nanT-inactivated mutant of T. forsythia was attenuated in its ability to uptake sialic acid. The mutant formed weaker biofilms compared to the wild-type strain in the presence of sialic acid and as co-biofilms with F. nucleatum. Moreover, compared to the wild-type T. forsythia nanT-inactivated mutant showed reduced survival when incubated on KB epithelial cells. Taken together, the data presented here demonstrate that NanT-mediated sialic transportation is essential for sialic acid utilization during biofilm growth and survival of the organism on epithelial cells and implies sialic acid might be key for its survival both in subgingival biofilms and during infection of human epithelial cells in vivo.

  17. Transcriptional Responses of Treponema denticola to Other Oral Bacterial Species

    PubMed Central

    Simanian, Emil J.; Shi, Wenyuan; Lux, Renate

    2014-01-01

    The classic organization by Socransky and coworkers categorized the oral bacteria of the subgingival plaque into different complexes. Treponema denticola, Porphyromonas gingivalis and Tannerella forsythia are grouped into the red complex that is highly correlated with periodontal disease. Socransky's work closely associates red with orange complex species such as Fusobacterium nucleatum and Prevotella intermedia but not with members of the other complexes. While the relationship between species contained by these complexes is in part supported by their ability to physically attach to each other, the physiological consequences of these interactions and associations are less clear. In this study, we employed T. denticola as a model organism to analyze contact-dependent responses to interactions with species belonging to the same complex (P. gingivalis and T. forsythia), the closely associated orange complex (using F. nucleatum and P. intermedia as representatives) and the unconnected yellow complex (using Streptococcus sanguinis and S. gordonii as representatives). RNA was extracted from T. denticola alone as well as after pairwise co-incubation for 5 hrs with representatives of the different complexes, and the respective gene expression profiles were determined using microarrays. Numerous genes related to motility, metabolism, transport, outer membrane and hypothetical proteins were differentially regulated in T. denticola in the presence of the tested partner species. Further analysis revealed a significant overlap in the affected genes and we identified a general response to the presence of other species, those specific to two of the three complexes as well as individual complexes. Most interestingly, many predicted major antigens (e.g. flagella, Msp, CTLP) were suppressed in responses that included red complex species indicating that the presence of the most closely associated species induces immune-evasive strategies. In summary, the data presented here provide

  18. Periodontal Pathogens and Risk of Incident Cancer in Postmenopausal Females: The Buffalo OsteoPerio Study

    PubMed Central

    Mai, Xiaodan; Genco, Robert J.; LaMonte, Michael J.; Hovey, Kathleen M.; Freudenheim, Jo L.; Andrews, Christopher A.; Wactawski-Wende, Jean

    2016-01-01

    Background Extraoral translocation of oral bacteria may contribute to associations between periodontal disease and cancer. The associations among the presence of three orange-complex periodontal pathogens (Fusobacterium nucleatum, Prevotella intermedia, and Campylobacter rectus), two red-complex periodontal pathogens (Porphyromonas gingivalis and Tannerella forsythia), and cancer risk were investigated. Methods A total of 1,252 postmenopausal females enrolled in the Buffalo Osteoporosis and Periodontal Disease Study were followed prospectively. Baseline subgingival plaque samples were assessed for the presence of periodontal pathogens using indirect immunofluorescence. Incident cancer cases were adjudicated by staff physicians via review of medical records. Cox proportional hazards regression was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for the associations of periodontal pathogens with total cancer and site-specific cancer risk in unadjusted and multivariable-adjusted models. Results Neither the presence of individual pathogens nor the presence of any red-complex pathogens was associated with total cancer or site-specific cancers. Borderline associations were seen among the presence of any orange-complex pathogens (F. nucleatum, P. intermedia, and C. rectus), total cancer risk (HR = 1.35, 95% CI = 1.00 to 1.84), and lung cancer risk (HR = 3.02, 95% CI = 0.98 to 9.29). Conclusions No associations were found between the presence of individual subgingival pathogens and cancer risk. However, there were suggestions of borderline positive associations of the presence of any orange-complex pathogens with total cancer and lung cancer risk. The study is limited by the small number of cancer cases and the assessment of only five oral bacteria. Additional research is needed to understand the possi ble role of periodontal disease in carcinogenesis. PMID:26513268

  19. In vitro growth-inhibitory effect of ethanol GRAS plant and supercritical CO₂ hop extracts on planktonic cultures of oral pathogenic microorganisms.

    PubMed

    Pilna, J; Vlkova, E; Krofta, K; Nesvadba, V; Rada, V; Kokoska, L

    2015-09-01

    Conventional chemical antiseptics used for treatment of oral infections often produce side-effects, which restrict their long-term use. Plants are considered as perspective sources of novel natural antiseptics. However, little is still known about their inhibitory properties against oral pathogens. The objective of this study was to test in vitro antimicrobial activities of generally recognized as safe (GRAS) species against planktonic cultures of cariogenic, periodontal and candidal microorganisms and identify active compounds of the most active extracts. Growth-inhibitory effects of ethanol extracts from 109 GRAS plant species, six Humulus lupulus cultivars and two hop supercritical CO2 extracts were evaluated using broth microdilution method. The chemical analysis was done through high-performance liquid chromatography. Best results were obtained for supercritical CO2 and ethanol extracts of H. lupulus with minimum inhibitory concentrations (MIC) ≥8 μg/mL and ≥16 μg/mL, respectively. The chemical analysis of supercritical CO2H. lupulus extracts revealed that α- and β-acids were their main constituents. Capsicum annuum and Capsicum frutescens showed antibacterial effect against Streptococcus sobrinus and Streptococcus salivarius (MIC=64-128 μg/mL). These strains were further inhibited by Zanthoxylum clava-herculis (MIC=64-128 μg/mL) and Myristica fragrans (both MIC≥128 μg/mL). The latter also exhibited antimicrobial activity against Fusobacterium nucleatum (MIC=64 μg/mL). Punica granatum possessed inhibitory effects against Candida albicans (MIC=128 μg/mL) and F. nucleatum (MIC=64 μg/mL). The results indicate that supercritical CO2H. lupulus extracts together with ethanol extracts of C. annuum, C. frutescens, M. fragrans, P. granatum and Z. clava-herculis are promising materials for further investigation on new antiseptic agents of oral care products.

  20. In vitro efficacy of cefovecin against anaerobic bacteria isolated from subgingival plaque of dogs and cats with periodontal disease.

    PubMed

    Khazandi, Manouchehr; Bird, Philip S; Owens, Jane; Wilson, Gary; Meyer, James N; Trott, Darren J

    2014-08-01

    Periodontal disease is a common disease of dogs and cats often requiring antimicrobial treatment as an adjunct to mechanical debridement. However, correct compliance with oral antimicrobial therapy in companion animals is often difficult. Cefovecin is a recently introduced veterinary cephalosporin that has demonstrated prolonged concentrations in extracellular fluid, allowing for dosing intervals of up to 14 days. Subgingival samples were collected from the oral cavity of 29 dogs and eight cats exhibiting grade 2 or grade 3 periodontal disease. Samples were cultivated on Wilkin Chalgrens agar and incubated in an anaerobic chamber for seven days. Selected anaerobic bacteria were isolated and identified to species level using 16S rRNA gene sequence analysis. Minimum inhibitory concentrations were determined for cefovecin and six additional antimicrobials using the agar dilution methodology recommended by the Clinical and Laboratory Standards Institute. The 65 clinical isolates were identified as Porphyromonas gulae (n = 45), Porphyromonas crevioricanis (n = 12), Porphyromonas macacae (n = 1), Porphyromonas cangingivalis (n = 1) Fusobacterium nucleatum (n = 2), Fusobacterium russii (n = 1) and Solobacterium moorei (n = 3). This is the first report of S. moorei being isolated from companion animals with periodontal disease. All isolates were highly susceptible to cefovecin, with a MIC90 of ≤0.125 μg/ml. Conversely, different resistance rates to ampicillin, amoxicillin and erythromycin between isolates were detected. Cefovecin is thus shown to be effective in vitro against anaerobic bacteria isolated from dogs and cats with periodontal disease.

  1. Preventive Effects of Houttuynia cordata Extract for Oral Infectious Diseases.

    PubMed

    Sekita, Yasuko; Murakami, Keiji; Yumoto, Hiromichi; Amoh, Takashi; Fujiwara, Natsumi; Ogata, Shohei; Matsuo, Takashi; Miyake, Yoichiro; Kashiwada, Yoshiki

    2016-01-01

    Houttuynia cordata (HC) (Saururaceae) has been used internally and externally as a traditional medicine and as an herbal tea for healthcare in Japan. Our recent survey showed that HC poultice (HCP) prepared from smothering fresh leaves of HC had been frequently used for the treatment of purulent skin diseases with high effectiveness. Our experimental study also demonstrated that ethanol extract of HCP (eHCP) has antibacterial, antibiofilm, and anti-inflammatory effects against S. aureus which caused purulent skin diseases. In this study, we focused on novel effects of HCP against oral infectious diseases, such as periodontal disease and dental caries. We determined the antimicrobial and antibiofilm effects of water solution of HCP ethanol extract (wHCP) against important oral pathogens and investigated its cytotoxicity and anti-inflammatory effects on human oral epithelial cells. wHCP had moderate antimicrobial effects against some oral microorganisms and profound antibiofilm effects against Fusobacterium nucleatum, Streptococcus mutans, and Candida albicans. In addition, wHCP had no cytotoxic effects and could inhibit interleukin-8 and CCL20 productions by Porphyromonas gingivalis lipopolysaccharide-stimulated human oral keratinocytes. Our findings suggested that wHCP may be clinically useful for preventing oral infectious diseases as a mouthwash for oral care.

  2. Coenzyme Recognition and Gene Regulation by a Flavin Mononucleotide Riboswitch

    SciTech Connect

    Serganov, A.; Huang, L; Patel, D

    2009-01-01

    The biosynthesis of several protein cofactors is subject to feedback regulation by riboswitches. Flavin mononucleotide (FMN)-specific riboswitches also known as RFN elements, direct expression of bacterial genes involved in the biosynthesis and transport of riboflavin (vitamin B2) and related compounds. Here we present the crystal structures of the Fusobacterium nucleatum riboswitch bound to FMN, riboflavin and antibiotic roseoflavin. The FMN riboswitch structure, centred on an FMN-bound six-stem junction, does not fold by collinear stacking of adjacent helices, typical for folding of large RNAs. Rather, it adopts a butterfly-like scaffold, stapled together by opposingly directed but nearly identically folded peripheral domains. FMN is positioned asymmetrically within the junctional site and is specifically bound to RNA through interactions with the isoalloxazine ring chromophore and direct and Mg{sup 2+}-mediated contacts with the phosphate moiety. Our structural data, complemented by binding and footprinting experiments, imply a largely pre-folded tertiary RNA architecture and FMN recognition mediated by conformational transitions within the junctional binding pocket. The inherent plasticity of the FMN-binding pocket and the availability of large openings make the riboswitch an attractive target for structure-based design of FMN-like antimicrobial compounds. Our studies also explain the effects of spontaneous and antibiotic-induced deregulatory mutations and provided molecular insights into FMN-based control of gene expression in normal and riboflavin-overproducing bacterial strains.

  3. In Vitro Evaluation of Planktonic Growth on Experimental Cement-Retained Titanium Surfaces

    PubMed Central

    Balci, Nur; Cakan, Umut; Aksu, Burak; Akgul, Oncu; Ulger, Nurver

    2016-01-01

    Background The purpose of this study was to compare the effects of selected cements, or their combination with titanium, on the growth of two periodontopathic bacteria: Prevotella intermedia (Pi) and Fusobacterium nucleatum (Fn). Material/Methods This study was comprised of several experimental groups: 1) Dental luting cements (glass ionomer cement, methacrylate-based resin cement, zinc-oxide eugenol cement, eugenol-free zinc oxide cement; 2) titanium discs; and 3) titanium combination cement discs. The disks were submerged in bacterial suspensions of either Fn or Pi. Planktonic bacterial growth within the test media was measured by determining the optical density of the cultures (OD600). Mean and standard deviations were calculated for planktonic growth from three separate experiments. Results Intergroup comparison of all experimental groups revealed increased growth of Pi associated with cement-titanium specimens in comparison with cement specimens. Regarding the comparison of all groups for Fn, there was an increased amount of bacterial growth in cement-titanium specimens although the increase was not statistically significant. Conclusions The combination of cement with titanium may exacerbate the bacterial growth capacity of Pi and Fn in contrast to their sole effect. PMID:27058704

  4. Role of oral microbiome on oral cancers, a review.

    PubMed

    Gholizadeh, Pourya; Eslami, Hosein; Yousefi, Mehdi; Asgharzadeh, Mohammad; Aghazadeh, Mohammad; Kafil, Hossein Samadi

    2016-12-01

    The oral cavity is inhibited by many of the bacterial species. Some of them have a key role in the development of oral disease. Interrelationships between oral microbiome and systemic conditions such as head-and-neck cancer have become increasingly appreciated in recent years. Emerging evidence also suggests a link between periodontal disease and oral cancer, and the explanation being that chronic inflammation could be a major factor in both diseases. Squamous cell carcinoma is that the most frequently occurring malignancy of the oral cavity and adjacent sites, representing over 90% of all cancers. The incidence of oral cancer is increasing, significantly among young people and women. Worldwide there are 350,000-400,000 new cases diagnosed every year. Bacteria, viruses, and fungi are strongly implicated as etiological factors in certain cancers. In this review we will discuss the association between the development of oral cancer in potentially malignant oral lesions with chronic periodontitis, chronic Porphyromonas gingivalis, Fusobacterium nucleatum, candida, other microbes and described mechanisms which may be involved in these carcinoma.

  5. Variations in the Oral Anaerobic Microbial Flora in Relation to Pregnancy

    PubMed Central

    Basavaraju, Anuradha; Durga S., Vijaya; Vanitha, B.

