Effect of thymoquinone on Fusobacterium nucleatum­associated biofilm and inflammation.

Periodontitis affects oral tissues and induces systemic inflammation, which increases the risk of cardiovascular disease and metabolic syndrome. Subgingival plaque accumulation is a trigger of periodontitis. Fusobacterium nucleatum (FN) contributes to subgingival biofilm complexity by intercalating with early and late bacterial colonizers on tooth surfaces. In addition, inflammatory responses to FN are associated with the progression of periodontitis. Nigella sativa Lin. seed, which is known as black cumin (BC), has been used as a herbal medicine to treat ailments such as asthma and infectious diseases. The current study examined the inhibitory effect of BC oil and its active constituents, thymol (TM) and thymoquinone (TQ), on FN­associated biofilm and inflammation. FN­containing biofilms were prepared by co­cultivation with an early dental colonizer, Actinomyces naeslundii (AN). The stability and biomass of FN/AN dual species biofilms were significantly higher compared with FN alone. This effect was retained even with prefixed cells, indicating that FN/AN co­aggregation is mediated by physicochemical interactions with cell surface molecules. FN/AN biofilm formation was significantly inhibited by 0.1% TM or TQ. Confocal laser scanning microscopy indicated that treatment of preformed FN/AN biofilm with 0.01% of BC, TM or TQ significantly reduced biofilm thickness, and TQ demonstrated a cleansing effect equivalent to that of isopropyl methylphenol. TQ dose­dependently suppressed TNF­α production from a human monocytic cell line, THP­1 exposed to FN, yet showed no toxicity to THP­1 cells. These results indicated that oral hygiene care using TQ could reduce FN­associated biofilm and inflammation in gingival tissue.

A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris.

Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membrane-bound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the Δvkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria.

Molecular studies of the structural ecology of natural occlusal caries.

Microbiological studies of occlusal dental biofilms have hitherto been hampered by inaccessibility to the sampling site and demolition of the original biofilm architecture. This study shows for the first time the spatial distribution of bacterial taxa in vivo at various stages of occlusal caries, applying a molecular methodology involving preparation of embedded hard dental tissue slices for fluorescence in situ hybridization (FISH) and confocal microscopy. Eleven freshly extracted teeth were classified according to their occlusal caries status. The teeth were fixed, embedded, sectioned and decalcified before FISH was performed using oligonucleotide probes for selected abundant species/genera associated with occlusal caries including Streptococcus, Actinomyces, Veillonella, Fusobacterium, Lactobacillus and Bifidobacterium. The sites showed distinct differences in the bacterial composition between different ecological niches in occlusal caries. Biofilm observed along the entrance of fissures showed an inner layer of microorganisms organized in palisades often identified as Actinomyces, covered by a more loosely structured bacterial layer consisting of diverse genera, similar to supragingival biofilm. Biofilm within the fissure proper seemed less metabolically active, as judged by low fluorescence signal intensity and presence of material of non-bacterial origin. Bacterial invasion (often Lactobacillus and Bifidobacterium spp.) into the dentinal tubules was seen only at advanced stages of caries with manifest cavity formation. It is concluded that the molecular methodology represents a valuable supplement to previous methods for the study of microbial ecology in caries by allowing analysis of the structural composition of the undisturbed biofilm in caries lesions in vivo.

Actinomyces neuii: review of an unusual infectious agent.

Actinomyces neuii, a species first described in 1994, has proven to be an exception in this genus on account of its aerobic growth, microscopic morphology (no branching), and the types and location of infections. Abscesses and infected atheromas are the most frequent types of infections, followed by infected skin structures, endophthalmitis, and bacteremias, including endocarditis. They are most likely of endogenous origin. To date, approximately 100 cases have been recorded in the literature. Intra-abdominal and intrathoracic infections, however, have not yet been described, and cases of classical actinomycosis seem to be extremely rare. Prognosis has generally been good with antibiotic and/or surgical treatment. Susceptibility to antibiotics has paralleled that of other Actinomyces spp.

Impact of hydrodynamics on oral biofilm strength.

