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Capnocytophaga ochracea possesses a type-IX secretion system that exports proteins which have a gliding motility-associated C-terminal (CTD) domain. This system is found in several species of the Bacteroidetes phylum. Hyalin, a large protein encoded by Coch_0033 in C. ochracea ATCC 27872, has a CTD domain and is posited to be involved in quorum sensing according to the database of the Kyoto Encyclopedia of Genes and Genomes. This suggests that it plays a role in biofilm formation via interbacterial communication. The aim of this study was to investigate the potential role of the hyalin-like protein coded by the Coch_0033 gene in gliding and biofilm formation of C. ochracea. A hyalin-like protein-deficient mutant strain of C. ochracea, designated mutant WR-1, was constructed through insertion of the ermF-ermAM cassette into the target gene. The spreading feature at the edge of the colony was lost in the mutant strain. Crystal violet and confocal laser scanning microscopy revealed no difference between the quantity of biofilm organized by the mutant and that organized by the wild-type strain. These data suggest that the hyalin-like protein encoded by the Coch_0033 gene is indeed involved in C. ochracea gliding activity.

Periodontal disease is a chronic infectious disease, which is characterized by the damaged dental hard tissue by lactic acid generated by microorganisms after the fermentation of carbohydrates rich diet. The risk of periodontal disease is known to be higher in diabetic patients. We compared the diversity of five commonly occurring dental bacteria including Porphyromonas gingivalis, Tannerella forsythia, Capnocytophaga ochracea, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans in 14 type-2 diabetic patients and equal numbers of healthy controls. The subgingival samples were collected using sterile paper points. We used 16S rRNA sequence specific primers for PCR-based identification of dental bacteria. Our results showed that A. actinomycetemcomitans was completely absent in control subjects but present in 43% of diabetic patients. C. ochracea was highly prevalent in diabetic patients (100%) as compared to controls (28.5%). The frequency of other three bacterial species was also higher in diabetic patients than control subjects. These findings indicate that dental bacteria are highly prevalent in subgingival pockets of diabetic patients. Therefore, proper monitoring of diabetic patients for dental care is important to prevent bacterial growth and its sequela in risky individuals. Further case-control studies using larger sample size would help in validating the association between oral diseases and diabetes. Keywords: Dental bacteria, Identification, PCR, Diabetes

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.

Pleural effusions and empyemas caused by Capnocytophaga spp. are uncommon with few cases previously reported. Here, we present the case of a 62-year-old man with untreated chronic lymphocytic leukemia (CLL) complicated by a pleural empyema caused by C. ochracea. The route of acquisition was likely the result of aspiration of C. ochracea coupled with the immune defects associated with untreated CLL.

BACKGROUND This study compared the composition of subgingival microbiota between obese and non-obese women with or without periodontal disease. METHODS Full-mouth periodontal clinical assessments were carried out in 76 obese women (17 periodontally healthy and 59 with periodontal disease), and 34 non-obese women (12 periodontally healthy, 22 with periodontal disease). Subgingival biofilm samples were individually obtained from seven sites of each individual, and the prevalence and counts of 40 bacterial taxa were determined by the checkerboard method. The frequency and counts of each species were computed for each individual and across the groups. Differences among and between groups were sought by the Kruskal-Wallis and Mann-Whitney tests, respectively. Possible correlations between obesity and clinical and microbiologic parameters were tested with Spearman correlation coefficient. RESULTS Streptococcus sanguinis, Streptococcus oralis, and Capnocytophaga ochracea were found in significantly higher levels in obese compared with non-obese women (P

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.

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

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.

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.

Introduction: Capnocytophaga species are recognized as part of human oral microbiota and implicated as periodontal pathogens associated with various periodontal diseases. The three original Capnocytophaga species - Capnocytophaga ochracea, Capnocytophaga sputigena and Capnocytophaga gingivalis were initially isolated from periodontitis in adults, but subsequent studies demonstrated their presence also at periodontally healthy sites in both children and adults. Their association with periodontal disease is a matter of controversy. Considering the differing virulence features of the respective isolate, it is crucial to identify these isolates to species level. Aims: The aim of this study is to investigate the prevalence of Capnocytophaga species by polymerase chain reaction (PCR) through restriction fragment length polymorphism in healthy individuals and patients with periodontal disease. Material and Methods: The study included a total of 300 individuals, 100 each with Gingivitis, Chronic periodontitis, and Healthy individuals. The plaque samples were collected using sterile curette in reduced transport fluid. DNA extraction was carried out for PCR analysis. Results: Of 300 individuals, Capnocytophaga species were identified from 237 (79%) participants in all groups. The prevalence was statistically analyzed using Chi-square test. The prevalence was more in males in gingivitis and healthy individuals (42% and 49% respectively), and females in periodontitis (40%). C. ochracea was observed in a higher proportion (36.33%), followed by C. granulosa (32.66%) and C. gingivalis (10%). They were identified more in the age group of 30–40 years in gingivitis and periodontitis, (30 and 21 individuals, respectively) and 39 individuals in 18–29 years in healthy individuals. They were present in 87% in healthy individuals, 77% in gingivitis and 73% in periodontitis. Conclusion: Capnocytophaga species are commonly present in healthy individuals and may be associated with periodontal disease. There is a need for further study to know the prevalence of other species of Capnocytophaga in health and disease.