    2012-01-01

    Introduction Pregnancy gingivitis is a major oral infection. Periodontium acts as a reservoir of inflammatory mediators and sub gingival biofilms of bacteria. Aim: To evaluate the anaerobic oral microbial flora in pregnant women before delivery and after delivery by comparing them with control group. Material and Methods: The study group included fifteen cases of pregnant women before and after delivery and healthy non-pregnant women of same age as control group. Sub gingival plaque samples were collected with the help of dentists. The samples were inoculated immediately into Thioglycollate broth (MV010), transported to the laboratory, inoculated on to selective media for anaerobes (Hi-media laboratories) incubated anaerobically (Gas pack). Results: Prevotella, Tanerella forsythia, Porphyromonas gingivalis and Fusobacterium nucleatum, Veillonella, Peptostreptococcus were isolated. Discussion: The anaerobic bacteria in pregnant women were Prevotella, Tanerella forsythia and Porphyromonas gingivalis. Viellonella and Peptostreptococcus were seen in control group and after delivery. Research suggests that periodontal pathogens may travel the blood stream from the oral cavity to the placenta. Conclusion: Pregnancy has significant effect on periodontal tissue. There is a significant alteration of bacterial flora during and after pregnancy. Oral health has to become a part of antenatal care /check up. PMID:23285437

  6. Molecular-level evaluation of selected periodontal pathogens from subgingival regions in canines and humans with periodontal disease

    PubMed Central

    Polkowska, Izabela; Bartoszcze-Tomaszewska, Małgorzata; Sobczyńska-Rak, Aleksandra; Matuszewski, Łukasz

    2017-01-01

    Dogs commonly serve as a model for various human conditions, including periodontal diseases. The aim of this study was to identify the anaerobic bacteria that colonize the subgingival areas in dogs and humans by using rapid real-time polymerase chain reaction (RT-PCR)-based tests and to compare the results obtained in each species. Bacterial microflora evaluations, both quantitative and qualitative, were performed by applying ready-made tests on twelve dogs and twelve humans. Five samples were collected from each subject's deepest gingival pockets and joined to form a collective sample. The results of the study revealed interspecies similarities in the prevalences of Porphyromonas (P.) gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum. Red complex bacteria comprised the largest portion of the studied bacterial complexes in all study groups, with P. gingivalis being the most commonly isolated bacterium. The results show similarities in the prevalence of bacterial microflora in dogs and humans. Microbiological analysis of gingival pockets by using rapid real-time PCR-based tests in clinical practice, both veterinary and human, can facilitate the choice of appropriate pharmacological treatment and can provide a basis for subsequent verification of the treatment's effectiveness. PMID:27297417

  7. Bacteriology of experimental gingivitis in young adult humans.

    PubMed Central

    Moore, W E; Holdeman, L V; Smibert, R M; Good, I J; Burmeister, J A; Palcanis, K G; Ranney, R R

    1982-01-01

    From replicate trials of experimental gingivitis in four periodontally healthy subjects, 166 bacterial species and subspecies were detected among 3,034 randomly selected isolates from 96 samples. Of these bacteria, Actinomyces naeslundii (serotype III and phenotypically similar strains that were unreactive with available antisera), Actinomyces odontolyticus (serotype I and phenotypically similar strains that were unreactive with available antisera), Fusobacterium nucleatum, Lactobacillus species D-2, Streptococcus anginosus, Veillonella parvula, and Treponema species A appeared to be the most likely etiological agents of gingivitis. Statistical interpretations indicated that the greatest source of microbiological variation of the total flora observed was person-to-person differences in the floras. The next greatest source of variation was the inflammatory status of the sample sites. Person-to-person differences were smallest at experimental day 4. The floras became more diverse with time and as gingivitis developed and progressed. Analyses indicated that sequential colonization by certain species was repeatable and therefore probably predictable. Variation was relatively small between replicate trials, between two sites on the same teeth sampled on the same day, and between the same sites sampled at the same relative time in a replicate trial. PMID:7141708

  8. Stress hormone epinephrine (adrenaline) and norepinephrine (noradrenaline) effects on the anaerobic bacteria.

    PubMed

    Boyanova, Lyudmila

    2017-01-08

    Microbial endocrinology is a relatively new research area that already encompasses the anaerobes. Stress hormones, epinephrine and norepinephrine, can affect the growth of anaerobic bacteria such as Fusobacterium nucleatum, Prevotella spp., Porhyromonas spp., Tanerella forsythia and Propionibacterium acnes and can increase virulence gene expression, iron acquisition and many virulence factors of some anaerobic species such as Clostridium perfringens, Porphyromonas gingivalis and Brachyspira pilosicoli. Epinephrine and norepinephrine effects can lead to a growth increase or decrease, or no effect on the growth of the anaerobes. The effects are species-specific and perhaps strain-specific. Discrepancies in the results of some studies can be due to the different methods and media used, catecholamine concentrations, measurement techniques and the low number of strains tested. Biological effects of the stress hormones on the anaerobes may range from halitosis and a worsening of periodontal diseases to tissue damages and atherosclerotic plaque ruptures. Optimizations of the research methods and a detailed assessment of the catecholamine effects in conditions mimicking those in affected organs and tissues, as well as the effects on the quorum sensing and virulence of the anaerobes and the full spectrum of biological consequences of the effects are interesting topics for further evaluation.

  9. Amphipathic polymers: tools to fold integral membrane proteins to their active form.

    PubMed

    Pocanschi, Cosmin L; Dahmane, Tassadite; Gohon, Yann; Rappaport, Fabrice; Apell, Hans-Jürgen; Kleinschmidt, Jörg H; Popot, Jean-Luc

    2006-11-28

    Among the major obstacles to pharmacological and structural studies of integral membrane proteins (MPs) are their natural scarcity and the difficulty in overproducing them in their native form. MPs can be overexpressed in the non-native state as inclusion bodies, but inducing them to achieve their functional three-dimensional structure has proven to be a major challenge. We describe here the use of an amphipathic polymer, amphipol A8-35, as a novel environment that allows both beta-barrel and alpha-helical MPs to fold to their native state, in the absence of detergents or lipids. Amphipols, which are extremely mild surfactants, appear to favor the formation of native intramolecular protein-protein interactions over intermolecular or protein-surfactant ones. The feasibility of the approach is demonstrated using as models OmpA and FomA, two outer membrane proteins from the eubacteria Escherichia coli and Fusobacterium nucleatum, respectively, and bacteriorhodopsin, a light-driven proton pump from the plasma membrane of the archaebacterium Halobacterium salinarium.

  10. Randomized in vivo evaluation of photodynamic antimicrobial chemotherapy on deciduous carious dentin

    NASA Astrophysics Data System (ADS)

    Steiner-Oliveira, Carolina; Longo, Priscila Larcher; Aranha, Ana Cecília Corrêa; Ramalho, Karen Müller; Mayer, Marcia Pinto Alves; de Paula Eduardo, Carlos

    2015-10-01

    The aim of this randomized in vivo study was to compare antimicrobial chemotherapies in primary carious dentin. Thirty-two participants ages 5 to 7 years underwent partial caries removal from deep carious dentin lesions in primary molars and were subsequently divided into three groups: control [chlorhexidine and resin-modified glass ionomer cement (RMGIC)], LEDTB [photodynamic antimicrobial chemotherapy (PACT) with light-emitting diode associated with toluidine blue solution and RMGIC], and LMB [PACT with laser associated with methylene blue solution and RMGIC]. The participants were submitted to initial clinical and radiographic examinations. Demographic features and biofilm, gingival, and DMFT/DMFS indexes were evaluated, in addition to clinical and radiographic followups at 6 and 12 months after treatments. Carious dentin was collected before and after each treatment, and the number of Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei, Fusobacterium nucleatum, Atopobium rimae, and total bacteria was established by quantitative polymerase chain reaction. No signs of pain or restoration failure were observed. All therapies were effective in reducing the number of microorganisms, except for S. sobrinus. No statistical differences were observed among the protocols used. All therapies may be considered as effective modern approaches to minimal intervention for the management of deep primary caries treatment.

  11. The OxyR homologue in Tannerella forsythia regulates expression of oxidative stress responses and biofilm formation.

    PubMed

    Honma, Kiyonobu; Mishima, Elina; Inagaki, Satoru; Sharma, Ashu

    2009-06-01

    Tannerella forsythia is an anaerobic periodontal pathogen that encounters constant oxidative stress in the human oral cavity due to exposure to air and reactive oxidative species from coexisting dental plaque bacteria as well as leukocytes. In this study, we sought to characterize a T. forsythia ORF with close similarity to bacterial oxidative stress response sensor protein OxyR. To analyse the role of this OxyR homologue, a gene deletion mutant was constructed and characterized. Aerotolerance, survival after hydrogen peroxide challenge and transcription levels of known bacterial antioxidant genes were then determined. Since an association between oxidative stress and biofilm formation has been observed in bacterial systems, we also investigated the role of the OxyR protein in biofilm development by T. forsythia. Our results showed that aerotolerance, sensitivity to peroxide challenge and the expression of oxidative stress response genes were significantly reduced in the mutant as compared with the wild-type strain. Moreover, the results of biofilm analyses showed that, as compared with the wild-type strain, the oxyR mutant showed significantly less autoaggregation and a reduced ability to form mixed biofilms with Fusobacterium nucleatum. In conclusion, a gene annotated in the T. forsythia genome as an oxyR homologue was characterized. Our studies showed that the oxyR homologue in T. forsythia constitutively activates antioxidant genes involved in resistance to peroxides as well as oxygen stress (aerotolerance). In addition, the oxyR deletion attenuates biofilm formation in T. forsythia.

  12. The surface layer of Tannerella forsythia contributes to serum resistance and oral bacterial coaggregation.

    PubMed

    Shimotahira, Naohiro; Oogai, Yuichi; Kawada-Matsuo, Miki; Yamada, Sakuo; Fukutsuji, Kenji; Nagano, Keiji; Yoshimura, Fuminobu; Noguchi, Kazuyuki; Komatsuzawa, Hitoshi

    2013-04-01

    Tannerella forsythia is an anaerobic, Gram-negative bacterium involved in the so-called "red complex," which is associated with severe and chronic periodontitis. The surface layer (S-layer) of T. forsythia is composed of cell surface glycoproteins, such as TfsA and TfsB, and is known to play a role in adhesion/invasion and suppression of proinflammatory cytokine expression. Here we investigated the association of this S-layer with serum resistance and coaggregation with other oral bacteria. The growth of the S-layer-deficient mutant in a bacterial medium containing more than 20% non-heat-inactivated calf serum (CS) or more than 40% non-heat-inactivated human serum was significantly suppressed relative to that of the wild type (WT). Next, we used confocal microscopy to perform quantitative analysis on the effect of serum. The survival ratio of the mutant exposed to 100% non-heat-inactivated CS (76% survival) was significantly lower than that of the WT (97% survival). Furthermore, significant C3b deposition was observed in the mutant but not in the WT. In a coaggregation assay, the mutant showed reduced coaggregation with Streptococcus sanguinis, Streptococcus salivarius, and Porphyromonas gingivalis but strong coaggregation with Fusobacterium nucleatum. These results indicated that the S-layer of T. forsythia plays multiple roles in virulence and may be associated with periodontitis.

  13. INFLAMMATORY INDEX AND TREATMENT OF BRAIN ABSCESS

    PubMed Central

    OYAMA, HIROFUMI; KITO, AKIRA; MAKI, HIDEKI; HATTORI, KENICHI; NODA, TOMOYUKI; WADA, KENTARO

    2012-01-01

    ABSTRACT This study retrospectively analyzed 12 patients with brain abscesses. Half of the patients were diagnosed inaccurately in the initial stage, and 7.2 days were required to achieve the final diagnosis of brain abscess. The patients presented only with a moderately elevated leukocyte count, serum CRP levels, or body temperatures during the initial stage. These markers changed, first with an increase in the leukocyte count, followed by the CRP and body temperature. The degree of elevation tended to be less prominent, and the time for each inflammatory index to reach its maximum value tended to be longer in the patients without ventriculitis than in those with it. The causative organisms of a brain abscess were detected in 10 cases. The primary causative organisms from dental caries were Streptococcus viridians or milleri, and Fusobacterium nucleatum. Nocardia sp. or farcinica were common when the abscess was found in other regions. The primary causative organisms of unrecognized sources of infection were Streptococcus milleri and Prolionibacterium sp. Nocardia is resistant to many antibiotics. However, carbapenem, tetracycline and quinolone were effective for Nocardia as well as many other kinds of bacteria. In summary, the brain abscesses presented with only mildly elevated inflammatory markers of body temperature, leukocyte and CRP. These inflammatory markers were less obvious in the patients without ventriculitis and/or meningitis. The source of infection tended to suggest some specific primary causative organism. It was reasonable to initiate therapy with carbapenem. PMID:23092104

  14. The predominant bacteria isolated from radicular cysts

    PubMed Central

    2013-01-01

    Purpose To detect predominant bacteria associated with radicular cysts and discuss in light of the literature. Material and methods Clinical materials were obtained from 35 radicular cysts by aspiration. Cultures were made from clinical materials by modern laboratory techniques, they underwent microbiologic analysis. Results The following are microorganisms isolated from cultures: Streptococcus milleri Group (SMG) (23.8%) [Streptococcus constellatus (19.1%) and Streptococcus anginosus (4.7%)], Streptococcus sanguis (14.3%), Streptococcus mitis (4.7%), Streptococcus cremoris (4.7%), Peptostreptococcus pevotii (4.7%), Prevotella buccae (4.7%), Prevotella intermedia (4.7%), Actinomyces meyeri (4.7%), Actinomyces viscosus (4.7%), Propionibacterium propionicum (4.7%), Bacteroides capillosus (4.7%), Staphylococcus hominis (4.7%), Rothia denticariosa (4.7%), Gemella haemolysans (4.7%), and Fusobacterium nucleatum (4.7%). Conclusions Results of this study demonstrated that radicular cysts show a great variety of anaerobic and facultative anaerobic bacterial flora. It was observed that all isolated microorganisms were the types commonly found in oral flora. Although no specific microorganism was found, Streptococcus spp. bacteria (47.5%) – especially SMG (23.8%) – were predominantly found in the microorganisms isolated. Furthermore, radicular cysts might be polymicrobial originated. Although radicular cyst is an inflammatory cyst, some radicular cyst fluids might be sterile. PMID:24011184

  15. A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    PubMed Central

    Schulz, Sarah; Iglesias-Cans, Marina; Krah, Alexander; Yildiz, Özkan; Leone, Vanessa; Matthies, Doreen; Cook, Gregory M.

    2013-01-01

    The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na+. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1Fo-ATP synthase with a novel Na+ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na+ specificity in physiological settings. Consistently, activity measurements showed Na+ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na+ ionophore monensin. Furthermore, Na+ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na+ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na+ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na+ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen. PMID:23824040

  16. Assessment of Periodontopathogens in Subgingival Biofilm of Banded and Bonded Molars in Early Phase of Fixed Orthodontic Treatment.

