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Bacterial outer membrane vesicles (OMVs) are spherical lipid bilayer nanostructures released by bacteria that facilitate oral biofilm formation via cellular aggregation and intercellular communication. Recent studies have revealed that Capnocytophaga ochracea is one of the dominant members of oral biofilms; however, their potential for OMV production has yet to be investigated. This study demonstrated the biogenesis of OMVs in C. ochracea associated with the concentration of unsaturated fatty acids of phosphatidylinositol (PI) and characterized the size and protein profile of OMVs produced at growth phases. Transmission electron microscopy showed isolated spherical structures from cells stained with heavy metals, indicating the production of OMVs with a size ranging from 25 to 100 nm. Lipidome analysis revealed the presence of phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, and PI as the main lipids. Some unsaturated fatty acids of PI were present specifically in OMV and little in the outer membrane, suggesting that OMVs are generated from a specific region of the membrane through blebbing rather than a random process such as cell lysis. Furthermore, the lack of similar PI accumulation in the OMV of Porphyromonas gingivalis suggests that C. ochracea has a different biogenesis mechanism. The blebbing mechanism was further supported by higher OMV production occurring at the exponential phase in comparison to the stationary phase, where cell lysis is more likely to occur. Further, comparative protein profile of OMVs isolated under different growth phases may indicate that the OMV cargo does not largely vary with growth phases. The present study provides a basis for further understanding the roles of C. ochracea OMVs in oral biofilms as well as systemic diseases that C. ochracea involves.

Objective The human oral cavity harbors several bacteria. Among them, Capnocytophaga ochracea, a facultative anaerobe, is responsible for the early phase of dental plaque formation. In this phase, the tooth surface or tissue is exposed to various oxidative stresses. For colonization in the dental plaque phase, a response by hydrogen peroxide (H2O2)-sensing transcriptional regulators, such as OxyR, may be necessary. However, to date, no study has elucidated the role of OxyR protein in C. ochracea. Methods Insertional mutagenesis was used to create an oxyR mutant, and gene expression was evaluated by reverse transcription-polymerase chain reaction and quantitative real-time reverse transcription-polymerase chain reaction. Bacterial growth curves were generated by turbidity measurement, and the sensitivity of the oxyR mutant to H2O2 was assessed using the disc diffusion assay. Finally, a two-compartment system was used to assess biofilm formation. Results The oxyR mutant grew slower than the wild-type under anaerobic conditions. The agar diffusion assay revealed that the oxyR mutant had increased sensitivity to H2O2. The transcript levels of oxidative stress defense genes, sod, ahpC, and trx, were lower in the oxyR mutant than in the wild-type strain. The turbidity of C. ochracea, simultaneously co-cultured with Streptococcus gordonii, was lower than that observed under conditions of homotypic growth. Moreover, the percentage decrease in growth of the oxyR mutant was significantly higher than that of the wild-type. Conclusions These results show that OxyR in C. ochracea regulates adequate in vitro growth and escapes oxidative stress.

A novel Gram-negative, capnophilic, fusiform bacterium, designated strain ChDC OS43T, was isolated from a human refractory periapical abscess in the left mandibular second molar and was characterized by polyphasic taxonomic analysis. The 16S rRNA gene sequence revealed that the strain belongs to the genus Capnocytophaga, as it showed sequence similarities to Capnocytophaga ochracea ATCC 27872T (96.30%) and C. sputigena ATCC 33612T (96.16%). The prevalent fatty acids of strain ChDC OS43T were isoC15:0 (57.54%), C16:0 (5.93%), C16:0 3OH (5.72%), and C18:1 cis 9 (4.41%). The complete genome of strain ChDC OS43T was 3,412,686 bp, and the G+C content was 38.2 mol%. The average nucleotide identity (ANI) value between strain ChDC OS43T and C. ochracea ATCC 27872T or C. sputigena ATCC 33612T was >92.01%. The genome-to-genome distance (GGD) value between strain ChDC OS43T and C. ochracea ATCC 27872T or C. sputigena ATCC 33612T was 32.0 and 45.7%, respectively. Based on the results of phenotypic, chemotaxonomic, and phylogenetic analysis, strain ChDC OS43T (= KCOM 1579T = KCTC 5562T = KCCM 42841T = JCM 32133T) should be classified as the type strain of a novel species of genus Capnocytophaga, for which the name Capnocytophaga endodontalis sp. nov. is proposed.

As one of the most complex human-associated microbial habitats, the oral cavity harbors hundreds of bacteria. Halitosis is a prevalent oral condition that is typically caused by bacteria. The aim of this study was to analyze the microbial communities and predict functional profiles in supragingival plaque from healthy individuals and those with halitosis. Ten preschool children were enrolled in this study; five with halitosis and five without. Supragingival plaque was isolated from each participant and 16S rRNA gene pyrosequencing was used to identify the microbes present. Samples were primarily composed of Actinobacteria, Bacteroidetes, Proteobacteria, Firmicutes, Fusobacteria, and Candidate phylum TM7. The α and β diversity indices did not differ between healthy and halitosis subjects. Fifteen operational taxonomic units (OTUs) were identified with significantly different relative abundances between healthy and halitosis plaques, and included the phylotypes of Prevotella sp., Leptotrichia sp., Actinomyces sp., Porphyromonas sp., Selenomonas sp., Selenomonas noxia, and Capnocytophaga ochracea. We suggest that these OTUs are candidate halitosis-associated pathogens. Functional profiles were predicted using PICRUSt, and nine level-3 KEGG Orthology groups were significantly different. Hub modules of co-occurrence networks implied that microbes in halitosis dental plaque were more highly conserved than microbes of healthy individuals' plaque. Collectively, our data provide a background for the oral microbiota associated with halitosis from supragingival plaque, and help explain the etiology of halitosis.

Retrospective analysis has already shown correlation between severe Chronic Periodontitis (CP) cases with human papiloma virus (HPV). Hence, we aimed to explore deep-seated infected granulation tissue removed during periodontal flap surgery procedures for residential bacterial species between HPV+ and HVP- CP cases, which may serve as good predisposition marker for oral cancer. All CP-granulation samples showed the prominence ofFirmicutes, Proteobacteria, andBacteroidetesphyla with an abundance of gram negative anaerobes, exceptStreptococcus. In Beta diversity nonmetric multidimensional scaling plot, the random distribution of species was observed between HPV+ and HPV- CP granulation-samples. However, an abundance ofCapnocytophaga ochraceawas observed in HPV+ CP samples (pPorphyromonas endodontalis,Macellibacteroides fermentas,Treponema phagedenis, andCampylobacter rectusspecies were highly abundant in HPV- CP samples (p

Background Microbial flora in several organs of HIV-infected individuals have been characterized; however, the palatine tonsil bacteriome and mycobiome and their relationship with each other remain unclear. Determining the palatine tonsil microbiome may provide a better understanding of the pathogenesis of oral and systemic complications in HIV-infected individuals. We conducted a cross-sectional study to characterize the palatine tonsil microbiome in HIV-infected individuals. Results Palatine tonsillar swabs were collected from 46 HIV-infected and 20 HIV-uninfected individuals. The bacteriome and mycobiome were analyzed by amplicon sequencing using Illumina MiSeq. The palatine tonsil bacteriome of the HIV-infected individuals differed from that of HIV-uninfected individuals in terms of the decreased relative abundances of the commensal genera Neisseria and Haemophilus. At the species level, the relative abundances and presence of Capnocytophaga ochracea, Neisseria cinerea, and Selenomonas noxia were higher in the HIV-infected group than those in the HIV-uninfected group. In contrast, fungal diversity and composition did not differ significantly between the two groups. Microbial intercorrelation analysis revealed that Candida and Neisseria were negatively correlated with each other in the HIV-infected group. HIV immune status did not influence the palatine tonsil microbiome in the HIV-infected individuals. Conclusions HIV-infected individuals exhibit dysbiotic changes in their palatine tonsil bacteriome, independent of immunological status. Electronic supplementary material The online version of this article (10.1186/s12866-018-1274-9) contains supplementary material, which is available to authorized users.