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.

Patient: Female, 17-days-old Final Diagnosis: Cardiac arrest Symptoms: Lower abdominal pain and contractions Medication: — Clinical Procedure: Prematurity treatment and Management of neonatal septicemia Specialty: Pediatrics and Neonatology Objective: Unknown ethiology Background: Capnocytophaga ochracea is a gram-negative anaerobic organism commonly found in human oral flora. It is characteristically sensitive to beta-lactams and resistant to aminoglycosides. Case Report: A 23-year-old woman presented with lower abdominal pain and was admitted for premature labor at 24-weeks of gestation. At presentation, the cervix was closed and the membrane was intact; however, contractions continued, the membrane subsequently ruptured before receiving any steroids or magnesium, and the mother gave birth to a 540-gram female baby. At birth, Apgar scores were 1 at 5 minutes, 1 at 10 minutes, and 2 at 15 minutes. On the fifth day of life, the blood culture grew Capnocytophaga species. Consequently, Cefotaxime was started and ampicillin continued for a total of 14 days; however, on the 6th day, the head ultrasound showed grade 4 intraventricular hemorrhage and a Do Not Resuscitate (DNR) order was placed in the chart. The patient’s health continued to deteriorate, having multiple episodes of bradycardia and desaturation until cardiac arrest on the 17th day. Conclusions: Capnocytophaga ochracea was isolated from the blood culture of a preterm neonate. It was thought to be the cause of the premature labor and subsequent neonatal septicemia. This case report suggests that the prevalence of Capnocytophaga infections is most likely underestimated and that additional premature labors and abortions could have been caused by Capnocytophaga infections that were never detected. Hence, more studies are needed to investigate the route of transmission.

Made available in DSpace on 2018-11-26T17:24:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-03-01. Added 1 bitstream(s) on 2019-10-09T18:26:38Z : No. of bitstreams: 1 S1678-77572017000200217.pdf: 199895 bytes, checksum: 0912d4979dd7e9af0e4d346708df9943 (MD5) Objective: The aim of this study was to compare the prevalence of periodontal pathogens, systemic inflammatory mediators and lipid profiles in type 1 diabetes children (DM) with those observed in children without diabetes (NDM), both with gingivitis. Material and methods: Twenty-four DM children and twenty-seven NDM controls were evaluated. The periodontal status, glycemic and lipid profiles were determined for both groups. Subgingival samples of periodontal sites were collected to determine the prevalence of periodontal microorganisms by PCR. Blood samples were collected for IL-1-beta, TNF-alpha. and IL-6 analysis using ELISA kits. Results: Periodontal conditions of DM and NDM patients were similar, without statistical differences in periodontal indices. When considering patients with gingivitis, all lipid parameters evaluated were highest in the DM group; Capnocytophaga sputigena and Capnocytophaga ochracea were more prevalent in the periodontal sites of DM children. Red complex bacteria were detected in few sites of DM and NDM groups. Fusobacterium nucleatum and Campylobacter rectus were frequently found in both groups. Similar levels of IL-1-beta, TNF-alpha. and IL-6 were detected in DM and NDM children. Conclusion: Clinical and immunological profiles are similar between DM and NDM children. The presence of Capnocytophaga sputigena and Capnocytophaga ochracea were associated with gingivitis in DM children. Univ Estadual Paulista, Fac Odontol Aracatuba, Dept Odontol Infantil & Social, Rua Jose Bonifacio 1193, BR-16015050 Sao Paulo, Brazil Univ Estadual Campinas, Fac Odontol Piracicaba, Dept Diagnost Oral, Sao Paulo, Brazil Univ Fed Fluminense, Fac Odontol Nova Friburgo, Rio De Janeiro, Brazil Univ Estadual Paulista, Fac Odontol Aracatuba, Dept Odontol Infantil & Social, Rua Jose Bonifacio 1193, BR-16015050 Sao Paulo, Brazil