    PubMed

    Mártha, Krisztina; Lőrinczi, Lilla; Bică, Cristina; Gyergyay, Réka; Petcu, Blanka; Lazăr, Luminița

    2016-03-01

    To assess the prevalence and occurrence of eleven periodontopathogens in subgingival biofilm of banded and bonded molars during the first period of fixed orthodontic treatment. Subjects were selected from patients referred to orthodontic treatment and were divided in two groups: group A comprised fifteen patients (14.4±2.45 years of age) who received orthodontic bands on first permanent molars and group B of ten patients (15.7±1.87 years of age) with directly bonded tubes on the labial surface of the same teeth. Subgingival sample collection was performed before bands and tubes application and 4-7 weeks after attachment placement. DNA-strip tehnique was used to assess the presence of eleven putative periodontopathogens at each time point. Fusobacterium nucleatum, Eikenella corrodens and Capnocytophaga spp. were found in a large number of samples, other periodontopathogens were present in a smaller rate. The 4-7 weeks after attachment placement a slight increase of putative species was observed in both groups. The presence of orthodontic tubes and bands influence the accumulation and composition of subgingival microbiota. Higher level of oral hygiene should be achieved before and during orthodontic treatment in order to prevent any side effects on periodontal tissues.

  17. Regulation of riboflavin biosynthesis and transport genes in bacteria by transcriptional and translational attenuation

    PubMed Central

    Vitreschak, Alexey G.; Rodionov, Dmitry A.; Mironov, Andrey A.; Gelfand, Mikhail S.

    2002-01-01

    The riboflavin biosynthesis in bacteria was analyzed using comparative analysis of genes, operons and regulatory elements. A model for regulation based on formation of alternative RNA structures involving the RFN elements is suggested. In Gram-positive bacteria including actinomycetes, Thermotoga, Thermus and Deinococcus, the riboflavin metabolism and transport genes are predicted to be regulated by transcriptional attenuation, whereas in most Gram-negative bacteria, the riboflavin biosynthesis genes seem to be regulated on the level of translation initiation. Several new candidate riboflavin transporters were identified (impX in Desulfitobacterium halfniense and Fusobacterium nucleatum; pnuX in several actinomycetes, including some Corynebacterium species and Strepto myces coelicolor; rfnT in Rhizobiaceae). Traces of a number of likely horizontal transfer events were found: the complete riboflavin operon with the upstream regulatory element was transferred to Haemophilus influenzae and Actinobacillus pleuropneumoniae from some Gram-positive bacterium; non-regulated riboflavin operon in Pyrococcus furiousus was likely transferred from Thermotoga; and the RFN element was inserted into the riboflavin operon of Pseudomonas aeruginosa from some other Pseudomonas species, where it had regulated the ribH2 gene. PMID:12136096

  18. High-level antimicrobial efficacy of representative Mediterranean natural plant extracts against oral microorganisms.

    PubMed

    Karygianni, Lamprini; Cecere, Manuel; Skaltsounis, Alexios Leandros; Argyropoulou, Aikaterini; Hellwig, Elmar; Aligiannis, Nektarios; Wittmer, Annette; Al-Ahmad, Ali

    2014-01-01

    Nature is an unexplored reservoir of novel phytopharmaceuticals. Since biofilm-related oral diseases often correlate with antibiotic resistance, plant-derived antimicrobial agents could enhance existing treatment options. Therefore, the rationale of the present report was to examine the antimicrobial impact of Mediterranean natural extracts on oral microorganisms. Five different extracts from Olea europaea, mastic gum, and Inula viscosa were tested against ten bacteria and one Candida albicans strain. The extraction protocols were conducted according to established experimental procedures. Two antimicrobial assays--the minimum inhibitory concentration (MIC) assay and the minimum bactericidal concentration (MBC) assay--were applied. The screened extracts were found to be active against each of the tested microorganisms. O. europaea presented MIC and MBC ranges of 0.07-10.00 mg mL(-1) and 0.60-10.00 mg mL(-1), respectively. The mean MBC values for mastic gum and I. viscosa were 0.07-10.00 mg mL(-1) and 0.15-10.00 mg mL(-1), respectively. Extracts were less effective against C. albicans and exerted bactericidal effects at a concentration range of 0.07-5.00 mg mL(-1) on strict anaerobic bacteria (Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Parvimonas micra). Ethyl acetate I. viscosa extract and total mastic extract showed considerable antimicrobial activity against oral microorganisms and could therefore be considered as alternative natural anti-infectious agents.

  19. [Usefulness and limit of Gram staining smear examination].

    PubMed

    Nagata, Kuniaki; Mino, Hirotoshi; Yoshida, Shunsuke

    2010-05-01

    Gram staining is one of the most simple and inexpensive methods for the rapid diagnosis of bacterial and fungal infections. It yields results much faster than culture, and provides important data for the patient's treatment and prognosis. However, a difference exists in the quality and quantity of information yielded by Gram staining smears based on the experience and knowledge of those conducting the tests. Therefore, a risk of misdiagnosis based on the information obtained from Gram staining smears is also present. The Gram staining conditions and morphology of bacteria sometimes change due to antimicrobial therapy. Species of Gram-negative rods sometimes become filamentous and pleomorphic. Gram-positive bacteria may become gram variable (change in staining condition) after antimicrobial therapy. Even bacteria that are easy to mis-identify exist, because the morphology of bacteria may be similar. Enterococcus faecalis is a Gram-positive diplococcus, forming Gram-positive clustered cocci in specimens from blood culture bottles, resembling Streptococcus pneumoniae. Acinetobacter baumannii is a Gram-negative diplococcus in sputum, resembling Moraxella (Branhamella) catarrhalis. Pasteurella multocida is a small-sized, Gram-negative short rod in the sputum, resembling Haemophilus influenzae. Prevotella intermedia is a small-sized, Gram-negative short rod in sputum, resembling Haemophilus influenzae. Capnocytophaga sp. is a Gram-negative fusiform (thin needle shape) rod present in clinical specimens, resembling Fusobacterium nucleatum.

  20. In Vitro Evaluation of Planktonic Growth on Experimental Cement-Retained Titanium Surfaces.

    PubMed

    Balci, Nur; Cakan, Umut; Aksu, Burak; Akgul, Oncu; Ulger, Nurver

    2016-04-08

    BACKGROUND The purpose of this study was to compare the effects of selected cements, or their combination with titanium, on the growth of two periodontopathic bacteria: Prevotella intermedia (Pi) and Fusobacterium nucleatum (Fn). MATERIAL AND METHODS This study was comprised of several experimental groups: 1) Dental luting cements (glass ionomer cement, methacrylate-based resin cement, zinc-oxide eugenol cement, eugenol-free zinc oxide cement; 2) titanium discs; and 3) titanium combination cement discs. The disks were submerged in bacterial suspensions of either Fn or Pi. Planktonic bacterial growth within the test media was measured by determining the optical density of the cultures (OD600). Mean and standard deviations were calculated for planktonic growth from three separate experiments. RESULTS Intergroup comparison of all experimental groups revealed increased growth of Pi associated with cement-titanium specimens in comparison with cement specimens. Regarding the comparison of all groups for Fn, there was an increased amount of bacterial growth in cement-titanium specimens although the increase was not statistically significant. CONCLUSIONS The combination of cement with titanium may exacerbate the bacterial growth capacity of Pi and Fn in contrast to their sole effect.

  1. Effects of Hangeshashinto on Growth of Oral Microorganisms

    PubMed Central

    Fukamachi, Haruka; Matsumoto, Chinami; Omiya, Yuji; Arimoto, Takafumi; Morisaki, Hirobumi; Kataoka, Hideo; Kadena, Miki; Funatsu, Takahiro; Fukutake, Masato; Kase, Yoshio; Kuwata, Hirotaka

    2015-01-01

    Oral mucositis (OM) in cancer patients induced by chemotherapy or radiotherapy has a significant impact on quality of life, and causes considerable morbidity. Oral microorganisms are likely to intensify the inflammatory process and aggravate the formation of ulcers. Hangeshashinto (HST), a Japanese kampo medicine, has been reported to be effective when used as a gargle for the treatment of OM. To clarify the effects of HST on oral microorganisms, we assessed its antimicrobial activity against 27 microbial species, including 19 oral bacteria and one fungus. HST extract inhibited the growth of Gram-negative bacteria, including Fusobacterium nucleatum, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, Prevotella melaninogenica, Tannerella forsythia, Treponema denticola, and Porphyromonas asaccharolytica, though inhibitory effects were less pronounced for Gram-positive bacteria and the fungal strain. We then investigated the effects of antibacterial activities on 15 purified ingredients of HST and determined that baicalein, berberine, coptisine, [6]-shogaol, and homogentisic acid actively inhibited the growth of these bacteria. These findings showed that HST inhibits the growth of specific Gram-negative periodontopathogenic bacteria, which are significant pathogens in OM, without disturbing the normal oral flora. Our data suggest that HST may be a useful treatment for OM in patients undergoing anticancer treatment. PMID:26170876

  2. Preventive Effects of Houttuynia cordata Extract for Oral Infectious Diseases

    PubMed Central

    Sekita, Yasuko; Murakami, Keiji; Amoh, Takashi; Ogata, Shohei; Matsuo, Takashi; Miyake, Yoichiro; Kashiwada, Yoshiki

    2016-01-01

    Houttuynia cordata (HC) (Saururaceae) has been used internally and externally as a traditional medicine and as an herbal tea for healthcare in Japan. Our recent survey showed that HC poultice (HCP) prepared from smothering fresh leaves of HC had been frequently used for the treatment of purulent skin diseases with high effectiveness. Our experimental study also demonstrated that ethanol extract of HCP (eHCP) has antibacterial, antibiofilm, and anti-inflammatory effects against S. aureus which caused purulent skin diseases. In this study, we focused on novel effects of HCP against oral infectious diseases, such as periodontal disease and dental caries. We determined the antimicrobial and antibiofilm effects of water solution of HCP ethanol extract (wHCP) against important oral pathogens and investigated its cytotoxicity and anti-inflammatory effects on human oral epithelial cells. wHCP had moderate antimicrobial effects against some oral microorganisms and profound antibiofilm effects against Fusobacterium nucleatum, Streptococcus mutans, and Candida albicans. In addition, wHCP had no cytotoxic effects and could inhibit interleukin-8 and CCL20 productions by Porphyromonas gingivalis lipopolysaccharide-stimulated human oral keratinocytes. Our findings suggested that wHCP may be clinically useful for preventing oral infectious diseases as a mouthwash for oral care. PMID:27413739

  3. Impact of growth conditions on susceptibility of five microbial species to alkaline stress.

    PubMed

    Brändle, Nathalie; Zehnder, Matthias; Weiger, Roland; Waltimo, Tuomas

    2008-05-01

    The effects of different growth conditions on the susceptibility of five taxa to alkaline stress were investigated. Enterococcus faecalis ATCC 29212, Streptococcus sobrinus OMZ 176, Candida albicans ATCC 90028, Actinomyces naeslundii ATCC 12104, and Fusobacterium nucleatum ATCC 10953 were grown as planktonic cells, allowed to adhere to dentin for 24 hours, grown as monospecies or multispecies biofilms on dentin under anaerobic conditions with a serum-enriched nutrient supply at 37 degrees C for 5 days. In addition, suspended biofilm microorganisms and 5-day old planktonic multispecies cultures were used. Microbial recovery upon direct exposure to saturated calcium hydroxide solution (pH 12.5) for 10 and 100 minutes was compared with control exposure to physiologic saline. Planktonic microorganisms were most susceptible; only E. faecalis and C. albicans survived in saturated solution for 10 minutes, the latter also for 100 minutes. Dentin adhesion was the major factor in improving the resistance of E. faecalis and A. naeslundii to calcium hydroxide, whereas the multispecies context in a biofilm was the major factor in promoting resistance of S. sobrinus to the disinfectant. In contrast, the C. albicans response to calcium hydroxide was not influenced by the growth condition. Adherence to dentin and interspecies interactions in a biofilm appear to differentially affect the sensitivity of microbial species to calcium hydroxide.

  4. The Surface Layer of Tannerella forsythia Contributes to Serum Resistance and Oral Bacterial Coaggregation

    PubMed Central

    Shimotahira, Naohiro; Oogai, Yuichi; Kawada-Matsuo, Miki; Yamada, Sakuo; Fukutsuji, Kenji; Nagano, Keiji; Yoshimura, Fuminobu; Noguchi, Kazuyuki

    2013-01-01

    Tannerella forsythia is an anaerobic, Gram-negative bacterium involved in the so-called “red complex,” which is associated with severe and chronic periodontitis. The surface layer (S-layer) of T. forsythia is composed of cell surface glycoproteins, such as TfsA and TfsB, and is known to play a role in adhesion/invasion and suppression of proinflammatory cytokine expression. Here we investigated the association of this S-layer with serum resistance and coaggregation with other oral bacteria. The growth of the S-layer-deficient mutant in a bacterial medium containing more than 20% non-heat-inactivated calf serum (CS) or more than 40% non-heat-inactivated human serum was significantly suppressed relative to that of the wild type (WT). Next, we used confocal microscopy to perform quantitative analysis on the effect of serum. The survival ratio of the mutant exposed to 100% non-heat-inactivated CS (76% survival) was significantly lower than that of the WT (97% survival). Furthermore, significant C3b deposition was observed in the mutant but not in the WT. In a coaggregation assay, the mutant showed reduced coaggregation with Streptococcus sanguinis, Streptococcus salivarius, and Porphyromonas gingivalis but strong coaggregation with Fusobacterium nucleatum. These results indicated that the S-layer of T. forsythia plays multiple roles in virulence and may be associated with periodontitis. PMID:23357386

  5. Bacterial diversity of symptomatic primary endodontic infection by clonal analysis.

    PubMed

    Nóbrega, Letícia Maria Menezes; Montagner, Francisco; Ribeiro, Adriana Costa; Mayer, Márcia Alves Pinto; Gomes, Brenda Paula Figueiredo de Almeida

    2016-10-10

    The aim of this study was to explore the bacterial diversity of 10 root canals with acute apical abscess using clonal analysis. Samples were collected from 10 patients and submitted to bacterial DNA isolation, 16S rRNA gene amplification, cloning, and sequencing. A bacterial genomic library was constructed and bacterial diversity was estimated. The mean number of taxa per canal was 15, ranging from 11 to 21. A total of 689 clones were analyzed and 76 phylotypes identified, of which 47 (61.84%) were different species and 29 (38.15%) were taxa reported as yet-uncultivable or as yet-uncharacterized species. Prevotella spp., Fusobacterium nucleatum, Filifactor alocis, and Peptostreptococcus stomatis were the most frequently detected species, followed by Dialister invisus, Phocaeicola abscessus, the uncharacterized Lachnospiraceae oral clone, Porphyromonas spp., and Parvimonas micra. Eight phyla were detected and the most frequently identified taxa belonged to the phylum Firmicutes (43.5%), followed by Bacteroidetes (22.5%) and Proteobacteria (13.2%). No species was detected in all studied samples and some species were identified in only one case. It was concluded that acute primary endodontic infection is characterized by wide bacterial diversity and a high intersubject variability was observed. Anaerobic Gram-negative bacteria belonging to the phylum Firmicutes, followed by Bacteroidetes, were the most frequently detected microorganisms.