Capnocytophaga ochracea is a Gram-negative, rod-shaped bacterium that demonstrates gliding motility when cultured on solid agar surfaces. C. ochracea possesses the ability to form biofilms; however, factors involved in biofilm formation by this bacterium are unclear. A type IX secretion system (T9SS) in Flavobacterium johnsoniae was shown to be involved in the transport of proteins (e.g., several adhesins) to the cell surface. Genes orthologous to those encoding T9SS proteins in F. johnsoniae have been identified in the genome of C. ochracea ; therefore, the T9SS may be involved in biofilm formation by C. ochracea . Here we constructed three ortholog-deficient C. ochracea mutants lacking sprB (which encodes a gliding motility adhesin) or gldK or sprT (which encode T9SS proteins in F. johnsoniae ). Gliding motility was lost in each mutant, suggesting that, in C. ochracea , the proteins encoded by sprB , gldK , and sprT are necessary for gliding motility, and SprB is transported to the cell surface by the T9SS. For the Δ gldK , Δ sprT , and Δ sprB strains, the amounts of crystal violet-associated biofilm, relative to wild-type values, were 49%, 34%, and 65%, respectively, at 48 h. Confocal laser scanning and scanning electron microscopy revealed that the biofilms formed by wild-type C. ochracea were denser and bacterial cells were closer together than in those formed by the mutant strains. Together, these results indicate that proteins exported by the T9SS are key elements of the gliding motility and biofilm formation of C. ochracea .

Gram-negative fusiform rods were detected in a blood culture obtained from a 63-year-old man who had been hospitalized for a long duration for severe heart failure. Although the organism could not be identified using a conventional method, it was finally identified as a bacterium of the Capnocytophaga ochracea group based on the results of biochemical testing, 16S rRNA sequencing and a matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. Although neutropenic patients with poor oral hygiene are exclusively vulnerable to Capnocytophaga bacteremia, this case was unique because such predisposing conditions were not noted. A multi-centered investigation is warranted for a better understanding of this clinically rare, but potentially pathogenic organism.

Objective Presently, a large number of individuals consider their companion animals as family members and have close contact with them in daily life. The purpose of the present study was to analyze the distribution of periodontopathic bacterial species in oral specimens taken from dogs and their owners. Design Dental plaque specimens were collected from 66 dogs and 81 members of 64 families who came to an animal clinic or dog training school in Okayama, Japan, in 2011. Bacterial DNA was extracted from each specimen and PCR analyses using primers specific for 11 periodontopathic species, Porphyromonas gingivalis , Porphyromonas gulae , Treponema denticola , Tannerella forsythia , Capnocytophaga ochracea , Capnocytophaga sputigena , Prevotella intermedia , Prevotella nigrescens , Aggregatibacter actinomycetemcomitans , Campylobacter rectus , and Eikenella corrodens were performed. Results P. gulae (71.2%), T. forsythia (77.3%), and C. rectus (66.7%) were frequently found in the dogs, whereas the detection rates of those species in humans were less frequent at 16.0%, 30.9%, and 21.0%, respectively. P. gulae was identified in 13 human subjects and each of their dogs was also positive for the species. Furthermore, E. corrodens and T. denticola in specimens obtained from dogs were correlated with their presence in specimens from owners who had close contact with them. Conclusions These results suggest that several periodontopathic species could be transmitted between humans and their companion dogs, though the distribution of periodontopathic species in both is generally different.

In order to study the effects of sonicated extracts from Prevotella intermedia, Actinobacillus actinomycetemcomit-ans, Porphyromonas gingivalis, and other oral-related bacteria, as well as Escherichia coli on bone formation, clone MC3T3-E1 cells, which have retained osteoblastic activity, were cultured with various bacterial extracts. The addition of the sonicated extracts from Prevotella intermedia and Actinobacillus actinomycetemcomitans decreased the alkaline phosphatase activity in a dose-dependent fashion over the concentration range of 1– 1000 ng ml-1 compared with the control. By contrast, the sonicated extracts from other oral bacteria including Porphyromonas gingivalis, Capnocytophaga ochracea, Streptococcus milleri and Streptococcus sanguis, and Escherichia coli did not decrease the alkaline phosphatase activity even in the presence of 100 ng ml-1 protein. The addition of Prevotella intermedia and Actinobacillus actinomycetemcomitans extracts that had been treated with heat and trypsin to the cell cultures also inhibited alkaline phosphatase activity in the cells, suggesting that inhibitory factors are not proteinaceouS. Polymyxin B did not change the alkaline phosphatase activity in the cells treated with the extracts from Prevotella intermedia and Actinobacillus actinomycetemcomitans, suggesting that the inhibitory activity of the extracts is not lipopolysaccha-ride. The inhibitory effect of both extracts was observed in the molecular mass over 290 kDa eluted from Sephadex G-200 column. The inhibitory substances of Prevotella intermedia were partially purified and showed broad band with estimated molecular weight of 170–190 kDa by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis. These results indicate that Prevotella intermedia and Actinobacillus actinomycetemcomitans may play an important role in inhibiting bone formation as well as in stimulating bone resorption.

Bacteria of the genus Capnocytophaga form part of the resident oral flora in children and adults. They are recognized as opportunistic pathogens of various extra-oral infections. The significance of individual species in periodontal and extra-oral diseases is unclear, due to the inability of conventional phenotypic tests to identify clinical isolates to species level. Aiming at a clear distinction between species, we undertook a phylogenetic study of a collection of 102 Capnocytophaga strains including 62 oral isolates from children and 40 reference strains from oral and extra-oral infections representing the five known, human, oral Capnocytophaga species. The phylogeny was estimated on the basis of multilocus enzyme electrophoresis (MLEE) of 12 intracellular, housekeeping enzymes and by partial 16S rRNA gene sequencing, and was compared to phenotypic characteristics. The clustering profiles in the MLEE and sequence-based dendrograms were concordant and allowed identification of isolates to species level, based on co-clustering with reference strains. The study confirmed Capnocytophaga ochracea and Capnocytophaga sputigena as separate species, and underlined the problems of distinguishing between them by conventional phenotypic tests. The presence of two distinct clusters of oral isolates from children indicated the existence of novel species, supported by analysis of near-full-length 16S rRNA gene sequences and by DNA-DNA hybridization results. One cluster of weakly saccharolytic isolates without the ability to ferment sucrose is proposed as Capnocytophaga leadbetteri sp. nov. (type strain AHN8855(T)=CCUG 51857(T)=NCTC 13375(T)). Another cluster not phenotypically distinguishable from C. ochracea and C. sputigena is designated Capnocytophaga genospecies AHN8471 (represented by strain AHN8471=CCUG 51856=NCTC 13374).