Made available in DSpace on 2018-12-11T17:12:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-08-01 Introduction This clinical study was conducted to correlate the microbiological profile and levels of endotoxins found in primary endodontic infection with the presence of clinical features and to evaluate the removal of microorganisms and endotoxins using rotary, reciprocating, and hybrid systems for biomechanical preparation. Methods Thirty single root canals with primary endodontic infection were evaluated with signs and symptoms and were randomly divided into 3 groups according to the instrumentation system used (n = 10) as follows: rotary Mtwo instruments (VDW, Munich, Germany) with 8 files, the reciprocating Reciproc system (VDW) with a single file, and Genius hybrid instruments with 3 files (1 rotary and 2 reciprocating files) with irrigation using 24 mL 2.5% sodium hypochlorite. Samples were collected before (S1) and after instrumentation (S2) before being submitted to microbiological culture (colony-forming units/mL) and the checkerboard DNA-DNA hybridization test. Endotoxins were quantified using the limulus amebocyte lysate assay. Results Microbiological culture showed statistical differences in the reduction of colony-forming units/mL with all systems tested (P

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.

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).

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 ochracea (C. ochracea), a known human microflora, has been reported to cause sepsis in immunocompromised patients and less severe infections such as intrauterine infections, endocarditis, peritonitis and septic arthritis in the immunocompetent patient. We present the first described case of C. ochracea causing severe sepsis and purpura fulminans in an immunocompetent host.

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.

Serum IgG responses to the cell envelope proteins (CEPs) from Capnocytophaga sputigena, Capnocytophaga ochracea, and Capnocytophaga gingivalis were examined in periodontally healthy and periodontitis subjects, both with and without type 1 diabetes (n = 60). Serum IgG responses to CEPs were determined by immunoblotting with biotin-goat anti-human IgG and an alkaline phosphatasestreptavidin system. Reactivity was analyzed by transmission densitometry, digitization, and computer manipulation. The patients with diabetes showed significantly (p < 0.01) fewer responses to 14 CEPs (from 81 to 10 kDa) from C. sputigena, 5 CEPs (from 90 to 17 kDa) from C. gingivalis, and the 27-kDa CEP from C. ochracea than in the non-diabetic group. The periodontitis patients showed significantly (p < 0.01) fewer responses to the 25-and 11-kDa CEPs from C. sputigena, the 125- and 17-kDa CEPs from C. gingivalis, and the 42-kDa CEP from C. ochracea than in the periodontally healthy group. HLA-DR4, HLA-DR53, and HLA-DQw3 were associated with periodontitis, while only HLA-DR4 was associated with diabetes (p < 0.02). Significant (p < 0.01) correlations were found between HLA-DR2 and IgG reactivity patterns associated with non-diabetics, and between HLA-DR4 and IgG reactivity patterns associated with diabetic and periodontitis subjects. These results indicate that both type 1 diabetics and periodontitis subjects have a depressed IgG antibody profile to Capnocytophaga, which may account for an increased susceptibility to periodontitis infection. Periodontitis in type 1 diabetes may be related more to the HLA-D type and altered immune function than to the diabetes itself.

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.

Background: Histopathologic studies of this unusual keratitis caused by Capnocytophaga species have not been reported previously. Methods: The authors report the light microscopic and ultrastructural findings of three patients with a distinctive necrotizing keratitis caused by an anaerobic gramnegative bacillus. In three patients, ages 19, 81, and 91 years, a necrotizing stromal keratitis developed; two of these patients had a previous penetrating keratoplasty for pseudophakic bullous keratopathy. The first patient did not have ocular surgery previously and was treated initially for presumed Acanthamoeba keratitis. Results: By light microscopy, all three keratectomy specimens were strikingly similar and showed a necrotizing and/or suppurative stromal keratitis displaying myriad slender, fusiform, gram-negative bacilli located anterior to Descemet's membrane and extending into the deep corneal stroma, assuming a "picket fence" appearance. Cultures of the cornea in case 1 grew Capnocytophaga ochracea . For the remaining two patients, a diagnosis presumptively was made based on characteristic histopathologic features. Results of electron microscopic examination showed numerous bacilli that were mostly extracellular; occasional organisms were phagocytosed by macrophages. Conclusion: The histopathologic features of Capnocytophaga keratitis are unique; therefore, a presumptive diagnosis can be made based on the morphology and location of the bacilli in the keratectomy specimens. To the authors' knowledge, this is the first study describing the typical histopathologic and electron microscopic findings of Capnocytophaga keratitis.