  6. Treponema denticola chymotrypsin-like proteinase (CTLP) integrates spirochaetes within oral microbial communities.

    PubMed

    Cogoni, Valentina; Morgan-Smith, Alex; Fenno, J Christopher; Jenkinson, Howard F; Dymock, David

    2012-03-01

    Treponema denticola is found ubiquitously in the human oral cavity and is mainly associated with bacterial communities implicated in the establishment and development of periodontal disease. The ability to become integrated within biofilm communities is crucial to the growth and survival of oral bacteria, and involves inter-bacterial coaggregation, metabolic cooperation, and synergy against host defences. In this article we show that the chymotrypsin-like proteinase (CTLP), found within a high-molecular-mass complex on the cell surface, mediates adherence of T. denticola to other potential periodontal pathogens, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia and Parvimonas micra. Proteolytic activity per se did not appear to be required for the interactions, and expression of the major outer-sheath protein (Msp) was not necessary, except for binding Parv. micra. Biofilms of densely packed cells and matrix, up to 40 µm in depth, were formed between T. denticola and P. gingivalis on salivary pellicle, with T. denticola cells enriched in the upper layers. Expression of CTLP, but not Msp, was critical for dual-species biofilm formation with P. gingivalis. T. denticola did not form dual-species biofilms with any of the other three periodontal bacterial species under various conditions. Synergy between T. denticola and P. gingivalis was also shown by increased inhibition of blood clotting, which was CTLP-dependent. The results demonstrate the critical role of CTLP in interactions of T. denticola with other oral micro-organisms, leading to synergy in microbial community development and host tissue pathogenesis.

  7. The Biofilm Community-Rebels with a Cause

    PubMed Central

    Aruni, A. Wilson; Dou, Yuetan; Mishra, Arunima; Fletcher, Hansel M.

    2015-01-01

    Oral Biofilms are one of the most complex and diverse ecosystem developed by successive colonization of more than 600 bacterial taxa. Development starts with the attachment of early colonizers such as Actinomyces species and oral streptococci on the acquired pellicle and tooth enamel. These bacteria not only adhere to tooth surface but also interact with each other and lay foundation for attachment of bridging colonizer such as Fusobacterium nucleatum followed by late colonizers including the red complex species: Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola-the founders of periodontal disease. As the biofilm progresses from supragingival sites to subgingival sites, the environment changes from aerobic to anaerobic thus favoring the growth of mainly Gram-negative obligate anaerobes while restricting the growth of the early Gram-positive facultative aerobes. Microbes present at supragingival level are mainly related to gingivitis and root-caries whereas subgingival species advance the destruction of teeth supporting tissues and thus causing periodontitis. This review summarizes our present understanding and recent developments on the characteristic features of supra- and subgingival biofilms, interaction between different genera and species of bacteria constituting these biofilms and draws our attention to the role of some of the recently discovered members of the oral community. PMID:26120510

  8. Emerging role of bacteria in oral carcinogenesis: a review with special reference to perio-pathogenic bacteria

    PubMed Central

    Perera, Manosha; Al-hebshi, Nezar Noor; Speicher, David J.; Perera, Irosha; Johnson, Newell W.

    2016-01-01

    Oral cancer, primarily oral squamous cell carcinoma (OSCC), continues to be a major global health problem with high incidence and low survival rates. While the major risk factors for this malignancy, mostly lifestyle related, have been identified, around 15% of oral cancer cases remain unexplained. In light of evidence implicating bacteria in the aetiology of some cancer types, several epidemiological studies have been conducted in the last decade, employing methodologies ranging from traditional culture techniques to 16S rRNA metagenomics, to assess the possible role of bacteria in OSCC. While these studies have demonstrated differences in microbial composition between cancerous and healthy tissues, they have failed to agree on specific bacteria or patterns of oral microbial dysbiosis to implicate in OSCC. On the contrary, some oral taxa, particularly Porphyromonas gingivalis and Fusobacterium nucleatum, show strong oral carcinogenic potential in vitro and in animal studies. Bacteria are thought to contribute to oral carcinogenesis via inhibition of apoptosis, activation of cell proliferation, promotion of cellular invasion, induction of chronic inflammation, and production of carcinogens. This narrative review provides a critical analysis of and an update on the association between bacteria and oral carcinogenesis and the possible mechanisms underlying it. PMID:27677454

  9. Prevotella intermedia induces severe bacteremic pneumococcal pneumonia in mice with upregulated platelet-activating factor receptor expression.

    PubMed

    Nagaoka, Kentaro; Yanagihara, Katsunori; Morinaga, Yoshitomo; Nakamura, Shigeki; Harada, Tatsuhiko; Hasegawa, Hiroo; Izumikawa, Koichi; Ishimatsu, Yuji; Kakeya, Hiroshi; Nishimura, Masaharu; Kohno, Shigeru

    2014-02-01

    Streptococcus pneumoniae is the leading cause of respiratory infection worldwide. Although oral hygiene has been considered a risk factor for developing pneumonia, the relationship between oral bacteria and pneumococcal infection is unknown. In this study, we examined the synergic effects of Prevotella intermedia, a major periodontopathic bacterium, on pneumococcal pneumonia. The synergic effects of the supernatant of P. intermedia (PiSup) on pneumococcal pneumonia were investigated in mice, and the stimulation of pneumococcal adhesion to human alveolar (A549) cells by PiSup was assessed. The effects of PiSup on platelet-activating factor receptor (PAFR) transcript levels in vitro and in vivo were analyzed by quantitative real-time PCR, and the differences between the effects of pneumococcal infection induced by various periodontopathic bacterial species were verified in mice. Mice inoculated with S. pneumoniae plus PiSup exhibited a significantly lower survival rate, higher bacterial loads in the lungs, spleen, and blood, and higher inflammatory cytokine levels in the bronchoalveolar lavage fluid (macrophage inflammatory protein 2 and tumor necrosis factor alpha) than those infected without PiSup. In A549 cells, PiSup increased pneumococcal adhesion and PAFR transcript levels. PiSup also increased lung PAFR transcript levels in mice. Similar effects were not observed in the supernatants of Porphyromonas gingivalis or Fusobacterium nucleatum. Thus, P. intermedia has the potential to induce severe bacteremic pneumococcal pneumonia with enhanced pneumococcal adhesion to lower airway cells.

  10. Proteomic profiling of host-biofilm interactions in an oral infection model resembling the periodontal pocket

    PubMed Central

    Bao, Kai; Belibasakis, Georgios N.; Selevsek, Nathalie; Grossmann, Jonas; Bostanci, Nagihan

    2015-01-01

    Periodontal infections cause inflammatory destruction of the tooth supporting tissues. We recently developed a dynamic, in vitro periodontal organotypic tissue model in a perfusion bioreactor system, in co-culture with an 11-species subgingival biofilm, which may recapitulate early events during the establishment of periodontal infections. This study aimed to characterize the global proteome regulations in this host-biofilm interaction model. Semi-quantitative shotgun proteomics were applied for protein identification and quantification in the co-culture supernatants (human and bacterial) and the biofilm lysates (bacterial). A total of 896 and 3363 proteins were identified as secreted in the supernatant and expressed in the biofilm lysate, respectively. Enriched gene ontology analysis revealed that the regulated secreted human tissue proteins were related to processes of cytoskeletal rearrangement, stress responses, apoptosis, and antigen presentation, all of which are commensurate with deregulated host responses. Most secreted bacterial biofilm proteins derived from their cytoplasmic domain. In the presence of the tissue, the levels of Fusobacterium nucleatum, Actinomyces oris and Campylobacter rectus proteins were significantly regulated. The functions of the up-regulated intracellular (biofilm lysate) proteins were associated with cytokinesis. In conclusion, the proteomic overview of regulated pathways in this host-biofilm interaction model provides insights to the early events of periodontal pathogenesis. PMID:26525412

  11. Clindamycin in Treatment of Aspiration Pneumonia in Children

    PubMed Central

    Brook, Itzhak

    1979-01-01

    Twenty-eight patients with anaerobic pleuropulmonary infections were treated with clindamycin alone or clindamycin with gentamicin. Sixteen of the patients presented with pneumonitis, nine with necrotizing pneumonia, and three with lung abscesses. The average length of treatment was 13.8 days, and the duration of temperature after initiation of therapy was 3.1 days. The predominant isolates were anaerobic gram-positive cocci (23 isolates), Bacteroides melaninogenicus (14), Bacteroides fragilis (9), and Fusobacterium nucleatum (11). The most frequent aerobic isolates were alpha-hemolytic streptococci (12), Diplococcus pneumoniae (12), Pseudomonas aeruginosa (9), Klebsiella pneumoniae (7), group A beta-hemolytic streptococci (5), Staphylococcus aureus (9), and Escherichia coli (6). All patients responded to the therapy and were cured of the infection. There were no side effects observed from the administration of clindamycin. None of the patients developed any blood dyscrasia, liver damage, diarrhea, or colitis. Clindamycin appears to be effective in the treatment of mixed aerobic and anaerobic pleuropulmonary infections in children, alone or with an aminoglycoside when indicated. PMID:380459

  12. 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.

  13. Axenic culture of a candidate division TM7 bacterium from the human oral cavity and biofilm interactions with other oral bacteria.

    PubMed

    Soro, Valeria; Dutton, Lindsay C; Sprague, Susan V; Nobbs, Angela H; Ireland, Anthony J; Sandy, Jonathan R; Jepson, Mark A; Micaroni, Massimo; Splatt, Peter R; Dymock, David; Jenkinson, Howard F

    2014-10-01

    The diversity of bacterial species in the human oral cavity is well recognized, but a high proportion of them are presently uncultivable. Candidate division TM7 bacteria are almost always detected in metagenomic studies but have not yet been cultivated. In this paper, we identified candidate division TM7 bacterial phylotypes in mature plaque samples from around orthodontic bonds in subjects undergoing orthodontic treatment. Successive rounds of enrichment in laboratory media led to the isolation of a pure culture of one of these candidate division TM7 phylotypes. The bacteria formed filaments of 20 to 200 μm in length within agar plate colonies and in monospecies biofilms on salivary pellicle and exhibited some unusual morphological characteristics by transmission electron microscopy, including a trilaminated cell surface layer and dense cytoplasmic deposits. Proteomic analyses of cell wall protein extracts identified abundant polypeptides predicted from the TM7 partial genomic sequence. Pleiomorphic phenotypes were observed when the candidate division TM7 bacterium was grown in dual-species biofilms with representatives of six different oral bacterial genera. The TM7 bacterium formed long filaments in dual-species biofilm communities with Actinomyces oris or Fusobacterium nucleatum. However, the TM7 isolate grew as short rods or cocci in dual-species biofilms with Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra, or Streptococcus gordonii, forming notably robust biofilms with the latter two species. The ability to cultivate TM7 axenically should majorly advance understanding of the physiology, genetics, and virulence properties of this novel candidate division oral bacterium.

  14. Influence of Biofilm Formation by Gardnerella vaginalis and Other Anaerobes on Bacterial Vaginosis.

    PubMed

    Machado, António; Cerca, Nuno

    2015-12-15

    Bacterial vaginosis (BV) is the worldwide leading vaginal disorder among women of reproductive age. BV is characterized by the replacement of beneficial lactobacilli and the augmentation of anaerobic bacteria. Gardnerella vaginalis is a predominant bacterial species, but BV is also associated with other numerous anaerobes, such as Atopobium vaginae, Mobiluncus mulieris, Prevotella bivia, Fusobacterium nucleatum, and Peptoniphilus species. Currently, the role of G. vaginalis in the etiology of BV remains a matter of controversy. However, it is known that, in patients with BV, a biofilm is usually formed on the vaginal epithelium and that G. vaginalis is typically the predominant species. So, the current paradigm is that the establishment of a biofilm plays a key role in the pathogenesis of BV. This review provides background on the influence of biofilm formation by G. vaginalis and other anaerobes, from the time of their initial adhesion until biofilm formation, in the polymicrobial etiology of BV and discusses the commensal and synergic interactions established between them to understand the phenotypic shift of G. vaginalis biofilm formation to BV establishment.

  15. Microbial dynamics during conversion from supragingival to subgingival biofilms in an in vitro model.

    PubMed

    Thurnheer, T; Bostanci, N; Belibasakis, G N

    2016-04-01

    The development of dental caries and periodontal diseases result from distinct shifts in the microbiota of the tooth-associated biofilm. This in vitro study aimed to investigate changes in biofilm composition and structure, during the shift from a 'supragingival' aerobic profile to a 'subgingival' anaerobic profile. Biofilms consisting of Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans and Veillonella dispar were aerobically grown in saliva-containing medium on hydroxyapatite disks. After 64 h, Campylobacter rectus, Prevotella intermedia and Streptococcus anginosus were further added along with human serum, while culture conditions were shifted to microaerophilic. After 96 h, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola were finally added and the biofilm was grown anaerobically for another 64 h. At the end of each phase, biofilms were harvested for species-specific quantification and localization. Apart from C. albicans, all other species gradually increased during aerobic and microaerophilic conditions, but remained steady during anaerobic conditions. Biofilm thickness was doubled during the microaerophilic phase, but remained steady throughout the anaerobic phase. Extracellular polysaccharide presence was gradually reduced throughout the growth period. Biofilm viability was reduced during the microaerophilic conversion, but was recovered during the anaerobic phase. This in vitro study has characterized the dynamic structural shifts occurring in an oral biofilm model during the switch from aerobic to anaerobic conditions, potentially modeling the conversion of supragingival to subgingival biofilms. Within the limitations of this experimental model, the findings may provide novel insights into the ecology of oral biofilms.

  16. High-throughput dental biofilm growth analysis for multiparametric microenvironmental biochemical conditions using microfluidics.