Capnocytophaga ochracea is present in the dental plaque biofilm of patients with periodontitis. Biofilm cells change their phenotype through quorum sensing in response to fluctuations in cell-population density. Quorum sensing is mediated by auto-inducers (AIs). AI-2 is involved in intercellular signaling, and production of its distant precursor is catalyzed by LuxS, an enzyme involved in the activated methyl cycle. Our aim was to clarify the role of LuxS in biofilm formation by C. ochracea. Two luxS-deficient mutants, TmAI2 and LKT7, were constructed from C. ochracea ATCC 27872 by homologous recombination. The mutants produced significantly less AI-2 than the wild type. The growth rates of these mutants were similar to that of the wild-type in both undiluted Tryptic soy broth and 0.5 × Tryptic soy broth. However, according to crystal violet staining, they produced significantly less biofilm than the wild type. Confocal laser scanning microscopy and scanning electron microscopy showed that the biofilm of the TmAI2 strain had a rougher structure than that of the wild type. Complementation of TmAI-2 with extrinsic AI-2 from the culture supernatant of wild-type strain did not restore biofilm formation by the TmAI2 strain, but complementation of LKT7 strain with luxS partially restored biofilm formation. These results indicate that LuxS is involved in biofilm formation by C. ochracea, and that the attenuation of biofilm formation by the mutants is likely caused by a defect in the activated methyl cycle rather than by a loss of AI-2.

Summary Objective We analyzed the distribution of 10 periodontal bacteria species ( Porphyromonas gingivalis, Tannerella forsythensis, Prevotella intermedia, Prevotella nigrescens, Campylobacter rectus, Eikenella corrodens, Actinobacillus actinomycetemcomitans, Capnocytophaga ochracea , Capnocytophaga sputigena, and Treponema denticola ) in children, and then compared their distribution in those children and their mothers, with special attention given to three of the species known as the red complex ( P. gingivalis , T. forsythensis , and T. denticola ) whose presence has been shown to be associated with conditions related to periodontal diseases. Methods One hundred thirteen pairs of children and their mothers were randomly selected from patients treated at the Pedodontic Clinic of Osaka University Dental Hospital. Saliva samples were taken at the second visit prior to receiving professional tooth brushing instruction. Genomic DNA was extracted from each saliva sample, followed by a polymerase chain reaction assay with species-specific sets of primers. Results A. actinomycetemcomitans was the most frequently detected species in the mothers, followed by C. sputigena , P. gingivalis , and T. forsythensis , while C. sputigena had the highest detection rate, followed by A. actinomycetemcomitans and T. denticola in the children. The detection rate of the red complex species in children whose mothers possessed the same species was significantly higher than in those whose mothers did not possess them. Conclusions Our results indicate a correlation between the presence of periodontal bacteria in children and their mothers, while the presence of red complex bacteria in children was highly associated with that in their mothers.

Toll-like receptors (TLRs) 2 and 4 have recently been identified as possible signal transducers for various bacterial ligands. To investigate the roles of TLRs in the recognition of periodontopathic bacteria by the innate immune system, a Chinese hamster ovary (CHO) nuclear factor-κB (NF-κB)-dependent reporter cell line, 7.7, which is defective in both TLR2- and TLR4-dependent signaling pathways was transfected with human CD14 and TLRs. When the transfectants were exposed to freeze-dried periodontopathic bacteria,Actinobacillus actinomycetemcomitans,Porphyromonas gingivalis,Capnocytophaga ochracea, andFusobacterium nucleatum, and a non-oral bacterium,Escherichia coli, all species of the bacteria induced NF-κB-dependent CD25 expression in 7.7/huTLR2 cells. Although freeze-driedA.actinomycetemcomitans,F.nucleatum, andE.colialso induced CD25 expression in 7.7/huTLR4 cells, freeze-driedP.gingivalisdid not. Similarly, lipopolysaccharides (LPS) extracted fromA.actinomycetemcomitans,F.nucleatum, andE.coliinduced CD25 expression in 7.7/huTLR4 cells, but LPS fromP.gingivalisandC.ochraceadid not. Furthermore, LPS fromP.gingivalisandC.ochraceaattenuated CD25 expression in 7.7/huTLR4 cells induced by repurified LPS fromE.coli. LPS fromP.gingivalisandC.ochraceaalso inhibited the secretion of interleukin-6 (IL-6) from U373 cells, the secretion of IL-1β from human peripheral blood mononuclear cells, and ICAM-1 expression in human gingival fibroblasts induced by repurified LPS fromE.coli. These findings indicated that LPS fromP.gingivalisandC.ochraceaworked as antagonists for human TLR4. The antagonistic activity of LPS from these periodontopathic bacteria may be associated with the etiology of periodontal diseases.

Introduction This clinical study aimed to determine the microbiological profile resistant to different intracanal medications in primary endodontic infections by using both microbiological culture and the checkerboard DNA-DNA hybridization technique. Methods Twenty primarily infected root canals were selected and then instrumented before being randomly divided into 2 groups according to the intracanal medications: calcium hydroxide (Ca[OH] 2 ) or Ca(OH) 2 + chlorhexidine (CHX). Samples were collected before and after root canal procedures, which consisted in submitting them to microbiological culture and processing them for checkerboard DNA-DNA hybridization. Results No differences were found between the Ca(OH) 2 (99.98%) and Ca(OH) 2 + CHX groups (99.76%) regarding the median percentage values for the reduction of cultivable bacteria. The most frequently detected species were Capnocytophaga ochracea (70%) and Fusobacterium nucleatum ssp. vincentii (70%) in the initial samples. After instrumentation, the most frequently detected species were E. faecium (60%). After root canal treatments using either Ca(OH) 2 or Ca(OH) 2 + CHX as intracanal medications, the most frequently detected species were F. nucleatum ssp. vincentii (90%) and Enterococcus faecium (40%), respectively. Both treatments significantly decreased the number of bacterial species compared with the initial sample. However, this reduction was significantly greater in the Ca(OH) 2 + CHX group ( P P Conclusions The use of Ca(OH) 2 associated with CHX as an intracanal medication showed better results by acting on gram-positive and gram-negative microorganisms although such an action to eradicate enterococci should also be sought.

Down's syndrome (DS) patients often develop severe early-onset marginal periodontitis in early adulthood; however, there is little information available on the microbiology of DS periodontitis.Subgingival plaque specimens were taken from 67 DS young adults and 41 age-matched systemically healthy individuals with mental disabilities (MD). The prevalence of 10 possible periodontopathic bacterial species, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Bacteroides forsythus, Treponema denticola, Prevotella intermedia, Prevotella nigrescens, Capnocytophaga ochracea, Capnocytophaga sputigena, Campylobacter rectus, and Eikenella corrodens, were investigated in their subgingival plaque samples using a polymerase chain reaction method. The detection of P. gingivalis fimA genotypes was also performed in P. gingivalis-positive samples.Although DS subjects generally develop an earlier and more extensive periodontal breakdown than those with MD, no significant differences were observed in the bacterial profiles. The profiles of subjects with periodontitis were significant in DS, but not in MD. The prevalence of P. gingivalis, B. forsythus, and P. intermedia were significant in the DS periodontitis group, compared to DS gingivitis group. Moreover, the occurrence of P. gingivalis with the type II fimA gene was significantly related to periodontitis in both DS and MD, with odds ratios of 6.32 and 12.03, respectively.These results suggest that early-onset periodontitis in DS is mainly due to the more susceptible host for the causative microbial agents including P. gingivalis with type II fimA.

Two hundred and thirty colonies from the caecal contents of six rabbits were picked up and, after a 2-d incubation, were microscopically characterized using Gram staining. Large Gram-negative (34%) and small Gram-negative (30%) irregular rods, Gram-negative (27%) and Gram-positive (8%) cocci were found. Eleven isolates (Bacteroides ovatus (6 strains), B. thetaiotamicron, B. caccae, B. stercoris, B. capillosus and Capnocytophaga ochracea) were identified using commercial tests for measuring their catalase activity, metabolite production, etc., and testing their growth in 20% bile. Bacteria belonging to the genus Bacteroides were demonstrated to be the principal pectinolytic organisms in the rabbit caecum.