Mechanical removal of oral biofilms is ubiquitously accepted as the best way to prevent caries and periodontal diseases. Removal effectiveness strongly depends on biofilm strength. To investigate the influence of hydrodynamics on oral biofilm strength, we grew single- and multi-species biofilms of Streptococcus oralis J22, Actinomyces naeslundii TV14-J1, and full dental plaque at shear rates ranging from 0.1 to 50 1/sec and measured their compressive strength. Subsequently, biofilm architecture was evaluated by confocal laser scanning microscopy. Multi-species biofilms were stronger than single-species biofilms, with strength values ranging from 6 to 51 Pa and from 5 to 17 Pa, respectively. In response to increased hydrodynamic shear, biofilm strength decreased, and architecture changed from uniform carpet-like to more "fluffy" with higher thickness. S. oralis biofilms grown under variable shear of 7 and 50 1/sec possessed properties intermediate of those measured at the respective single shears.

Actinomyces naeslundii in initial dental biofilm formation.

The combined use of confocal laser scanning microscopy (CLSM) and fluorescent in situ hybridization (FISH) offers new opportunities for analysis of the spatial relationships and temporal changes of specific members of the microbiota of intact dental biofilms. The purpose of this study was to analyse the patterns of colonization and population dynamics of Actinomyces naeslundii compared to streptococci and other bacteria during the initial 48 h of biofilm formation in the oral cavity. Biofilms developed on standardized glass slabs mounted in intra-oral appliances worn by ten individuals for 6, 12, 24 and 48 h. The biofilms were subsequently labelled with probes against A. naeslundii (ACT476), streptococci (STR405) or all bacteria (EUB338), and were analysed by CLSM. Labelled bacteria were quantified by stereological tools. The results showed a notable increase in the number of streptococci and A. naeslundii over time, with a tendency towards a slower growth rate for A. naeslundii compared with streptococci. A. naeslundii was located mainly in the inner part of the multilayered biofilm, indicating that it is one of the species that attaches directly to the acquired pellicle. The participation of A. naeslundii in the initial stages of dental biofilm formation may have important ecological consequences.

Direct visualization of spatial and temporal patterns of antimicrobial action within model oral biofilms.

A microscopic method for noninvasively visualizing the action of an antimicrobial agent inside a biofilm was developed and applied to describe spatial and temporal patterns of mouthrinse activity on model oral biofilms. Three species biofilms of Streptococcus oralis, Streptococcus gordonii, and Actinomyces naeslundii were grown in glass capillary flow cells. Bacterial cells were stained with the fluorogenic esterase substrate Calcien AM (CAM). Loss of green fluorescence upon exposure to an antimicrobial formulation was subsequently imaged by time-lapse confocal laser scanning microscopy. When an antimicrobial mouthrinse containing chlorhexidine digluconate was administered, a gradual loss of green fluorescence was observed that began at the periphery of cell clusters where they adjoined the flowing bulk fluid and progressed inward over a time period of several minutes. Image analysis was performed to quantify a penetration velocity of 4 mum/min. An enzyme-based antimicrobial formulation led to a gradual, continually slowing loss of fluorescence in a pattern that was qualitatively different from the behavior observed with chlorhexidine. Ethanol at 11.6% had little effect on the biofilm. None of these treatments resulted in the removal of biomass from the biofilm. Most methods to measure or visualize antimicrobial action in biofilms are destructive. Spatial information is important because biofilms are known for their structural and physiological heterogeneity. The CAM staining technique has the potential to provide information about the rate of antimicrobial penetration, the presence of tolerant subpopulations, and the extent of biomass removal effected by a treatment.

Host-derived pentapeptide affecting adhesion, proliferation, and local pH in biofilm communities composed of Streptococcus and Actinomyces species.

Salivary proline-rich proteins (PRPs) attach commensal Actinomyces and Streptococcus species to teeth. Here, gel filtration, mass spectrometry and Edman degradation were applied to show the release of a pentapeptide, RGRPQ, from PRP-1 upon proteolysis by Streptococcus gordonii. Moreover, synthetic RGRPQ and derivatives were used to investigate associated innate properties and responsible motifs. The RGRPQ peptide increased 2.5-fold the growth rate of S. gordonii via a Q-dependent sequence motif and selectively stimulated oral colonization of this organism in a rat model in vivo. In contrast, the growth of Streptococcus mutans, implicated in caries, was not affected. While the entire RGRPQ sequence was required to block sucrose-induced pH-decrease by S. gordonii and S. mutans, the N-terminal Arg residue mediated the pH increase (i.e., ammonia production) by S. gordonii alone (which exhibits Arg catabolism to ammonia). Strains of commensal viridans streptococci exhibited PRP degradation and Arg catabolism, whereas cariogenic species did not. The RGRPQ peptide mediated via a differential Q-dependent sequence motif, adhesion inhibition, and desorption of PRP-1-binding strains of A. naeslundii genospecies 2 (5 of 10 strains) but not of S. gordonii (n=5). The inhibitable A. naeslundii strains alone displayed the same binding profile as S. gordonii to hybrid peptides terminating in RGRPQ or GQSPQ, derived from the middle or C-terminal segments of PRP-1. The present findings indicate the presence of a host-bacterium interaction in which a host peptide released by bacterial proteolysis affects key properties in biofilm formation.