    PubMed

    Lam, Raymond H W; Cui, Xin; Guo, Weijin; Thorsen, Todd

    2016-04-26

    Dental biofilm formation is not only a precursor to tooth decay, but also induces more serious systematic health problems such as cardiovascular disease and diabetes. Understanding the conditions promoting colonization and subsequent biofilm development involving complex bacteria coaggregation is particularly important. In this paper, we report a high-throughput microfluidic 'artificial teeth' device offering controls of multiple microenvironmental factors (e.g. nutrients, growth factors, dissolved gases, and seeded cell populations) for quantitative characteristics of long-term dental bacteria growth and biofilm development. This 'artificial teeth' device contains multiple (up to 128) incubation chambers to perform parallel cultivation and analyses (e.g. biofilm thickness, viable-dead cell ratio, and spatial distribution of multiple bacterial species) of bacteria samples under a matrix of different combinations of microenvironmental factors, further revealing possible developmental mechanisms of dental biofilms. Specifically, we applied the 'artificial teeth' to investigate the growth of two key dental bacteria, Streptococci species and Fusobacterium nucleatum, in the biofilm under different dissolved gas conditions and sucrose concentrations. Together, this high-throughput microfluidic platform can provide extended applications for general biofilm research, including screening of the biofilm properties developing under combinations of specified growth parameters such as seeding bacteria populations, growth medium compositions, medium flow rates and dissolved gas levels.

  17. The effect of sodium hypochlorite on Enterococcus faecalis when grown on dentine as a single- and multi-species biofilm.

    PubMed

    Yap, Benlee; Zilm, Peter S; Briggs, Nancy; Rogers, Anthony H; Cathro, Peter C

    2014-12-01

    Enterococcus faecalis is often involved in the aetiology of apical periodontitis after endodontic treatment. This project aimed to establish, on dentine in vitro, a multi-species biofilm containing E. faecalis, and to determine if the organism had an increased resistance to sodium hypochlorite compared with an axenic biofilm. Biofilms were established on dentine discs in flow cells with either E. faecalis alone (axenic) or together with Fusobacterium nucleatum and Streptococcus sanguinis. Following treatment with either 0.9% sodium hypochlorite or saline, the viability of E. faecalis was determined by serial plating and qualitative analysis was performed by scanning electron microscopy and confocal laser scanning microscopy. Viable counts indicated that 0.9% NaOCl is highly effective against E. faecalis grown alone and as part of a multi-species biofilm (P = 0.0005 and P = 0.001, respectively). No significant difference in its survival in the two biofilm types was found (P = 0.8276).

  18. Novel Therapeutic Approach for the Treatment of Periodontitis by Curcumin

    PubMed Central

    Bhatia, Madhu; Pentyala, Kishore Babu; Urolagin, Sarvesh Basavaraj; K B, Menaka; Bhoi, Shreedevi

    2014-01-01

    Aims and objectives: The aim of the present study was to evaluate the clinical and microbiological efficacy of locally delivered 1% curcumin gel as an adjunct to scaling and root planing in the treatment of chronic periodontitis. Materials and Methods: The study group consisted of 25 patients, belonging to both sex, aged between 21-45 years. All patients diagnosed as chronic periodontitis with periodontal pockets of depth >5mm bilaterally were randomly selected. A split mouth design was followed and the patients received a complete prophylaxis including scaling and root planing. Examination of plaque index, bleeding index, probing pocket depth and clinical attachment level were measured for each patient. The test group received 1% curcumin gel along with scaling and root planing whereas the control group received scaling and root planing alone followed by microbiological samples taken at baseline, 1, 3 and 6 months interval. Results: The 1% curcumin gel appeared to provide significant improvements in clinical parameters. Microbiological counts of Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and capnocytophaga showed significant reduction in periopathogens at the test sites after six months when compared with that of control sites. Conclusion: Locally delivered 1% curcumin gel was more effective in inhibiting the growth of oral bacteria when used as an adjunct to SRP in the treatment of chronic periodontitis. PMID:25654035

  19. Selective responses of human gingival fibroblasts and bacteria on carbon fiber reinforced polyetheretherketone with multilevel nanostructured TiO2.

    PubMed

    Wang, Xiao; Lu, Tao; Wen, Jin; Xu, Lianyi; Zeng, Deliang; Wu, Qianju; Cao, Lingyan; Lin, Shuxian; Liu, Xuanyong; Jiang, Xinquan

    2016-03-01

    The long-term success of dental implants relies not only on stable osseointegration but also on the integration of implant surfaces with surrounding soft tissues. In our previous work, titanium plasma immersion ion implantation (PIII) technique was applied to modify the carbon-fiber-reinforced polyetheretherketone (CFRPEEK) surface, constructing a unique multilevel TiO2 nanostructure thus enhancing certain osteogenic properties. However, the interactions between the modified surface and soft-tissue cells are still not clear. Here, we fully investigate the biological behaviors of human gingival fibroblasts (HGFs) and oral pathogens on the structured surface, which determine the early peri-implant soft tissue integration. Scanning electron microscopy (SEM) shows the formation of nanopores with TiO2 nanoparticles embedded on both the sidewall and bottom. In vitro studies including cell adhesion, viability assay, wound healing assay, real-time PCR, western blot and enzyme-linked immunosorbent assay (ELISA) disclose improved adhesion, migration, proliferation, and collagen secretion ability of HGFs on the modified CFRPEEK. Moreover, the structured surface exhibits sustainable antibacterial properties towards Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis. Our results reveal that the multilevel TiO2 nanostructures can selectively enhance soft tissue integration and inhibit bacterial reproduction, which will further support and broaden the adoption of CFRPEEK materials in dental fields.

  20. Fusobacterium polysaccharolyticum sp.nov., a gram-negative rod from the rumen that produces butyrate and ferments cellulose and starch.

    PubMed

    van Gylswyk, N O

    1980-01-01

    A new Gram-negative, non-sporulating, rod-shaped, anaerobic bacterium capable of fermenting cellulose and starch was isolated from the rumens of sheep fed supplemented maize stover diets. The organism fermented few carbohydrates, showing a preference for polysaccharides. The main acid products of carbohydrate fermentation were butyrate and formate. Acetate was utilized.

  1. 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

  2. Three-species biofilm model onto plasma-treated titanium implant surface.

    PubMed

    Matos, Adaias O; Ricomini-Filho, Antônio P; Beline, Thamara; Ogawa, Erika S; Costa-Oliveira, Bárbara E; de Almeida, Amanda B; Nociti Junior, Francisco H; Rangel, Elidiane C; da Cruz, Nilson C; Sukotjo, Cortino; Mathew, Mathew T; Barão, Valentim A R

    2017-04-01

    In this study, titanium (Ti) was modified with biofunctional and novel surface by micro-arc oxidation (MAO) and glow discharge plasma (GDP) and we tested the development of a three-species periodontopatogenic biofilm onto the treated commercially-pure titanium (cpTi) surfaces. Machined and sandblasted surfaces were used as control group. Several techniques for surface characterizations and monoculture on bone tissue cells were performed. A multispecies biofilm composed of Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum was developed onto cpTi discs for 16.5h (early biofilm) and 64.5h (mature biofilm). The number of viable microorganisms and the composition of the extracellular matrix (proteins and carbohydrates) were determined. The biofilm organization was analyzed by scanning electron microscopy (SEM) and Confocal laser scanning microscopy (CLSM). In addition, MC3T3-E1 cells were cultured on the Ti surfaces and cell proliferation (MTT) and morphology (SEM) were assessed. MAO treatment produced oxide films rich in calcium and phosphorus with a volcano appearance while GDP treatment produced silicon-based smooth thin-film. Plasma treatments were able to increase the wettability of cpTi (p<0.05). An increase of surface roughness (p<0.05) and formation of anatase and rutile structures was noted after MAO treatment. GDP had the greatest surface free energy (p<0.05) while maintaining the surface roughness compared to the machined control (p>0.05). Plasma treatment did not affect the viable microorganisms counts, but the counts of F. nucleatum was lower for MAO treatment at early biofilm phase. Biofilm extracellular matrix was similar among the groups, excepted for GDP that presented the lowest protein content. Moreover, cell proliferation was not significantly affected by the experimental, except for MAO at 6days that resulted in an increased cell proliferative. Together, these findings indicate that plasma treatments are a viable and

  3. Surface structures (peritrichous fibrils and tufts of fibrils) found on Streptococcus sanguis strains may be related to their ability to coaggregate with other oral genera.

    PubMed Central

    Handley, P S; Carter, P L; Wyatt, J E; Hesketh, L M

    1985-01-01

    We screened 36 strains of Streptococcus sanguis biotype I and 8 strains of S. sanguis biotype II for the presence of surface structures and for their ability to coaggregate with Actinomyces viscosus, Actinomyces naeslundii, and Fusobacterium nucleatum. Negative staining under an electron microscope revealed detectable surface structures on all S. sanguis strains. The majority of strains (38 of 44) carried peritrichous fibrils, which have an irregular profile and no distinct width. They usually appeared as a fringe with a constant width around the cell. Strains selected for measurement had a fringe with an average length of 72.4 +/- 8.5 nm on biotype I strains and 51.6 +/- 3.3 nm on biotype II strains. Some fibrillar biotype I strains carried an additional, longer (158.7 +/- 33.1 nm) type of fibril projecting through the shorter fibrils. Fibrillar density was characteristic for each strain, ranging from very dense on all cells in a population to very sparse on a few cells in a population. A small group of six strains carried tufts of fibrils in a lateral or polar position on the cell. Either one or two lengths of fibril were present in the tuft depending on the strain. One strain carried both peritrichous fibrils and fimbriae. Fimbriae are flexible structures with a constant width (4.5 to 5.0 nm) all along their length but very variable lengths (less than or equal to 0.7 micron) on each cell. S. sanguis I and II both included strains with peritrichous fibrils and tufts of fibrils, but the mixed morphotype strain was confined to biotype II. Fibrils were present on cells at all stages throughout the growth cycle for the strains tested. Freshly isolated fibrillar strains coaggregated consistently well with A. viscosus and A. naeslundii, although some fibrillar reference strains lacked the ability. In addition, all tufted strains could not coaggregate, but the strains with the mixed morphotype coaggregated well. Coaggregation with F. nucleatum was very strong for the

  4. Evaluation of Antimicrobial-Antibiofilm Activity of a Hydrogen Peroxide Decontaminating System Used in Dental Unit Water Lines

    PubMed Central

    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 H2O2 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 H2O2 ppm (also including M. marinum and C. albicans). However, H2O2 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% H2O2, 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. PMID:21113279

  5. Extensive Identification of Bacterial Riboflavin Transporters and Their Distribution across Bacterial Species.

    PubMed

    Gutiérrez-Preciado, Ana; Torres, Alfredo Gabriel; Merino, Enrique; Bonomi, Hernán Ruy; Goldbaum, Fernando Alberto; García-Angulo, Víctor Antonio

    2015-01-01

    Riboflavin, the precursor for the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide, is an essential metabolite in all organisms. While the functions for de novo riboflavin biosynthesis and riboflavin import may coexist in bacteria, the extent of this co-occurrence is undetermined. The RibM, RibN, RfuABCD and the energy-coupling factor-RibU bacterial riboflavin transporters have been experimentally characterized. In addition, ImpX, RfnT and RibXY are proposed as riboflavin transporters based on positional clustering with riboflavin biosynthetic pathway (RBP) genes or conservation of the FMN riboswitch regulatory element. Here, we searched for the FMN riboswitch in bacterial genomes to identify genes encoding riboflavin transporters and assessed their distribution among bacteria. Two new putative riboflavin transporters were identified: RibZ in Clostridium and RibV in Mesoplasma florum. Trans-complementation of an Escherichia coli riboflavin auxotroph strain confirmed the riboflavin transport activity of RibZ from Clostridium difficile, RibXY from Chloroflexus aurantiacus, ImpX from Fusobacterium nucleatum and RfnT from Ochrobactrum anthropi. The analysis of the genomic distribution of all known bacterial riboflavin transporters revealed that most occur in species possessing the RBP and that some bacteria may even encode functional riboflavin transporters from two different families. Our results indicate that some species possess ancestral riboflavin transporters, while others possess transporters that appear to have evolved recently. Moreover, our data suggest that unidentified riboflavin transporters also exist. The present study doubles the number of experimentally characterized riboflavin transporters and suggests a specific, non-accessory role for these proteins in riboflavin-prototrophic bacteria.

  6. Antimicrobial and immunomodulatory properties of PGLa-AM1, CPF-AM1, and magainin-AM1: potent activity against oral pathogens.

    PubMed

    McLean, Denise T F; McCrudden, Maelíosa T C; Linden, Gerard J; Irwin, Christopher R; Conlon, J Michael; Lundy, Fionnuala T

    2014-11-01

    Cationic amphipathic α-helical peptides are intensively studied classes of host defence peptides (HDPs). Three peptides, peptide glycine-leucine-amide (PGLa-AM1), caerulein-precursor fragment (CPF-AM1) and magainin-AM1, originally isolated from norepinephrine-stimulated skin secretions of the African volcano frog Xenopus amieti (Pipidae), were studied for their antimicrobial and immunomodulatory activities against oral and respiratory pathogens. Minimal effective concentrations (MECs), determined by radial diffusion assay, were generally lower than minimal inhibitory concentrations (MICs) determined by microbroth dilution. PGLa-AM1 and CPF-AM1 were particularly active against Streptococcus mutans and all three peptides were effective against Fusobacterium nucleatum, whereas Enterococcus faecalis and Candida albicans proved to be relatively resistant micro-organisms. A type strain of Pseudomonas aeruginosa was shown to be more susceptible than the clinical isolate studied. PGLa-AM1 displayed the greatest propensity to bind lipopolysaccharide (LPS) from Escherichia coli, P. aeruginosa and Porphyromonas gingivalis. All three peptides showed less binding to P. gingivalis LPS than to LPS from the other species studied. Oral fibroblast viability was unaffected by 50 μM peptide treatments. Production of the pro-inflammatory cytokine IL-8 by oral fibroblasts was significantly increased following treatment with 1 or 10 μM magainin-AM1 but not following treatment with PGLa-AM1 or CPF-AM1. In conclusion, as well as possessing potent antimicrobial actions, the X. amieti peptides bound to LPS from three human pathogens and had no effect on oral fibroblast viability. CPF-AM1 and PGLa-AM1 show promise as templates for the design of novel analogues for the treatment of oral and dental diseases associated with bacteria or fungi.

  7. High Individuality of Respiratory Bacterial Communities in a Large Cohort of Adult Cystic Fibrosis Patients under Continuous Antibiotic Treatment

    PubMed Central

    Kramer, Rolf; Sauer-Heilborn, Annette; Welte, Tobias; Jauregui, Ruy; Brettar, Ingrid; Guzman, Carlos A.; Höfle, Manfred G.