The purpose of this study was to detect the presence of Capnocytophaga sputigena, C. ochracea, and C. gingivalis in plaque samples from the toothbrushes of 122 children, using a polymerase chain reaction (PCR) method. The subjects were 25, 85, and 12 children with healthy gingiva, gingivitis, and periodontitis, respectively, ranging in age from 2-12 years old. Plaque samples were collected from all erupted tooth sites using a sterile toothbrush. The mean amount of DNA recovered from the samples was approximately 19.3 microg, which was deemed sufficient for performing a PCR-based survey. C. sputigena prevalence in healthy, gingivitis, and periodontitis subjects was 48.0%, 36.5% and 25.0%, respectively, that for C. ochracea was 100%, 89.4%, and 50.0%, respectively, and that for C. gingivalis was 96.0%, 84.7%, and 75.0%, respectively. The lowest age of positive subjects was approximately 2 years. Our results showed that C. sputigena was moderately prevalent, whereas C. ochracea and C. gingivalis were commonly detected in the oral cavities of the tested children, suggesting that all of these species become established in the early years.

Background, aims: The purpose of the present study was to compare and relate the microbial composition of supra and subgingival plaque in 23 adult periodontitis subjects (mean age 51±14 years). Methods: A total of 1,170 samples of supra and subgingival plaque were collected from the mesial aspect of every tooth (up to 28 supra and 28 subgingival samples) from each subject and evaluated for the presence and levels of 40 bacterial taxa using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Clinical assessments including dichotomous measures of gingival redness, bleeding on probing, plaque accumulation and suppuration, as well as duplicate measures of pocket depth and attachment level, were made at 6 sites per tooth. The counts (levels), % DNA probe count (proportion) and % of sites colonized (prevalence) of each species in supra and separately in subgingival plaque were computed for each subject. Significance of differences between supra and subgingival plaque for each species was sought using the Wilcoxon signed ranks test and adjusted for multiple comparisons. Results: All 40 taxa were detected in both supra and subgingival plaque. Actinomyces species were the most prevalent taxa in both habitats. 75 to 100% of supra and 62 to 100% of subgingival sites were colonized by at least one of the 5 Actinomyces species. Supragingival samples exhibited significantly higher counts of Actinomyces naeslundii genospecies 1, Actinomyces israelii, Actinomyces odontolyticus, Neisseria mucosa, Streptococcus gordonii, Capnocytophaga ochracea and Capnocytophaga sputigena when compared with mean counts in subgingival samples taken from the same tooth surfaces. Subgingival plaque samples presented significantly higher counts of Prevotella nigrescens, Prevotella intermedia, Bacteroides forsythus and Porphyromonas gingivalis. Subgingival samples exhibited a significantly higher proportion of “red” and “orange complex” species, while supragingival plaque exhibited higher proportions of “green” and “purple” complex species as well as Actinomyces species. Suspected periodontal pathogens could be detected in supragingival plaque from sites where subgingival samples were negative for the same species. Conclusions: The data indicate that supragingival plaque can harbor putative periodontal pathogens, suggesting a possible role of this environment as a reservoir of such species for the spread or reinfection of subgingival sites.

It is widely known that individuals with Down syndrome (DS) often develop severe early-onset periodontal diseases. In this study, we examined the prevalence of periodontopathic bacteria in DS children to determine if specific pathogens are acquired in their childhood.The subjects were 60 DS children (2 to 13 years old, 5 in each age bracket) and 60 age-matched controls. Ten pathogens, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Bacteroides forsythus, Treponema denticola, Prevotella intermedia, P nigrescens, Capnocytophaga ochracea, C. sputigena, Campyrobacter rectus, and Eikenella corrodens were surveyed in subgingival plaque samples using a polymerase chain reaction. Periodontal status was evaluated by probing depth, bleeding on probing, and gingival index.No significant difference in periodontal status was observed between the DS and control groups, however, all of the pathogens were detected with greater frequency in the DS children. B. forsythus, T. denticola, P. nigrescens, and C. rectus were significantly prevalent throughout all age brackets of the DS children (P0.01 or 0.05). The occurrence of P. gingivalis was also significant in the DS subjects over 5 years old. A cluster analysis of the microbial profiles of the DS subjects showed that gingivitis severity was associated with increased varieties of the harboring pathogens and the distribution of P. gingivalis.These results suggest that various periodontopathogens can colonize in the very early childhood of DS patients and maturation of subgingival components, including P. gingivalis, plays an important role in the initiation of gingival inflammation.

We examined the levels of anti-thymocyte/T lymphocyte autoantibody (ATA) in the serum of mice injected intraperitoneally with lipopolysaccharides (LPS) from periodontopathic bacteria; Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Capnocytophaga ochracea, and non-oral Escherichia coli. All of the LPS induced IgM-ATA. Among these, LPS from C. ochracea induced the highest level of IgM-ATA, whereas that of P. gingivalis induced the lowest. The peritoneal T lymphocytes of mice injected with LPS were bound by IgM-ATA. Peritoneal B-1 (CD5+B) cells stimulated by each LPS produced much more IgM-ATA than splenic B-2 (CD5-B) cells, suggesting that B-1 cells might be responsible for the production of these antibodies. Serum of mice injected with C. ochracea and F. nucleatum LPS showed cytotoxicity against thymocytes in the presence of rabbit complements. Binding and cytotoxicity were confirmed by IgM purified from serum of the mice injected with C. ochracea LPS. Furthermore, serum of mice treated with C. ochracea, F. nucleatum or A. actinomycetemcomitans LPS inhibited the proliferation of thymocytes. However, purified IgM from the serum of mice treated with C. ochracea LPS failed to produce the same inhibition. Our results suggest that LPS from certain species of periodontopathic bacteria can induce IgM-ATA in the serum and these antibodies may modulate the local immune network in periodontal tissues.

Capnocytophaga, one of the genera of oral bacteria, has been implicated in the pathogenesis of several diseases, including endocarditis, septicaemia and disorders of the oral cavity such as abscesses and periodontal disease. This study examined sonic extracts (SE) of Capnocytophaga strains for their ability to alter lymphocyte function. The SE of tested Capnocytophaga caused dose-dependent suppression of spleen cells in response to mitogen. This suppressive effect was heat-labile and sensitive to the proteolytic enzyme pronase E. The suppressive factor (SF) was purified from SE of C. ochrasea by a combination of ultrogel-AcA34, high-pressure liquid DEAE ion-exchange chromatography and hydroxyapatite columns, which revealed a single band of 14 kDa by SDS-PAGE. Rabbit anti-serum against the purified SF inhibited the immunosuppression induced by SE of C. ochracea with the recovery of lymphocyte proliferation.

Thirty-one strains of 23 gram-negative oral bacterial species were examined for dextran-degrading activity on agar plates containing blue dextran. One strain each of Capnocytophaga ochracea, Capnocytophaga sputigena, Prevotella loescheii, Prevotella melaninogenica and Prevotella oralis had detectable dextranase activity. The culture supernatants of P. melaninogenica and P. oralis cells contained dextranases of multiple sizes, but those of the other three species had a single size of enzyme. A 56-kDa dextranase was purified from the culture supernatant of P. oralis and the antiserum against the enzyme was prepared with a rabbit. The Ouchterlony test showed that the antibody reacted with the supernatants of both P. melaninogenica and P. oralis but not with the others. Dot-blot hybridization using the dextranase gene of Streptococcus mutans as a probe revealed that there was no significantly homologous sequence in the chromosomal DNA of the five species.