Uso del DIU asociado a la infección secundaria por Actinomyces en tracto genital femenino / The use of IUD related to secondary infection by Actinomyces in the female genital tract

Nuestro objetivo principal es que los médicos tengan en mente que toda usuaria de DIU debe ser constantemente supervisada, pues se han reportado casos de actinomicosis pélvica en relación al DIU, en especial a los modelos plásticos (Asa de Lippes); el agente causante en la mayoría de casos es Actinomyces israelii, pero ahora también se ha encontrado A. naeslundii por las nuevas conductas sexuales (sexo oral). La evolución de la actinomicosis es lenta, si no se descubre a tiempo, invade varios órganos. Se deben recalcar la importancia de los métodos diagnósticos más eficaces como son: la inmunofluorescencia y la citología exfoliativa cervico-vaginal; pues se han reportado casos en la literatura de confusiones de la actinomicosis pélvica con una neoplasia maligna, lo que lleva a un manejo enteramente diferente de la enfermedad. El tratamiento adecuado es penicilina G y drenaje de los abscesos actinomicóticos

Our main objective is to encourage physicians in the constant supervision of all IUD users, because of reported cases of pelvic actinomycosis, specially related to plastic models (Lippes Handle); The most common causal agents are A. israelii and A. naeslundii, the latter specially related to new sexual behaviors (oral sex). The evolution of actinomycosis is slow, and if not diagnosed on time the microorganism invades several organs. The importance of efficient diagnostic tools such as inmmunofluorescence in cervico-vaginal smear must be insisted upon, specially in cases where actinomycosis was misdiagnosed as neoplasms, implying an entirely different treatment of the illness. The appropriate treatment is G penicillin and drainage of the actinomycotic abscesses

Actinomyces hongkongensis sp. nov. a novel Actinomyces species isolated from a patient with pelvic actinomycosis.

A bacterium was isolated from the pus of a patient with pelvic actinomycosis. The cells were strictly anaerobic, straight, non-sporulating, Gram-positive rods. It grows on sheep blood agar as non-haemolytic, pinpoint colonies after 24 hours of incubation at 37 degrees C in anaerobic environment. It is non-motile and does not produce catalase. 16S ribosomal RNA (rRNA) gene sequencing showed that there were 6.6% difference between the 16S rRNA gene sequence of the bacterium that of Actinomyces marimammalium (GenBank Accession no. AJ276405), a new species described in 2001, isolated from two seals and a porpoise. For these reasons a new species, Actinomyces hongkongensis sp. nov., is proposed, for which HKU8(T) is the type strain. Further studies should be performed to ascertain the potential of this bacterium to become an important cause of actinomycosis.

Surface characteristics and in vitro biofilm formation on glass ionomer and composite resin.

In the initial stages of dental plaque formation, early colonizing bacteria bind to receptor structures in the pellicle, a proteinaceous film formed instantly after cleaning of the tooth surface. Dental restorative materials with surface characteristics different from the tooth might affect pellicle formation and the ability of bacteria to colonize the oral cavity. In this study (i) roughness and chemical composition of glass ionomer and composite resin surfaces before and after polishing, and (ii) the adsorption of salivary proteins and bacterial adherence to the pellicle-coated surfaces were examined. Compared with unpolished composite resin, unpolished glass ionomer had higher surface roughness, contained more inorganic, positively charged components, collected more proteins, and promoted better bacterial adherence. Polishing had the most pronounced effect on the composite resin, giving an enlarged and a rougher surface with a more inorganic character. Polishing the composite resin also led to increased biofilm formation.

Esophageal actinomycosis in a patient with AIDS.

Actinomycosis has been rarely reported in patients with HIV/AIDS in contrast to other opportunistic and common pathogens. We report a case of esophageal ulcer disease, secondary to actinomycosis occurring in a patient with recurrent odynophagia. The diagnosis was made histologically only after repeated upper endoscopy with biopsies.

Actinomyces bowdenii sp. nov., isolated from canine and feline clinical specimens.