    2015-01-01

    Background Routine clinical diagnostics of CF patients focus only on a restricted set of well-known pathogenic species. Recent molecular studies suggest that infections could be polymicrobial with many bacteria not detected by culture-based diagnostics. Methodology and Principal Findings A large cohort of 56 adults with continuous antibiotic treatment was studied and different microbial diagnostic methods were compared, including culture-independent and culture-based bacterial diagnostics. A total of 72 sputum samples including longitudinal observations was analysed by 16S rRNA gene sequence comparison. Prevalence of known pathogens was highly similar among all methods but the vast spectrum of bacteria associated with CF was only revealed by culture-independent techniques. The sequence comparison enabled confident determination of the bacterial community composition and revealed a high diversity and individuality in the communities across the cohort. Results of microbiological analyses were further compared with individual host factors, such as age, lung function and CFTR genotype. No statistical relationship between these factors and the diversity of the entire community or single bacterial species could be identified. However, patients with non-ΔF508 mutations in the CFTR gene often had low abundances of Pseudomonas aeruginosa. Persistence of specific bacteria in some communities was demonstrated by longitudinal analyses of 13 patients indicating a potential clinical relevance of anaerobic bacteria, such as Fusobacterium nucleatum and Streptococcus millerii. Conclusions The high individuality in community composition and the lack of correlation to clinical host factors might be due to the continuous treatment with antibiotics. Since this is current practice for adult CF patients, the life-long history of the patient and the varying selection pressure on the related microbial communities should be a focus of future studies and its relation to disease progression

  8. Effects of stress hormones on the production of volatile sulfur compounds by periodontopathogenic bacteria.

    PubMed

    Calil, Caroline Morini; Oliveira, Gisele Mattos; Cogo, Karina; Pereira, Antonio Carlos; Marcondes, Fernanda Klein; Groppo, Francisco Carlos

    2014-01-01

    Little is known about the effects of stress hormones on the etiologic agents of halitosis. Thus, the aim of this study was to evaluate in vitro the effects of adrenaline (ADR), noradrenaline (NA) and cortisol (CORT) on bacteria that produce volatile sulfur compounds (VSC), the major gases responsible for bad breath. Cultures of Fusobacterium nucleatum (Fn), Porphyromonas endodontalis (Pe), Prevotella intermedia (Pi) and Porphyromonas gingivalis (Pg) were exposed to 50 µM ADR, NA and CORT or equivalent volumes of sterile water as controls for 12 and 24 h. Growth was evaluated based on absorbance at 660 nm. Portable gas chromatography was used to measure VSC concentrations. Kruskal-Wallis and the Dunn post-hoc test were used to compare the groups. For Fn, ADR, NA and CORT significantly reduced bacterial growth after 12 h and 24 h (p<0.05). All the substances tested increased hydrogen sulfide (H2S) production (p<0.05). For Pe, all the substances tested reduced bacterial development after 24 h (p<0.05), and NA significantly increased the H2S concentration after 12 h (p<0.05). In the Pg and Pi cultures, no effects on bacterial growth were observed (p>0.05). In the Pi cultures, ADR, NA and CORT increased H2S (p<0.05). Catecholamines and cortisol can interfere with growth and H2S production of sub-gingival species in vitro. This process appears to be complex and supports the association between stress and the production of VSC.

  9. Changes in microflora in dental plaque from cancer patients undergoing chemotherapy and the relationship of these changes with mucositis: A pilot study

    PubMed Central

    Caldarazzo, Vito; Ottolenghi, Livia

    2015-01-01

    Background To assess changes in oral micro flora in dental plaque from cancer patients within 7 days of the first course of chemotherapy, and the relationship of the changes with mucositis. Material and Methods Thirty cancer patients, divided into a test group undergoing chemotherapy and a control group no undergoing chemotherapy, were enrolled in this pilot study. Oral micro flora were cultured from three samples of dental plaque at t0 (before chemotherapy), t1 (1 day after chemotherapy) and t2 (7 days after chemotherapy). Single and crossed descriptive analyses were used to establish prevalence, and the χ2 test was used to establish the statistical significance of the differences observed in distributions (significance level: P<0.05. Results In most patients (57%), oral micro flora consisted mainly of Gram-positive cocci, while the remaining 43% of the bacterial flora also had periodontal-pathogenic species. No Porphyromonas gingivalis appeared in the test group. Actinobacillus was the least frequently found bacterium among periodontal pathogens in the test group, while Fusobacterium nucleatum was the most frequently found. No significant differences were found in quantitative bacterial changes between t0, t1 and t2 in either the test or control groups, or between the two groups. According to World Health Organization scores, oral mucositis developed in 10 patients (66.6%) in the test group. Conclusions The results of this pilot study indicate that there were no changes in microflora in dental plaque in cancer patients within 7 days of the first course of chemotherapy. No correlations between oral mucositis and specific microorganisms were assessed. Key words: Oral microflora, dental plaque, cancer patients, chemotherapy. PMID:25662538

  10. High-Level Antimicrobial Efficacy of Representative Mediterranean Natural Plant Extracts against Oral Microorganisms

    PubMed Central

    Cecere, Manuel; Skaltsounis, Alexios Leandros; Argyropoulou, Aikaterini; Hellwig, Elmar; Aligiannis, Nektarios

    2014-01-01

    Nature is an unexplored reservoir of novel phytopharmaceuticals. Since biofilm-related oral diseases often correlate with antibiotic resistance, plant-derived antimicrobial agents could enhance existing treatment options. Therefore, the rationale of the present report was to examine the antimicrobial impact of Mediterranean natural extracts on oral microorganisms. Five different extracts from Olea europaea, mastic gum, and Inula viscosa were tested against ten bacteria and one Candida albicans strain. The extraction protocols were conducted according to established experimental procedures. Two antimicrobial assays—the minimum inhibitory concentration (MIC) assay and the minimum bactericidal concentration (MBC) assay—were applied. The screened extracts were found to be active against each of the tested microorganisms. O. europaea presented MIC and MBC ranges of 0.07–10.00 mg mL−1 and 0.60–10.00 mg mL−1, respectively. The mean MBC values for mastic gum and I. viscosa were 0.07–10.00 mg mL−1 and 0.15–10.00 mg mL−1, respectively. Extracts were less effective against C. albicans and exerted bactericidal effects at a concentration range of 0.07–5.00 mg mL−1 on strict anaerobic bacteria (Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Parvimonas micra). Ethyl acetate I. viscosa extract and total mastic extract showed considerable antimicrobial activity against oral microorganisms and could therefore be considered as alternative natural anti-infectious agents. PMID:25054150

  11. Antibacterial activity of sphingoid bases and fatty acids against Gram-positive and Gram-negative bacteria.

    PubMed

    Fischer, Carol L; Drake, David R; Dawson, Deborah V; Blanchette, Derek R; Brogden, Kim A; Wertz, Philip W

    2012-03-01

    There is growing evidence that the role of lipids in innate immunity is more important than previously realized. How lipids interact with bacteria to achieve a level of protection, however, is still poorly understood. To begin to address the mechanisms of antibacterial activity, we determined MICs and minimum bactericidal concentrations (MBCs) of lipids common to the skin and oral cavity--the sphingoid bases D-sphingosine, phytosphingosine, and dihydrosphingosine and the fatty acids sapienic acid and lauric acid--against four Gram-negative bacteria and seven Gram-positive bacteria. Exact Kruskal-Wallis tests of these values showed differences among lipid treatments (P < 0.0001) for each bacterial species except Serratia marcescens and Pseudomonas aeruginosa. D-sphingosine (MBC range, 0.3 to 19.6 μg/ml), dihydrosphingosine (MBC range, 0.6 to 39.1 μg/ml), and phytosphingosine (MBC range, 3.3 to 62.5 μg/ml) were active against all bacteria except S. marcescens and P. aeruginosa (MBC > 500 μg/ml). Sapienic acid (MBC range, 31.3 to 375.0 μg/ml) was active against Streptococcus sanguinis, Streptococcus mitis, and Fusobacterium nucleatum but not active against Escherichia coli, Staphylococcus aureus, S. marcescens, P. aeruginosa, Corynebacterium bovis, Corynebacterium striatum, and Corynebacterium jeikeium (MBC > 500 μg/ml). Lauric acid (MBC range, 6.8 to 375.0 μg/ml) was active against all bacteria except E. coli, S. marcescens, and P. aeruginosa (MBC > 500 μg/ml). Complete killing was achieved as early as 0.5 h for some lipids but took as long as 24 h for others. Hence, sphingoid bases and fatty acids have different antibacterial activities and may have potential for prophylactic or therapeutic intervention in infection.

  12. 2-hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum.

    PubMed

    Hans, M; Sievers, J; Müller, U; Bill, E; Vorholt, J A; Linder, D; Buckel, W

    1999-10-01

    Component D (HgdAB) of 2-hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum was purified to homogeneity. It is able to use component A from Acidaminococcus fermentans (HgdC) to initiate catalysis together with ATP, Mg2+ and a strong reducing agent such as Ti(III)citrate. Component D from C. symbiosum has a 6 x higher specific activity compared with that from A. fermentans and contains a second [4Fe-4S] cluster but the same amount of riboflavin 5'-phosphate (1.0 per heterodimeric enzyme, m = 100 kDa). Mössbauer spectroscopy revealed symmetric cube-type structures of the two [4Fe-4S]2+ clusters. EPR spectroscopy showed the resistance of the clusters to reducing agents, but detected a sharp signal at g = 2. 004 probably due to a stabilized flavin semiquinone. Three genes from C. symbiosum coding for components D (hgdA and hgdB) and A (hgdC) were cloned and sequenced. Primer extension experiments indicated that the genes are transcribed in the order hgdCAB from an operon only half the size of that from A. fermentans. Sequence comparisons detected a close relationship to the dehydratase system from A. fermentans and HgdA from Fusobacterium nucleatum, as well as to putative proteins of unknown function from Archaeoglobus fulgidus. Lower, but significant, identities were found with putative enzymes from several methanogenic Archaea and Escherichia coli, as well as with the mechanistically related benzoyl-CoA reductases from the Proteobacteria Rhodopseudomonas palustris and Thauera aromatica.

  13. Broad-Range PCR Coupled with Electrospray Ionization Time of Flight Mass Spectrometry for Detection of Bacteremia and Fungemia in Patients with Neutropenic Fever.

    PubMed

    Desmet, S; Maertens, J; Bueselinck, K; Lagrou, K

    2016-10-01

    Infection is an important complication in patients with hematologic malignancies or solid tumors undergoing intensive cytotoxic chemotherapy. In only 20 to 30% of the febrile neutropenic episodes, an infectious agent is detected by conventional cultures. In this prospective study, the performance of broad-range PCR coupled with electrospray ionization time of flight mass spectrometry (PCR/ESI-MS) technology was compared to conventional blood cultures (BC) in a consecutive series of samples from high-risk hematology patients. In 74 patients, BC and a whole-blood sample for PCR/ESI-MS (Iridica BAC BSI; Abbott, Carlsbad, CA, USA) were collected at the start of each febrile neutropenic episode and, in case of persistent fever, also at day 5. During 100 different febrile episodes, 105 blood samples were collected and analyzed by PCR/ESI-MS. There was evidence of a bloodstream infection (BSI) in 36/105 cases (34%), based on 14 cases with both PCR/ESI-MS and BC positivity, 17 cases with BC positivity only, and 5 cases with PCR/ESI-MS positivity only. The sensitivity of PCR/ESI-MS was 45%, specificity was 93%, and the negative predictive value was 80% compared to blood culture. PCR/ESI-MS detected definite pathogens (Fusobacterium nucleatum and Streptococcus pneumoniae) missed by BC, whereas it missed both Gram-negative and Gram-positive organisms detected by BC. PCR/ESI-MS testing detected additional microorganisms but showed a low sensitivity (45%) compared to BC in neutropenic patients. Our results indicate a lower concordance between BC and PCR/ESI-MS in the neutropenic population than what has been previously reported in other patient groups with normal white blood cell distribution, and a lower sensitivity than other PCR-based methods.

  14. IspH-RPS1 and IspH-UbiA: "Rosetta Stone" Proteins.

    PubMed

    Rao, Guodong; O'Dowd, Bing; Li, Jikun; Wang, Ke; Oldfield, Eric

    2015-12-01

    The protein IspH, (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMPPP) reductase, is an essential 4Fe-4S cluster-containing protein in the methylerythritol phosphate pathway for isoprenoid biosynthesis. Using a sequence similarity network we found that there are >400 IspH proteins that are about twice as large as most of the IspHs studied to date since their IspH domains are fused to either the ribosomal protein S1 (RPS1), or to a UbiA (4-hydroxybenzoate octaprenyltransferase)-like protein. Many of the IspH-RPS1 proteins are present in anaerobes found in the human gut and some, such as Clostridium botulinum, C. tetani and Fusobacterium nucleatum, are pathogens. The IspH-UbiAs are all found in sulfate-reducing anaerobes. The IspH domains in IspH-RPS1 are fused to 4 and in a few cases 6 tandem repeats in RPS1 that, in most organisms, bind to mRNA or form part of the bacterial ribosome. Mutants in which the four RPS1 domains were sequentially eliminated had similar IspH activity as wild-type protein, indicating they are not essential for IspH catalysis. Overall, the results are of interest since they represent the first isolation of a catalytically active IspH-RPS1, as well as the identification of IspH-UbiA hybrids, two "Rosetta stone" proteins that are likely to be functionally related-IspH producing the isoprenoids required for a UbiA-like prenyl transferase; the IspH-RPS1 hybrids, perhaps, being involved in the stringent response or as Fe/O2 sensors.