The purpose of this study was to examine the composition of subgingival plaque of 140 periodontal lesions in 35 patients with cardiovascular disorders who were administered nifedipine and manifested nifedipine-induced gingival overgrowth (GO). Age was inversely associated with the GO. Plaque index and bleeding index showed a significant association with GO, while nifedipine dosage and duration of nifedipine therapy were not found to be significant predictors of GO. The gingival inflammation as expressed in the logistic regression model by the interaction term color x tone was found to be significantly associated with the GO. Statistically significant differences between the groups of comparable probing depth and different degrees of GO were detected for Propionibacterium acnes, Capnocytophaga gingivalis, Capnocytophaga ochracea, Capnocytophaga sputigena, Bacteroides gracilis, Fusobacterium mortiferum, Fusobacterium nucleatum, Fusobacterium varium and Selenomonas sputigena in deep and enlarged lesions. Significantly more frequently isolated were the bacterial species Eubacterium alactolyticum, Campylobacter concisus, C. gingivalis, C. ochracea, C. sputigena, F. mortiferum, F. nucleatum, and F. varium from the more enlarged lesions (GO >3).

Fusobacterium nucleatum has been identified as significantly associated with sites with active periodontal disease and, as a group, the oral fusobacteria coaggregate with members of all oral bacteria genera tested. Monoclonal antibodies were prepared and used in conjunction with other potential inhibitors, such as simple sugars and amino acids, to characterize coaggregation interactions, of F. nucleatum PK1594. Four unique monoclonal antibodies, 5H11, 14C7, 19F2 and 29C12, were obtained by their ability to inhibit coaggregation of F. nucleatum PK1594 with Actinomyces israelii PK16. They were also capable of inhibiting other coaggregations including Streptococcus oralis H1, S. oralis J22, Capnocytophaga ochracea ATCC33596, Prevotella denticola PK1277 and Prevotella intermedia PK1511. All of these interactions were completely inhibited by N-acetylneuraminic acid. Neither N-acetylneuraminic acid nor monoclonal antibody 5H11 had any inhibitory effect on other F. nucleatum PK1594 interactions, including all galactose-inhibitable coaggregations. The results indicate that F. nucleatum PK1594 expresses upon its surface a distinct type of adhesin that mediates coaggregation interactions that are inhibited by N-acetylneuraminic acid.

The purpose of this investigation was to identify bacterial species present on or in crevicular epithelial cells in healthy and diseased sites using DNA probes. In order to achieve this aim, further improvements were made in the separation of unattached bacteria from those adherent to epithelial cells isolated from the human gingival crevice or periodontal pocket. Then the DNA probes were used to determine the prevalence of detectable DNA from 15 microbial species on or in crevicular epithelial cells. One sample was taken from a single subgingival site in each of 51 individuals ranging in age from 19 to 45 years. Samples were taken from 27 sites of clinically healthy subjects and 24 samples were taken from subjects having periodontally diseased sites. DNA-DNA hybridization indicated that a majority of epithelial cells from healthy sites (63%) were in contact with or harbored Streptococcus oralis. On the other hand, species such as Bacteroides forsythus, Prevotella intermedia, Capnocytophaga ochracea and Campylobacter rectus were more frequently detected in elevated numbers in periodontally diseased sites. Cluster analysis of the microbial profiles generally aggregated subjects with and without periodontitis into separate cluster groups. The cluster patterns suggest the possibility that microbial complexes will be, in part, determined by the receptors available on the epithelial cells.

The purpose of this study was to determine if endodontic bacterial act in vitro on human gingival fibroblast functions via extracellular products. The bacteria used were Prevotella nigrescens, Capnocytophaga ochracea, Peptostreptoccocus micros and Actinobacillus actinomycetemcomitans. Supernatants were collected from bacterial cultures at the beginning of the stationary phase when their density was similar. Toxins that inhibited fibroblast proliferation were found in all culture supernatants of Gram-positive or Gram-negative bacterial strains, except for Prev. nigrescens. The cytotoxicity of A. actinomycetemcomitans supernatant was about 1000 fold higher than the others. This supernatant diluted to 1/1000 led to total fibroblast growth inhibition whereas only 25% growth inhibition was obtained with Capn. ochracea and Pept. micros diluted to 1/10. Bacterial supernatant proteolytic activity was investigated in confluent fibroblast cultures that were incubated for 48 hr with each of the supernatants diluted to 1/2 except for A. actinomycetemcomitans supernatant diluted to 1/20. Indirect immunofluorescence studies of extracellular-matrix molecules, followed by immunoelectrophoretic analysis of extracts of whole-cell layers, demonstrated that only conditioned medium of Prev. nigrescens had a proteolytic activity capable of degrading the greater part of type I collagen and fibronectin fibres in the extracellular matrix.

International audience; OBJECTIVES: To determine macrolide-lincosamide-streptogramin (MLS) resistance determinants in the Capnocytophaga genus and to describe the prevalence of β-lactam resistance genes in human oral Capnocytophaga species. METHODS: Forty-eight Capnocytophaga isolates identified by analysis of 16S rRNA sequences were isolated from subgingival samples from 14 haematology patients (HPs), 11 periodontitis patients (PPs) and 17 healthy volunteers (HVs). MICs of β-lactam and MLS antibiotics were obtained for all isolates. blaCfxA, blaCSP-1 (encoding a new class A β-lactamase) and MLS resistance genes [erm(F), erm(B), erm(Q), erm(D), erm(C) and erm(A)] were evaluated using specific PCR and sequencing. RESULTS: In HVs, which had the lowest prevalence of β-lactamase-producing isolates in comparison with the other groups (16%; P

The degradation of human lactoferrin by putative periodontopathogenic bacteria was examined. Fragments of lactoferrin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measured by densitometry. The degradation of lactoferrin was more extensive by Porphyromonas gingivalis and Capnocytophaga sputigena, slow by Capnocytophaga ochracea, Actinobacillus actinomycetemcomitans and Prevotella intermedia, and very slow or absent by Prevotella nigrescens, Campylobacter rectus, Campylobacter sputorum, Fusobacterium nucleatum ssp. nucleatum, Capnocytophaga gingivalis, Bacteroides forsythus and Peptostreptococcus micros. All strains of P. gingivalis tested degraded lactoferrin. The degradation was sensitive to protease inhibitors, cystatin C and albumin. The degradation by C. sputigena was not affected by the protease inhibitors and the detected lactoferrin fragments exhibited electrophoretic mobilities similar to those ascribed to deglycosylated forms of lactoferrin. Furthermore a weak or absent reactivity of these fragments with sialic acid-specific lectin suggested that they are desialylated. The present data indicate that certain bacteria colonizing the periodontal pocket can degrade lactoferrin. The presence of other human proteins as specific inhibitors and/or as substrate competitors may counteract this degradation process.

A beta-lactamase gene ( cfxA3 , 966 bp) was isolated from a beta-lactam-resistant Capnocytophaga ochracea clinical isolate and amplified using primers from the cfxA gene of Bacteroides vulgatus . The MICs of third-generation cephalosporins were much higher than those of the transconjugant Escherichia coli strain. The deduced protein sequence, by comparison with CfxA2 of Prevotella intermedia , had a Y239D substitution and possessed the characteristics of a class A, group 2e beta-lactamase.