Four strains of a previously undescribed Actinomyces-like bacterium were isolated from canine and feline clinical specimens. Phenotypic studies indicated the strains were members of the genus Actinomyces, and most closely resembled Actinomyces viscosus serotype I and Actinomyces slackii. Comparative 16S rRNA gene sequencing studies demonstrated the unknown bacterium constitutes a new subline within a group of Actinomyces species, which includes Actinomyces bovis, the type species of the genus. Based on phylogenetic and phenotypic evidence it is proposed that the unknown bacterium be classified as Actinomyces bowdenii sp. nov. The type strain of Actinomyces bowdenii is CCUG 37421T.

Actinomycosis and other bronchopulmonary infections with bacterial granules.

Pulmonary infections with formation of bacterial granules are rare. We reviewed the clinical and pathologic data from 18 cases diagnosed using surgical specimens in our department during the last 10 years. Three clinicopathologic forms were observed: endobronchial infections complicating tuberculous sequelae or bronchiectases (n = 7), tumor-like lesions (n = 8), and diffuse pneumonia (n = 3). The two latter forms contrasted with the former by a male predominance, association with general debilitating conditions and inflammatory syndrome, and pathologically by smaller granules often located in parenchymal abscesses or excavations. The pathologic examination of the bacteria forming the granules permitted the diagnoses of actinomycosis (n = 10), botryomycosis (n = 7), or nocardiosis (n = 1). The latter case corresponded to an endobronchial infection. Both actinomycosis and botryomycosis were encountered in every clinicopathologic form. At present, pulmonary actinomycosis and related infections rarely seems to present with chest wall invasion. On the contrary, purely endobronchial forms represented a large proportion of our cases. Cultures are often difficult and the clinical appearance is not specific. However, pathologic examination with special stains must indicate the type of involved microorganism.

Experimental actinomycosis in mice induced by alginate gel particles containing Actinomyces israelii.

The present study aimed to make an animal model for investigating chronic infection. Bacterial cells of Actinomyces israelii (ATCC 10048) were packed in alginate gel particles and injected intra-peritoneally into BALB/c mice. Actinomycotic lesions were induced efficiently in 9 mice out of 12 after 3 or 9 weeks. Actinomycotic lesions were not produced by injecting a bacterial suspension of A. israelii except in one animal. Neither did injection of gel particles without bacteria induce lesions. Bacteria survived in the lesions for at least 9 weeks after the injection, and serum IgG levels against the bacteria were significantly elevated in the animals, indicating that the bacteria were protected from immunological elimination and activated humoral immunity in the animals. Histopathological observation of the lesion by staining revealed that the bacteria in the lesions were acid-fast and seemed to become resistant to phagocytosis. The bacterial masses were surrounded by inflammatory cells, including polymorphonuclear leukocytes and macrophages, and were separated from the host cells by a fringe-like structure similar to the capsular structure of natural sulfur granules. The present study indicated that the use of an alginate gel was effective in inducing actinomycotic lesions in mice.

The importance of fimbriae in the virulence and ecology of some oral bacteria.

Cumulative evidence indicates that bacterial adherence to mucosal and tooth surfaces as well as bacterial coaggregation are essential steps for colonization of various oral bacterial species. Bacterial fimbriae have been shown to play an important role in the interaction between bacteria and host cells or among bacterial cells. The properties of fimbriae from selected species of oral bacteria are discussed in terms of virulence traits and ecological significance. Among others, Porphyromonas gingivalis fimbriae have been most extensively studied. The fimbrial structure is composed of 41-kDa fimbrillin proteins. DNA sequencing of the fimbrillin gene (fimA) from nine strains of P. gingivalis suggests intraspecies variation in the structure of fimA, while retaining common immunochemical specificities. P. gingivalis fimbriae exhibit a wide variety of biological activities including immunogenicity, binding to various host proteins, stimulation of cytokine production and promotion of bone resorption, Actinobacillus actinomycetemcomitans also possesses fimbriae; however, little is known concerning their chemical, genetical, and biological properties. Fimbriae of Prevotella intermedia are shown to induce hemagglutination reaction, while those of Prevotella loescheii are found to cause coaggregation with other bacteria, i.e., Actinomyces viscosus and sanguis streptococci. Fimbriae from gram-positive oral bacteria such as oral Actinomyces and sanguis streptococci are described. These fimbriae may participate in coaggregation, binding to saliva-coated hydroxyapatite or glycoprotein of the surface layer of oral epithelial cells. Taken together, fimbriae are key components in cell-to-surface and cell-to-cell adherence of oral bacteria and pathogenesis of some oral and systemic diseases.