  15. Effect of an oxygenating agent on oral bacteria in vitro and on dental plaque composition in healthy young adults

    PubMed Central

    Fernandez y Mostajo, Mercedes; van der Reijden, Wil A.; Buijs, Mark J.; Beertsen, Wouter; van der Weijden, Fridus; Crielaard, Wim; Zaura, Egija

    2014-01-01

    Oral bacteria live in symbiosis with the host. Therefore, when mouthwashes are indicated, selective inhibition of taxa contributing to disease is preferred instead of broad-spectrum antimicrobials. The potential selectivity of an oxygenating mouthwash, Ardox-X® (AX), has not been assessed. The aim of this study was to determine the antimicrobial potential of AX and the effects of a twice-daily oral rinse on dental plaque composition. Material and methods: In vitro, 16 oral bacterial strains were tested using agar diffusion susceptibility, minimum inhibitory and minimum bactericidal concentration tests. A pilot clinical study was performed with 25 healthy volunteers. Clinical assessments and microbiological sampling of supragingival plaque were performed at 1 month before the experiment (Pre-exp), at the start of the experiment (Baseline) and after the one-week experimental period (Post-exp). During the experiment individuals used AX mouthwash twice daily in absence of other oral hygiene measures. The microbiological composition of plaque was assessed by 16S rRNA gene amplicon sequencing. Results: AX showed high inter-species variation in microbial growth inhibition. The tested Prevotella strains and Fusobacterium nucleatum showed the highest sensitivity, while streptococci and Lactobacillus acidophilus were most resistant to AX. Plaque scores at Pre-exp and Baseline visits did not differ significantly (p = 0.193), nor did the microbial composition of plaque. During a period of 7-days non-brushing but twice daily rinsing plaque scores increased from 2.21 (0.31) at Baseline to 2.43 (0.39) Post-exp. A significant microbial shift in composition was observed: genus Streptococcus and Veillonella increased while Corynebacterium, Haemophilus, Leptotrichia, Cardiobacterium and Capnocytophaga decreased (p ≤ 0.001). Conclusion: AX has the potential for selective inhibition of oral bacteria. The shift in oral microbiome after 1 week of rinsing deserves further research

  16. An investigation of the presence of specific anaerobic species in necrotic primary teeth.

    PubMed

    Gomes, Genara Brum; Sarkis-Onofre, Rafael; Bonow, Maria Laura Menezes; Etges, Adriana; Jacinto, Rogério Castilho

    2013-01-01

    Different microbial identification methods have shown that the microbial community profiles in endodontic infections are diverse and assorted. The aim of this study was to evaluate the frequency of selected endodontic pathogens in the pulp chambers (PCs) and root canals (RCs) of infected primary teeth using PCR methods. Paired PC and RC samples were collected from 15 subjects and analyzed by PCR for the presence of Filifactor alocis, Fusobacterium nucleatum, Parvimonas micra, Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Prevotella tannerae, Tanerella forsythia, Treponema denticola, and Treponema socranskii. The frequency of each species was determined in the PC and RC of each case. The species most frequently detected in PCs were P. nigrescens (86.7%), P. gingivalis (73.3%), and F. alocis (73.3%). Of the PC samples, 13.3% contained P. micra and T. denticola, and 6.7% contained T. forsythia. The species most frequently detected in RCs were P. gingivalis (100%) and P. nigrescens (93.3%). P. tannerae, P. micra, and T. denticola were found in 40% of the RC samples; T. forsythia was found in 26.7% of the RC samples. The "red complex", which comprises P. gingivalis, T. denticola, and T. forsythia, was not found in the PC of any tooth but was found in 30% of the RC samples. The detection of P. nigrescens in the PC was statistically associated with the presence of P. nigrescens in the RC (p = 0.04). The results suggest high heterogeneity among the samples, even among those from the same subject.

  17. Pathogenic bacterial species associated with endodontic infection evade innate immune control by disabling neutrophils.

    PubMed

    Matsui, Aritsune; Jin, Jun-O; Johnston, Christopher D; Yamazaki, Hajime; Houri-Haddad, Yael; Rittling, Susan R

    2014-10-01

    Endodontic infections, in which oral bacteria access the tooth pulp chamber, are common and do not resolve once established. To investigate the effects of these infections on the innate immune response, we established a mouse subcutaneous chamber model, where a mixture of four oral pathogens commonly associated with these infections (endodontic pathogens [EP]), i.e., Fusobacterium nucleatum, Streptococcus intermedius, Parvimonas micra, and Prevotella intermedia, was inoculated into subcutaneously implanted titanium chambers. Cells that infiltrated the chamber after these infections were primarily neutrophils; however, these neutrophils were unable to control the infection. Infection with a nonpathogenic oral bacterial species, Streptococcus mitis, resulted in well-controlled infection, with bacterial numbers reduced by 4 to 5 log units after 7 days. Propidium iodide (PI) staining of the chamber neutrophils identified three distinct populations: neutrophils from EP-infected chambers were intermediate in PI staining, while cells in chambers from mice infected with S. mitis were PI positive (apoptotic) or negative (live). Strikingly, neutrophils from EP-infected chambers were severely impaired in their ability to phagocytose and to generate reactive oxygen species in vitro after removal from the chamber compared to cells from S. mitis-infected chambers. The mechanism of neutrophil impairment was necrotic cell death as determined by morphological analyses. P. intermedia alone could induce a similar neutrophil phenotype. We conclude that the endodontic pathogens, particularly P. intermedia, can efficiently disable and kill infiltrating neutrophils, allowing these infections to become established. These results can help explain the persistence of endodontic infections and demonstrate a new virulence mechanism associated with P. intermedia.

  18. Microbial diversity of the supra- and subgingival biofilm of healthy individuals after brushing with chlorhexidine- or silver-coated toothbrush bristles.

    PubMed

    do Nascimento, Cássio; Paulo, Diana Ferreira; Pita, Murillo Sucena; Pedrazzi, Vinícius; de Albuquerque Junior, Rubens Ferreira

    2015-02-01

    Nanoparticulate silver has recently been reported as an effective antimicrobial agent. The aim of this clinical study was to investigate the potential changes on the oral microbiota of healthy individuals after controlled brushing with chlorhexidine- or silver-coated toothbrush bristles. Twenty-four healthy participants were enrolled in this investigation and randomly submitted to 3 interventions. All the participants received, in a crossover format, the following toothbrushing interventions: (i) chlorhexidine-coated bristles, (ii) silver-coated bristles, and (iii) conventional toothbrush (Control). All the interventions had a duration of 30 days. The DNA checkerboard hybridization method was used to identify and quantify up to 43 microbial species colonizing the supra- and subgingival biofilm. The supragingival samples presented higher genome counts than the subgingival samples (p < 0.0001). The total genome counts from the Control group showed the highest values, followed by the silver and chlorhexidine groups (p < 0.0001). After 4 weeks of brushing, the silver-coated and chlorhexidine-coated bristles were capable of reducing or maintaining lower levels of the bacterial counts of the putative periodontal pathogens Tanerella forsythia, Treponema denticola, and Porphyromonas gingivalis. Other major periodontal pathogens, such as Prevotella intermedia, Fusobacterium nucleatum, Prevotella nigrescens, and Parvimonas micra, were also detected at lower levels. The toothbrush bristles impregnated with silver nanoparticles reduced the total and individual genome count in the supra- and subgingival biofilm after 4 weeks of brushing. Chlorhexidine was not effective in reducing the total genome counts in both supra- or subgingival biofilm after 4 weeks of brushing. Chlorhexidine reduced the individual genome counts in the supragingival biofilm for most of the target species, including putative periodontal pathogens.

  19. Development of a Cavity Disinfectant Containing Antibacterial Monomer MDPB.

    PubMed

    Hirose, N; Kitagawa, R; Kitagawa, H; Maezono, H; Mine, A; Hayashi, M; Haapasalo, M; Imazato, S

    2016-12-01

    An experimental cavity disinfectant (ACC) that is intended to be used for various direct and indirect restorations was prepared by adding an antibacterial monomer 12-methacryloyloxydodecylpyridinum bromide (MDPB) at 5% into 80% ethanol. The antibacterial effectiveness of ACC and its influences on the bonding abilities of resin cements were investigated. To examine the antibacterial activity of unpolymerized MDPB, the minimum inhibitory and bactericidal concentrations (MIC and MBC) were determined for Streptococcus mutans, Lactobacillus casei, Actinomyces naeslundii, Parvimonas micra, Enterococcus faecalis, Fusobacterium nucleatum, and Porphyromonas gingivalis Antibacterial activities of ACC and the commercial cavity disinfectant containing 2% chlorhexidine and ethanol (CPS) were evaluated by agar disk diffusion tests through 7 bacterial species and by MIC and MBC measurement for S. mutans The effects of ACC and CPS to kill bacteria in dentinal tubules were compared with an S. mutans-infected dentin model. Shear bond strength tests were used to examine the influences of ACC on the dentin-bonding abilities of a self-adhesive resin cement and a dual-cure resin cement used with a primer. Unpolymerized MDPB showed strong antibacterial activity against 7 oral bacteria. ACC produced inhibition zones against all bacterial species similar to CPS. For ACC and CPS, the MIC value for S. mutans was identical, and the MBC was similar with only a 1-step dilution difference (1:2). Treatment of infected dentin with ACC resulted in significantly greater bactericidal effects than CPS (P < 0.05, analysis of variance and Tukey's honest significant difference test). ACC showed no negative influences on the bonding abilities to dentin for both resin cements, while CPS reduced the bond strength of the self-adhesive resin cement (P < 0.05). This study clarified that the experimental cavity disinfectant containing 5% MDPB is more effective in vitro than the commercially available

  20. Effect of estradiol on planktonic growth, coaggregation, and biofilm formation of the Prevotella intermedia group bacteria.

    PubMed

    Fteita, Dareen; Könönen, Eija; Söderling, Eva; Gürsoy, Ulvi Kahraman

    2014-06-01

    Alterations in the quantity and quality of biofilms at gingival margin are considered to play a role in the initiation and development of pregnancy-related gingivitis. Prevotella intermedia sensu lato is able to consume estradiol, the major sex hormone secreted during pregnancy, in the absence of vitamin K. The aim of the study was to examine the effect of estradiol on the planktonic growth, coaggregation, polysaccharide production, and biofilm formation of the P. intermedia group bacteria, namely P. intermedia, Prevotella nigrescens, and Prevotella pallens. In all experiments, the type strain (ATCC) and a clinical strain (AHN) of P. intermedia, P. nigrescens, and P. pallens were incubated with the concentrations of 0, 30, 90, and 120 nmol/L of estradiol. Planktonic growth was assessed by means of the colony forming unit method, while coaggregation and biofilm formation were assessed by spectrophotometric methods. In the determination of protein and polysaccharide levels, the Bradford and phenol-sulfuric acid methods were used, respectively. P. pallens AHN 9283 and P. nigrescens ATCC 33563 increased their numbers at planktonic stage with increasing estradiol concentrations. In 48-h biofilm tests, elevated protein levels were found for both strains of P. intermedia, and the strains P. nigrescens ATCC 33563 and P. pallens AHN 9283 in the presence of estradiol. The P. intermedia strains also increased the levels of polysaccharide formation in the biofilm. Coaggregation of the P. intermedia group organisms with Fusobacterium nucleatum was enhanced only in P. intermedia AHN 8290. In conclusion, our in vitro experiments indicate that estradiol regulates planktonic growth, coaggregation, polysaccharide production, and biofilm formation characteristics of P. intermedia, P. nigrescens, and P. pallens differently. These results may, at least partly, explain the differences seen in their contribution to the pathogenesis of pregnancy-related gingivitis.

  1. Shifts in Campylobacter species abundance may reflect general microbial community shifts in periodontitis progression

    PubMed Central

    Henne, Karsten; Fuchs, Felix; Kruth, Sebastian; Horz, Hans-Peter; Conrads, Georg

    2014-01-01

    Background Oral Campylobacter species have been found to be associated with periodontitis progression. While the etiological significance of Campylobacter rectus is quite established, the association of C. gracilis, C. concisus, and C. curvus with health or disease remains contradictory. Objectives This study hypothesizes that the proportion of species within the Campylobacter genus rather than the absolute abundance of a single species is a suitable indicator for periodontitis progression. Design Subgingival plaque from 90 periodontitis patients and gingival sulcus fluid of 32 healthy individuals were subjected to a newly developed nested PCR approach, in which all Campylobacter spp. were amplified simultaneously. The resulting mixture of 16S-rRNA-gene-amplicons were separated by single-stranded conformation polymorphism (SSCP) gel electrophoresis, followed by sequencing and identification of excised bands and relative quantification of band intensities. In all samples, the abundance of selected periodontitis marker species was determined based on DNA hybridization on a microarray. Results The highly prevalent Campylobacter community was composed of varying proportions of C. rectus, C. gracilis, C. concisus, and C. curvus. Cluster analysis based on SSCP-banding pattern resulted in distinct groups which in turn coincided with significant differences in abundance of established periodontitis marker species (Tannerella forsythia, Porphyromonas gingivalis, and Fusobacterium nucleatum) and progression. Conclusions The shift in the Campylobacter community composition seems to display the general microbial community shift during clinical progression in a simplified manner. The focus on members of the Campylobacter in this study suggests that this genus can be an indicator of ecological changes in the subgingival oral microflora. PMID:25412608

  2. Bacterial community development in experimental gingivitis.

    PubMed

    Kistler, James O; Booth, Veronica; Bradshaw, David J; Wade, William G

    2013-01-01

    Current knowledge of the microbial composition of dental plaque in early gingivitis is based largely on microscopy and cultural methods, which do not provide a comprehensive description of oral microbial communities. This study used 454-pyrosequencing of the V1-V3 region of 16S rRNA genes (approximately 500 bp), and bacterial culture, to characterize the composition of plaque during the transition from periodontal health to gingivitis. A total of 20 healthy volunteers abstained from oral hygiene for two weeks, allowing plaque to accumulate and gingivitis to develop. Plaque samples were analyzed at baseline, and after one and two weeks. In addition, plaque samples from 20 chronic periodontitis patients were analyzed for cross-sectional comparison to the experimental gingivitis cohort. All of the healthy volunteers developed gingivitis after two weeks. Pyrosequencing yielded a final total of 344,267 sequences after filtering, with a mean length of 354 bases, that were clustered into an average of 299 species-level Operational Taxonomic Units (OTUs) per sample. Principal coordinates analysis (PCoA) plots revealed significant shifts in the bacterial community structure of plaque as gingivitis was induced, and community diversity increased significantly after two weeks. Changes in the relative abundance of OTUs during the transition from health to gingivitis were correlated to bleeding on probing (BoP) scores and resulted in the identification of new health- and gingivitis-associated taxa. Comparison of the healthy volunteers to the periodontitis patients also confirmed the association of a number of putative periodontal pathogens with chronic periodontitis. Taxa associated with gingivitis included Fusobacterium nucleatum subsp. polymorphum, Lachnospiraceae [G-2] sp. HOT100, Lautropia sp. HOTA94, and Prevotella oulorum, whilst Rothia dentocariosa was associated with periodontal health. Further study of these taxa is warranted and may lead to new therapeutic approaches

  3. Extensive Identification of Bacterial Riboflavin Transporters and Their Distribution across Bacterial Species

    PubMed Central

    Merino, Enrique; Bonomi, Hernán Ruy; Goldbaum, Fernando Alberto; García-Angulo, Víctor Antonio