The bacterial genus Bartonella (Rochalimaea) includes emerging human pathogens with five recognized species. These are fastidious gram-negative bacteria, exhibiting few phenotypic characteristics and whose identification relies upon serotyping, cellular fatty acid analysis, and molecular typing. Most of the isolates have been recovered from the blood of patients, and three of the four pathogenic Bartonella species are associated with infectious endocarditis. We performed PCR-restriction fragment length polymorphism (RFLP) analysis of the blood culture bottle supernatant for the routine identification of Bartonella species among fastidious gram-negative bacteria. The amplification of the citrate-synthase gene with primers previously reported (R. L. Regnery, C. L. Spruill, and B. D. Plikaytis, J. Bacteriol. 173:1576-1589, 1991) yielded a 379-bp product from Bartonella species and a 382-bp product for Capnocytophaga ochracea but no product from any of the other 15 genotypically or phenotypically related species tested. We determined the sequences of the citrate-synthase gene-amplified products for Bartonella species and C. ochracea in order to predict the optimal restriction enzyme to be used in RFLP analysis. TaqI and AciI allowed identification of Bartonella species and C. ochracea. We propose that acridine orange and Gram staining, followed by PCR-RFLP analysis of the blood bottle supernatant, be included in the examination of blood samples from patients with suspected infectious endocarditis.

Introduction The aim of this study was to evaluate, by checkerboard DNA-DNA hybridization, the composition of the microbiota of primary endodontic infections in cases associated with exposed (n = 30) and unexposed (n = 30) pulp space. Methods Samples were collected by means of a #15 H-type file and 2 sterile paper points from 60 single-rooted teeth with necrotic pulp and periapical lesions. The presence, levels, and proportions of 40 bacterial species were determined by checkerboard DNA-DNA hybridization. Results The species found in higher counts (×10 5 ) in exposed pulp space cases were Eubacterium saburreum , Fusobacterium nucleatum ssp. vincentii , Tannerella forsythia , Enterococcus faecalis , Neisseria mucosa , Campylobacter gracilis , and Prevotella nigrescens , and in unexposed pulp space cases they were F. nucleatum ssp. vincentii , N. mucosa , E. faecalis , E. saburreum, C. gracilis , and Porphyromonas gingivalis . Counts of F. nucleatum ssp. vincentii , Campylobacter sputigena, Capnocytophaga showae, Treponema socrenskii, Porphyromonas endodontalis, Eikenella corrodens , and Capnocytophaga ochracea were significantly higher in unexposed pulp space cases ( P Conclusions The data of the present investigation suggested specific differences between the composition of the microbiota in cases with exposed and unexposed pulp space and an association between higher levels of some specific species and unexposed pulp space cases.

Several subgingival microorganisms were tested for their ability to utilize human immunoglobulin G (IgG) as a substrate for growth. This was done using a protein-free chemically defined medium, supplemented with IgG. Stimulation of growth was observed for Capnocytophaga ochracea, Porphyromonas asaccharolytica, Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, Prevotella oralis, Lactobacillus catenaforme and Streptococcus intermedius. Immunoelectrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a protein assay demonstrated that P. intermedia and P. endodontalis completely degraded the protein chains of IgG. Partial breakdown of IgG was observed for P. asaccharolytica and C. ochracea, whereas P. oralis cleaved the IgG heavy chain, yielding Fc and Fab fragments. All these bacteria utilized IgG as a substrate for growth. Binding studies using an enzyme-linked immunosorbent assay, revealed complete loss of in vitro antigen-antibody binding capacity after incubation of specific IgG with P. endodontalis and partial loss of binding with P. intermedia, P. gingivalis, C. ochracea or Fusobacterium nucleatum. Degradation or inactivation of IgG by oral bacteria is thought to be important in the causation of polymicrobial infections.

Previous studies have shown increased levels of proteolytic enzymes in affected periodontal sites. The aim of the present investigation was to evaluate the effect of proteolytic environments on the lysis and growth of selected oral bacteria associated with either healthy or diseased periodontal sites. The effect of trypsin, chymotrypsin and proleinase K on cell lysis was determined following incubation with bacteria, whereas the effect of the same proteolytic enzymes on bacterial growth was tested using a disc-plate technique. Overall, gram-positive bacteria appeared to be more resistant to lysis than gram-negative bacteria. The most susceptible bacteria were Actinomyces spp., Eubacterium saburreum, Prerotella intermedia, Capnocytophaga ochracea, Fitsobacterium nucleatum, Prevotella loescheii, Treponema clenticola and Actinobacilhts actinomycetemcomitans. The disc-plate procedure indicated that the growth of Actinomyces spp., E. sabwreum, C. ochracea, P. intermedia, P. loescheii, Porphyromonas gingivalis and T. dentieola were the most affected, more particularly by chymotrypsin and proteinase K. Interestingly, the growth of F. nucleatum was rather stimulated by proteolytic enzymes. The observations reported in this investigation indicate that specific and general proteolytic activities have the ability to lyse some oral bacterial species and to interfere with their growth. It is suggested that such effects could represent new mechanisms by which the bacterial ecology of subgingival sites may be affected.

The formation of dental plaque biofilm by specific Gram-negative rods and spirochetes plays an important role in the development of periodontal disease. The aim of this study was to characterize biofilm formation by Fusobacterium nucleatum and Capnocytophaga ochracea. Coaggregation between F. nucleatum and Capnocytophaga species was determined by visual assay. Biofilm formation was assessed by crystal violet staining. Enhancement of biofilm formation by F. nucleatum via soluble factor of C. ochracea was evaluated by addition of culture supernatant and a two-compartment separated co-culture system. Production of autoinducer-2 by the tested organisms was evaluated using Vibrio harveyi BB170. F. nucleatum strains coaggregated with C. ochracea ATCC 33596 or ONO-26 strains. Ethylenediamine tetraacetic acid, N-acetyl-d-galactosamine or lysine inhibited coaggregation. Heating or proteinase K treatment of F. nucleatum cells affected coaggregation, whereas the same treatment of C. ochracea cells did not. Co-culture of F. nucleatum with C. ochracea in the same well resulted in a statistically significant increase in biofilm formation. Enhancement of F. nucleatum biofilm formation by a soluble component of C. ochracea was observed using the two-compartment co-culture system (P < 0.05) and confirmed by addition of culture supernatant of C. ochracea (P < 0.01). The present findings indicate that induction of coaggregation and intracellular interaction by release of a diffusible molecule by C. ochracea play a significant role in the formation of biofilm by F. nucleatum and C. ochracea.

The ability of representative oral microorganisms to degrade biopolymers, such as collagen, chondroitin-4-sulphate, hyaluronic acid, heparin and proteins was investigated using both native and synthetic substrates. Porphyromonas gingivalis and Bacteroides heparinolyticus were the only species which had heparinase activity. Hydrolysis of hyaluronic acid and chondroitin-4-sulphate was detected in P. gingivalis irrespective of the method used. Both enzymes were largely cell associated and increased up to 120 h. Hyaluronic acid and chondroitin-4-sulphate degradation was detected in B. melaninogenicus, B. oralis, Fusobacterium nucleatum, Capnocytophaga ochracea and Veillonella parvula by a spectrophotometric assay but not by a plate method. Collagenase activity was mainly associated with P. gingivalis ; activity was largely extracellular and reached a maximum after 96 h. Several other species such as the black-pigmented bacteroides, F. nucleatum and C. ochracea showed very weak activity. All species tested except B. oralis possessed some proteolytic activity against gelatin, casein and skimmed milk. Fluorometnc and chromogenic substrates which contained an arginine residue were readily hydrolysed. Keywords: Oral flora; Hydrolytic enzymes; Periodontal disease; Gram-negative anaerobes.

The present study examined the IgG antibody activity in sera from a case:control population against whole cell proteins of Porphyromonas gingivalis and four other presumed periodontal pathogens: Bacteroides intermedius ; Capnocytophaga ochracea ; Wolinella recta ; Fusobacterium nucleatum . Whole cell enzyme-linked immunosorbent assays (ELISA) were performed to provide quantitative data on levels of IgG antibody and immunoblotting of whole cell lysates was carried out on 10 case:control pairs to identify the major individual antigens of each organism. For all organisms the mean level of IgG antibody activity against whole cells was higher in the cases compared to controls. For P. gingivalis the difference was highly significant ( p

Previous reports have indicated the association of periodontal diseases with elevated levels of serum immunoglobulin G (IgG) antibodies to periodontally relevant bacteria. Recent results from this laboratory suggest that enzymes proteolytic for immunoglobulins are important virulence factors of several periodontal bacteria. Specifically, enzymes from Porphyromonas (Bacteroides) gingivalis culture supernatant fluid (SF) cleaved human IgG (4 subclasses), IgA1 and IgA2, IgM, IgD and IgE. Proteolytic enzymes from Actinobacillus actinomycetemcomitans culture SF cleaved IgG, IgA and IgM. An enriched Ig proteolytic preparation from Capnocytophaga ochracea culture SF was shown to extensively cleave all 4 subclasses of human IgG. Extensive degradation of IgG and IgA in crevicular fluid samples on SDS-PAGE from periodontal disease sites of localized juvenile periodontitis (LJP) patients in comparison to little degradation in healthy sites indicated the potential role the proteolytic enzymes from periodontopathogenic bacteria may play in situ. Treatment of IgG with P. gingivalis, A. actinomycetemcomitans and C. ochracea SF resulted in similar patterns of degradation. LJP patients had significantly higher levels of IgG and IgA proteolytic activity in whole saliva than age-, sex-, and race-matched periodontal disease-free controls. However, not all of the proteolytic activity could be ascribed to bacterial proteases since neutrophils are also present in large numbers at diseased sites. Using similar techniques, lysates of neutrophils from healthy controls cleaved IgG, IgA and IgM. The observation of enhanced Ig cleavage activity in crevicular fluid and saliva in LJP patients suggest a role for Ig proteolytic enzymes in LJP.

A plasmid-encoded extended-spectrum TEM β-lactamase with a pI of 5.5 was detected in a Capnocytophaga ochracea clinical isolate. The bla gene was associated with a strong TEM-2 promoter and was derived from bla TEM-1a with a single-amino-acid substitution: Glu 104 →Lys, previously assigned to TEM-17, which is thus the first TEM β-lactamase to be reported in the phylum Flavobacter-Bacteroides .

The incidence of potential periodontal pathogens (Aggregatibacter actinomycetemcomitans, formerly Actinobacillus actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, Prevotella nigrescens, Prevotella intermedia and Capnocytophaga ochracea) was monitored in patients with chronic periodontitis and in healthy control subjects. Two types of studies were carried out in which the composition of the bacterial communities in different niches of the same oral cavity ecosystem was investigated. Fluctuation or at least pronounced quantitative changes in the incidence of individual species in time were documented in the long-term study as well as after the local administration of antibacterial drug Chlo-Site or Metronidazole. Even within two weeks, a turnover of the monitored bacteria in separate niches of the oral biotope can be detected. A relatively high incidence of the tested periopathogens in the clinically healthy teeth of patients implies that even the “healthy” niches in the periodontal biotope function as a dynamic reservoir of periopathogenic microorganisms. This should be kept in mind when a local application of antibacterial compounds is used in the therapy of periodontal disease.

Capnocytophaga ochracea (Prévot et al. 1956) Leadbetter et al. 1982 is the type species of the genus Capnocytophaga. It is of interest because of its location in the Flavobacteriaceae, a genomically not yet charted family within the order Flavobacteriales. The species grows as fusiform to rod shaped cells which tend to form clumps and are able to move by gliding. C. ochracea is known as a capnophilic (CO(2)-requiring) organism with the ability to grow under anaerobic as well as aerobic conditions (oxygen concentration larger than 15%), here only in the presence of 5% CO(2). Strain VPI 2845(T), the type strain of the species, is portrayed in this report as a gliding, Gram-negative bacterium, originally isolated from a human oral cavity. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence from the flavobacterial genus Capnocytophaga, and the 2,612,925 bp long single replicon genome with its 2193 protein-coding and 59 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

The present study was undertaken to evaluate the effect of Porphyromonas gingivalis outer membrane vesicles on the bactericidal activity of human serum. Human serum was pretreated with extracellular vesicles and then incubated with a cell suspension of Capnocytophaga ochracea. After 2 h at 37 degrees C, the percent viability of C. ochracea was determined by cultivation on blood agar plates. At a final concentration of 0.3 mg/ml, outer membrane vesicles completely inhibited the serum bactericidal activity against C. ochracea. Boiling the vesicles prevented this inhibition. However, partial inhibition of the serum lethal action was obtained when a higher concentration (1.5 mg/ml) of boiled vesicles was used, which indicates the involvement of both heat-labile and heat-stable components associated with vesicles. Combining vesicles at a suboptimal concentration (0.1 mg/ml) with a reducing agent brought back inhibition of the bactericidal activity, whereas combining vesicles at an optimal concentration (0.3 mg/ml) with a thiol-blocking reagent caused a restoration of the bactericidal activity. When a purified preparation of P. gingivalis lipopolysaccharides was used instead of vesicles, inhibition of the bactericidal activity was also observed. These results indicate that the lipopolysaccharides and the proteolytic enzyme(s) associated with P. gingivalis outer membrane vesicles are likely to represent the heat-stable and the heat-labile components, respectively. It is possible that outer membrane vesicles released by P. gingivalis protect other bacterial species from complement action, thus favoring the pathogenic process of periodontal disease.

Human polymorphonuclear leukocytes (PMN) were able to generate and release superoxide anions upon stimulation of Streptococcus mutans, Bacteroides gingivalis, and Capnocytophaga ochracea when incubated aerobically but not when incubated anaerobically. Lysozyme release and phagocytosis by PMN were independent of oxygen, and no difference between PMN incubated aerobically or anaerobically was observed (PMN stimulated by B. gingivalis released 7.6% total lysozyme when aerobic and 6.9% when anaerobic). There were variations in lysozyme release and phagocytosis for the three organisms, particularly for phagocytosis. B. gingivalis and C. ochracea yielded lower phagocytosis values than those for S. mutans, e.g., at 1 h 67% of the initial inoculum of S. mutans was phagocytosed (versus only 40% for B. gingivalis). Transmission electron microscopy showed that both S. mutans and B. gingivalis were internalized into classical phagolysosomes. In contrast, C. ochracea showed two forms of internalization; C. ochracea either formed a classical phagolysosome or was tightly bound in the cytoplasm with no surrounding cell membrane. Intracellular killing of S. mutans and C. ochracea was unaffected by anaerobiosis, but killing of C. ochracea was much lower than that of S. mutans (1 x 10(7) to 2 x 10(7) bacteria killed compared with 5.1 x 10(7) bacteria killed at 6 h). In contrast, a greater number of B. gingivalis was killed in the presence of oxygen (5.3 x 10(7) bacteria were killed when aerobically incubated and 1.9 x 10(7) bacteria were killed when anaerobically incubated). These results suggest that the ability to survive anaerobically may enable some bacteria to evade PMN killing; however, abnormal phagocytosis may represent a more efficient way to evade both oxygen-dependent and -independent killing mechanisms, leading to enhanced virulence of the organism.