    2015-01-01

    Riboflavin, the precursor for the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide, is an essential metabolite in all organisms. While the functions for de novo riboflavin biosynthesis and riboflavin import may coexist in bacteria, the extent of this co-occurrence is undetermined. The RibM, RibN, RfuABCD and the energy-coupling factor-RibU bacterial riboflavin transporters have been experimentally characterized. In addition, ImpX, RfnT and RibXY are proposed as riboflavin transporters based on positional clustering with riboflavin biosynthetic pathway (RBP) genes or conservation of the FMN riboswitch regulatory element. Here, we searched for the FMN riboswitch in bacterial genomes to identify genes encoding riboflavin transporters and assessed their distribution among bacteria. Two new putative riboflavin transporters were identified: RibZ in Clostridium and RibV in Mesoplasma florum. Trans-complementation of an Escherichia coli riboflavin auxotroph strain confirmed the riboflavin transport activity of RibZ from Clostridium difficile, RibXY from Chloroflexus aurantiacus, ImpX from Fusobacterium nucleatum and RfnT from Ochrobactrum anthropi. The analysis of the genomic distribution of all known bacterial riboflavin transporters revealed that most occur in species possessing the RBP and that some bacteria may even encode functional riboflavin transporters from two different families. Our results indicate that some species possess ancestral riboflavin transporters, while others possess transporters that appear to have evolved recently. Moreover, our data suggest that unidentified riboflavin transporters also exist. The present study doubles the number of experimentally characterized riboflavin transporters and suggests a specific, non-accessory role for these proteins in riboflavin-prototrophic bacteria. PMID:25938806

  4. Pathogenic Bacterial Species Associated with Endodontic Infection Evade Innate Immune Control by Disabling Neutrophils

    PubMed Central

    Matsui, Aritsune; Jin, Jun-O; Johnston, Christopher D.; Yamazaki, Hajime; Houri-Haddad, Yael

    2014-01-01

    Endodontic infections, in which oral bacteria access the tooth pulp chamber, are common and do not resolve once established. To investigate the effects of these infections on the innate immune response, we established a mouse subcutaneous chamber model, where a mixture of four oral pathogens commonly associated with these infections (endodontic pathogens [EP]), i.e., Fusobacterium nucleatum, Streptococcus intermedius, Parvimonas micra, and Prevotella intermedia, was inoculated into subcutaneously implanted titanium chambers. Cells that infiltrated the chamber after these infections were primarily neutrophils; however, these neutrophils were unable to control the infection. Infection with a nonpathogenic oral bacterial species, Streptococcus mitis, resulted in well-controlled infection, with bacterial numbers reduced by 4 to 5 log units after 7 days. Propidium iodide (PI) staining of the chamber neutrophils identified three distinct populations: neutrophils from EP-infected chambers were intermediate in PI staining, while cells in chambers from mice infected with S. mitis were PI positive (apoptotic) or negative (live). Strikingly, neutrophils from EP-infected chambers were severely impaired in their ability to phagocytose and to generate reactive oxygen species in vitro after removal from the chamber compared to cells from S. mitis-infected chambers. The mechanism of neutrophil impairment was necrotic cell death as determined by morphological analyses. P. intermedia alone could induce a similar neutrophil phenotype. We conclude that the endodontic pathogens, particularly P. intermedia, can efficiently disable and kill infiltrating neutrophils, allowing these infections to become established. These results can help explain the persistence of endodontic infections and demonstrate a new virulence mechanism associated with P. intermedia. PMID:25024367

  5. Distinct polymicrobial populations in a chronic foot ulcer with implications for diagnostics and anti-infective therapy

    PubMed Central

    2014-01-01

    Background Polymicrobial infections caused by combinations of different bacteria are being detected with an increasing frequency. The evidence of such complex infections is being revealed through the use of novel molecular and culture-independent methods. Considerable progress has been made in the last decade regarding the diagnostic application of such molecular techniques. In particular, 16S rDNA-based sequencing and even metagenomic analyses have been successfully used to study the microbial diversity in ecosystems and human microbiota. Here, we utilized denaturing high-performance liquid chromatography (DHPLC) as a diagnostic tool for identifying different bacterial species in complex clinical samples of a patient with a chronic foot ulcer. Case presentation A 45-year-old female suffered from a chronic 5x5cm large plantar ulcer located in the posterior calcaneal area with subcutaneous tissue infection and osteomyelitis. The chronic ulcer developed over a period of 8 years. Culture and DHPLC revealed a distinct and location-dependent polymicrobial infection of the ulcer. The analysis of a superficial biopsy revealed a mixture of Staphylococcus aureus, Proteus vulgaris, and Fusobacterium nucleatum, whereas the tissue-deep biopsy harbored a mixture of four different bacterial species, namely Gemella morbillorum, Porphyromonas asaccharolytica, Bacteroides fragilis, and Arcanobacterium haemolyticum. Conclusions This clinical case highlights the difficulties in assessing polymicrobial infections where a mixture of fastidious, rapid and slow growing bacteria as well as anaerobes exists as structured communities within the tissue architecture of chronic wound infections. The diagnosis of this multilayered polymicrobial infection led to a microbe-adapted antibiotic therapy, targeting the polymicrobial nature of this infection in addition to a standard local wound treatment. However, a complete wound closure could not be achieved due to the long-lasting extensive

  6. Characterization of Bacteroides forsythus isolates.

    PubMed Central

    Takemoto, T; Kurihara, H; Dahlen, G

    1997-01-01

    Fifteen Bacteroides forsythus strains freshly isolated from patients with periodontitis were used together with three collection strains and one type strain for characterization of growth on various media; determination of enzymatic profiles, antibiotic susceptibility profiles, 16S rRNA ribotypes, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) outer membrane protein profiles, and pathogenicity; and gas chromatography analysis by using a wound chamber model in rabbits. All strains were stimulated by N-acetylmuramic acid, while one strain needed a further supplement such as yeast extract for optimal growth. All strains showed trypsin-like activity. While 10 different ribotypes were found, the SDS-PAGE profiles revealed similar patterns for all strains. All strains were sensitive to penicillin G (MICs, <0.5 microg/ml), ampicillin (MICs, <1.0 microg/ml), amoxicillin (MICs, <0.38 microg/ml), metronidazole (MICs, <0.005 microg/ml), tetracycline (MICs, <0.19 microg/ml), doxycycline (MICs, 0.05 microg/ml), erythromycin (MICs, <0.4 microg/ml), and clindamycin (MICs, <0.016 microg/ml), while they were less sensitive to ciprofloxacin (MICs, <4 microg/ml). B. forsythus did not cause abscess formation by monoinoculation. B. forsythus coinoculated with Fusobacterium nucleatum ATCC 10953 caused abscess formation in 75% of rabbits, while it caused abscess formation in 100% of rabbits when it was coinoculated with Porphyromonas gingivalis FDC 381. In the case of the latter combination, four of six rabbits died of sepsis after 6 to 7 days, and P. gingivalis and B. forsythus were recovered from the heart blood at a proportion of 10:1. B. forsythus strains were highly virulent and invasive in combination with P. gingivalis. PMID:9163447

  7. Periodontal pathogens invade gingiva and aortic adventitia and elicit inflammasome activation in αvβ6 integrin-deficient mice.

    PubMed

    Velsko, Irina M; Chukkapalli, Sasanka S; Rivera-Kweh, Mercedes F; Zheng, Donghang; Aukhil, Ikramuddin; Lucas, Alexandra R; Larjava, Hannu; Kesavalu, Lakshmyya

    2015-12-01

    The American Heart Association supports an association between periodontal diseases and atherosclerosis but not a causal association. This study explores the use of the integrin β6(-/-) mouse model to study the causality. We investigated the ability of a polymicrobial consortium of Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum to colonize the periodontium and induce local and systemic inflammatory responses. Polymicrobially infected Itgβ6(-/-) mice demonstrate greater susceptibility to gingival colonization/infection, with severe gingival inflammation, apical migration of the junctional epithelium, periodontal pocket formation, alveolar bone resorption, osteoclast activation, bacterial invasion of the gingiva, a greater propensity for the bacteria to disseminate hematogenously, and a strong splenic T cell cytokine response. Levels of atherosclerosis risk factors, including serum nitric oxide, oxidized low-density lipoprotein, serum amyloid A, and lipid peroxidation, were significantly altered by polybacterial infection, demonstrating an enhanced potential for atherosclerotic plaque progression. Aortic gene expression revealed significant alterations in specific Toll-like receptor (TLR) and nucleotide-binding domain- and leucine-rich-repeat-containing receptor (NLR) pathway genes in response to periodontal bacterial infection. Histomorphometry of the aorta demonstrated larger atherosclerotic plaques in Itgβ6(-/-) mice than in wild-type (WT) mice but no significant difference in atherosclerotic plaque size between mice with polybacterial infection and mice with sham infection. Fluorescence in situ hybridization demonstrated active invasion of the aortic adventitial layer by P. gingivalis. Our observations suggest that polybacterial infection elicits distinct aortic TLR and inflammasome signaling and significantly increases local aortic oxidative stress. These results are the first to demonstrate the mechanism of

  8. Multi-centre evaluation of mass spectrometric identification of anaerobic bacteria using the VITEK® MS system.

    PubMed

    Garner, O; Mochon, A; Branda, J; Burnham, C-A; Bythrow, M; Ferraro, M; Ginocchio, C; Jennemann, R; Manji, R; Procop, G W; Richter, S; Rychert, J; Sercia, L; Westblade, L; Lewinski, M

    2014-04-01

    Accurate and timely identification of anaerobic bacteria is critical to successful treatment. Classic phenotypic methods for identification require long turnaround times and can exhibit poor species level identification. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an identification method that can provide rapid identification of anaerobes. We present a multi-centre study assessing the clinical performance of the VITEK(®) MS in the identification of anaerobic bacteria. Five different test sites analysed a collection of 651 unique anaerobic isolates comprising 11 different genera. Multiple species were included for several of the genera. Briefly, anaerobic isolates were applied directly to a well of a target plate. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry. Mass spectra results were generated with the VITEK(®) MS, and the comparative spectral analysis and organism identification were determined using the VITEK(®) MS database 2.0. Results were confirmed by 16S rRNA gene sequencing. Of the 651 isolates analysed, 91.2% (594/651) exhibited the correct species identification. An additional eight isolates were correctly identified to genus level, raising the rate of identification to 92.5%. Genus-level identification consisted of Actinomyces, Bacteroides and Prevotella species. Fusobacterium nucleatum, Actinomyces neuii and Bacteroides uniformis were notable for an increased percentage of no-identification results compared with the other anaerobes tested. VITEK(®) MS identification of clinically relevant anaerobes is highly accurate and represents a dramatic improvement over other phenotypic methods in accuracy and turnaround time.

  9. Characterization of the transduction pathway involved in c-fos and c-jun expression induced by Aggregatibacter actinomycetemcomitans lipopolysaccharides in human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Castillo-Alemán, Ramiro

    2008-11-01

    Periodontal disease is an inflammatory disease caused by infection with oral bacteria that results in tooth exfoliation. Lipopolysaccharides (LPS) are a major component of the outer membrane of Gram-negative microorganisms and are involved in the inflammatory response. c-fos and c-jun are involved in pathological conditions such as inflammatory disorders. Inflammatory signaling cascades leading to c-fos activation in human gingival fibroblasts (HGFs) are not well characterized. Thus, we have investigated the kinase pathways involved in c-fos and c-jun activation induced by LPS in human gingival fibroblasts. LPS promoted a dose- and time-dependent increase in c-fos transcription. Phosphoinositide-phospholipase C inhibitor (U-73122), protein kinase A inhibitor (H89), MEK1 inhibitor (PD 98,059), p38 inhibitor (SB203580), and tyrosine kinase inhibitors (genistein and herbimycin) attenuated the LPS effect, while the PI-3 K inhibitor (Wortmannin) had no effect on LPS-induced c-fos transcription. Protein kinase C inhibitors (Ro 31-8220, calphostin C, staurosporine, and chelerythrine chloride) also inhibited LPS-induced c-fos transcription. However, long-term treatment (24 -h) with the PKC activator tetradecanoyl phorbol-13-acetate (PMA) had no significant effect on LPS-induced transcription in HGFs. We also found an increase in c-jun expression in HGF stimulated with LPS. In addition, experiments using pharmacological inhibitors of individual mitogen-activated protein kinases (MAPK) and of protein kinase C (PKC) revealed that p38, ERK 1/2, and PKC are involved in LPS-induced c-jun expression. Our results indicate that LPS-induced c-fos and c-jun expressions are mediated by two different signaling pathways: one through phosphoinositide-phospholipase C via an upstream protein tyrosine kinase, which activates PKC isoforms that are insensitive to phorbol stress, and the second through activation of protein kinase A (PKA). Both kinases regulate the phosphorylation of the extracellular signal-regulated kinase ERK 1/2 and p38.

  10. 21 CFR 520.2260b - Sulfamethazine sustained-release boluses.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... rot) (Fusobacterium necrophorum), colibacillosis (bacterial scours) (Escherichia coli), calf... Fusobacterium necrophorum; colibacillosis (bacterial scours) caused by Escherichia coli; coccidiosis caused by....; colibacillosis (bacterial scours) caused by Escherichia coli; coccidiosis caused by Eimeria bovis and E....

  11. 21 CFR 520.2260b - Sulfamethazine sustained-release boluses.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... rot) (Fusobacterium necrophorum), colibacillosis (bacterial scours) (Escherichia coli), calf... Fusobacterium necrophorum; colibacillosis (bacterial scours) caused by Escherichia coli; coccidiosis caused by....; colibacillosis (bacterial scours) caused by Escherichia coli; coccidiosis caused by Eimeria bovis and E....

  12. 21 CFR 520.2260b - Sulfamethazine sustained-release boluses.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... rot) (Fusobacterium necrophorum), colibacillosis (bacterial scours) (Escherichia coli), calf... Fusobacterium necrophorum; colibacillosis (bacterial scours) caused by Escherichia coli; coccidiosis caused by....; colibacillosis (bacterial scours) caused by Escherichia coli; coccidiosis caused by Eimeria bovis and E....

  13. 21 CFR 520.2260b - Sulfamethazine sustained-release boluses.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... rot) (Fusobacterium necrophorum), colibacillosis (bacterial scours) (Escherichia coli), calf... Fusobacterium necrophorum; colibacillosis (bacterial scours) caused by Escherichia coli; coccidiosis caused by....; colibacillosis (bacterial scours) caused by Escherichia coli; coccidiosis caused by Eimeria bovis and E....

  14. 21 CFR 520.2260b - Sulfamethazine sustained-release boluses.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... rot) (Fusobacterium necrophorum), colibacillosis (bacterial scours) (Escherichia coli), calf... Fusobacterium necrophorum; colibacillosis (bacterial scours) caused by Escherichia coli; coccidiosis caused by....; colibacillosis (bacterial scours) caused by Escherichia coli; coccidiosis caused by Eimeria bovis and E....

  15. Oral bacterial DNA findings in pericardial fluid

    PubMed Central

    Louh