Neutrophils play a major role in defending the periodontium against infection by oral, gram-negative, facultative bacteria, such as Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. We examined the sensitivity of these bacteria to a mixture of low-molecular-weight peptides and highly purified individual defensin peptides (HNP-1, HNP-2, and HNP-3) isolated from human neutrophils. Whereas the Capnocytophaga spp. strains were killed significantly by the mixed human neutrophil peptides, the A. actinomycetemcomitans and E. corrodens strains were resistant. Killing was attributable to the defensins. The bactericidal activities of purified defensins HNP-1 and HNP-2 were equal, and both of these activities were greater than HNP-3 activity against strains of Capnocytophaga sputigena and Capnocytophaga gingivalis. The strain of Capnocytophaga ochracea was more sensitive to defensin-mediated bactericidal activity than either C. sputigena or C. gingivalis was. The three human defensins were equipotent in killing C. ochracea. C. ochracea was killed under aerobic and anaerobic conditions and over a broad pH range. Killing was most effective under hypotonic conditions but also occurred at physiologic salt concentrations. We concluded that Capnocytophaga spp. are sensitive to oxygen-independent killing by human defensins. Additional studies will be required to identify other components that may equip human neutrophils to kill A. actinomycetemcomitans, E. corrodens, and other oral gram-negative bacteria.

A filter paper spot test with 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid as a substrate was used to study the prevalence of sialidase activity among gram-negative anaerobic and capnophilic bacteria. A total of 567 isolates representing four genera of obligate anaerobes and four genera of capnophilic organisms was tested. Sialidase activity was detected in 94% of 66 isolates from the Bacteroides fragilis group, 98% of 66 B. bivius isolates, and all isolates of the following species (number of isolates follows species name): B. capillosus, 4; B. levii, 2; B. denticola, 22; B. loescheii, 23; B. melaninogenicus, 32; B. forsythus, 44; and B. buccalis, 2. However, sialidase activity was detected in only 29% of 7 B. buccae isolates, 79% of 14 B. disiens isolates, and 55% of 11 B. oralis isolates. Sialidase activity was not detected among any of 13 isolates of B. gracilis, 12 isolates of B. ureolyticus, 61 isolates of B. intermedius, or 26 isolates of B. corporis. Porphyromonas (Bacteroides) asaccharolytica (20 isolates) and P. endodontalis (8 isolates) did not demonstrate sialidase activity, while 25 isolates of P. gingivalis were sialidase positive. Sialidase activity was found in 10 (100%) of 10 isolates of Capnocytophaga ochracea of C. sputigena but not in any of 4 C. gingivalis isolates. Other gram-negative anaerobic or capnophilic bacteria, including the following, were negative for sialidase activity: Fusobacterium nucleatum, 39 isolates; Wolinella recta, 19 isolates; Eikenella corrodens, 17 isolates; Haemophilus aphrophilus, 10 isolates; and Actinobacillus actinomycetemcomitans, 10 isolates. These data demonstrate sialidase activity in several species of the genera Bacteroides and Porphyromonas and suggest that this characteristic may be useful for identification.

The coaggregation of the Gram-negative microoganism Capnocytophaga ochracea ATCC 33596 with several Gram-positive bacteria, including streptococci, actinomyces and rothia, is mediated by rhamnose-sensitive adhesin(s). In the present study, MAbs against the C. ochracea adhesin(s) were prepared. These antibodies inhibited all the rhamnose-sensitive interactions, indicating that they recognize epitopes at or near the rhamnose binding site of the adhesin. The monoclonals served as probes in immunoblot analysis and recognized a polypeptide of Mr 155 K present in the wild-type organism but absent in a coaggregation defective mutant. It is concluded that the rhamnose-sensitive interactions of C. ochracea are mediated by a 155 K Mr polypeptide present on its outer surface.

The clinical and microbiological features of 7 cases of bacteremia due to Capnocytophaga (Capnocytophaga ochracea group) are reported. They were diagnosed during 1991-93 at three hospital clinics. Five patients were10 years old and all had hematological disorders, 4 acute lymphoblastic leukemia and 1 each had aplastic anemia, non-Hodgkin lymphoma, and myelodysplastic syndrome. All were profoundly granulocytopenic with an absolute granulocyte count0.13 x 10(9)/l, and all but 1 had oral lesions as a possible portal of entry. A favourable response to antibiotic therapy was recorded in all patients but one who, being profoundly granulocytopenic, rapidly succumbed to Pseudomonas aeruginosa septicemia. None of the isolates were beta-lactamase producers. In addition to penicillin the isolates were susceptible to broad-spectrum cephalosporins and ciprofloxacin, but resistant to aminoglycosides.

Little information is available on periodontopathic bacterial infection in childhood. We assessed the prevalence by age of 10 putative periodontopathic microorganisms in periodontally healthy children using a polymerase chain reaction (PCR) assay.Plaque samples were collected from the buccal-mesial sulcus of the first molar or second primary molar in the right upper quadrant of 144 children (2 to 13 years old, 12 subjects from each year of age) who showed negligible periodontal inflammation. Using species-specific primers of Porphyromonas gingivalis, Bacteroides forsythus, Prevotella intermedia, Prevotella nigrescens, Campylobacter rectus, Eikenella corrodens, Actinobacillus actinomycetemcomitans, Capnocytophaga ochracea, Capnocytophaga sputigena, and Treponema denticola, PCR amplification was performed with bacterial genomic DNA from plaque samples.The results indicated that C. rectus, E. corrodens, A. actinomycetemcomitans, C. ochracea, and C. sputigena were found in about 50% of the plaque samples from all age groups, while B. forsythus and P. intermedia were detected less frequently, and P. gingivalis and T. denticola were not found. In contrast, the percentage of P. nigrescens-positive subjects increased with age in primary dentition, and reached about 50% at 7 years of age and older. Subject-based analyses suggested that the number of bacterial species in the plaque samples increased gradually with age until 5 years old, and then reached a plateau after the mixed dentition period.The colonization of many putative periodontopathic microorganisms can occur quite early in childhood without clinical signs of periodontal disease. However, colonization by P. gingivalis and T. denticola was not detected in periodontally healthy children.

Intergeneric coaggregation is responsible for the complexity of the microbiota in human dental plaque and is believed to be important in the initial bacterial colonization of the human oral cavity. Actinomyces naeslundii, an carly colonizer of the tooth surface, may enhance subsequent colonization by Porphyromonas gingivalis which is associated with adult periodontitis. The purpose of this study was to isolate and characterize the A. naeslundii aggregation factor (AnAF) that mediates coaggregation with P. gingivalis. AnAF was isolated from A. naeslundii sonic extract (SE) by gel filtration on a Sephacryl S-400HR, by hydrophobic interaction chromatography on a HiTrap Octyl Sepharose 4FF, and by ion exchange chromatography on a HiTrap Q. The specific activity increased 12-fold with a yield of 2.5%. SDS-PAGE analysis of AnAF revealed a protein band of high molecular weight in excess of 200 kDa. Carbohydrate was detected as the only material coinciding with the protein band, indicating that the AnAF was a glycoprotein. Immunoblotting analysis indicated that AnAF directly bound to P. gingivalis cells. AnAF was sensitive to sodium metaperiodate treatment but not to heat or protease treatments. These results suggest that the AnAF carbohydrate component mediated coaggregation with P. gingivalis cells. AnAF also inhibited coaggregation with other periodontal disease-associated bacteria such as Prevotella intermedia, Fusobacterium nucleatum, Capnocytophaga ochracea, but not streptococci.