Evaluation of oral microbiota in undernourished and eutrophic children using checkerboard DNA-DNA hybridization.

The aim of this study was to evaluate the relationship among nutritional status, gingival health and the composition of oral microbiota in children of a public school from a very poor area of San Miguel de Tucuman. Forty-five children ranging in age from 6 to 14 years old, 13 males and 32 females were studied. Twenty of these children were undernourished (Lejarraga-Morasso Table) and twenty-five were eutrophic. A clinical study that included DMF and dmf indexes, Löe Silness Plaque Index and bleeding on probing was performed. For microbiological study, saliva samples without stimulation were taken; aliquots of them were immediately placed in TAE buffer pH 7.6, adding NaOH (N and keeping at -70 °C until processed by checkerboard DNA-DNA hybridization method to check the presence of 40 oral microorganism species. Positive bleeding on probing was present in more than 80% of children, without significant differences between eutrophic and undernourished groups. Same result were obtain for the other clinical indexes (p > 0.05, Two Way ANOVA). Significant differences were found for some oral microorganism species, with a higher percentage of undernourished children harboring them. That was the case of S. gordonii (p < 0.05), Capnocitophaga gingivalis and C. ochraceae (p < 0.01 and p < 0.10, respectively), F. nucleatum ss nucleatum (p < 0.05), P. nigrescens (p < 0.10), Campylobacter gracilis (p < 0,05), and T. denticola (p < 0.10, multiple logistic regression). Significant differences were also found between children groups for E. saborreum (p < 0.001), P. acnes (p < 0.10), G. morbillorum (p < 0.05) and L. buccalis (p < 0.10). Gingivitis and bleeding on probing would not be related to nutritional status in the groups of children studied. There were significant differences for the presence of some of the main periodontal pathogen species between eutrophic and undernourished children. It would be important to study the meaning of significant differences found for the other microorganisms more deeply.

Arthritis-induced alveolar bone loss is associated with changes in the composition of oral microbiota.

Rheumatoid arthritis (RA) and periodontitis (PD) are chronic inflammatory disorders that cause bone loss. PD tends to be more prevalent and severe in RA patients. Previous experimental studies demonstrated that RA triggers alveolar bone loss similarly to PD. The aim of this study was to investigate if arthritis-induced alveolar bone loss is associated with modification in the oral microbiota. Checkerboard DNA-DNA hybridization was employed to analyze forty oral bacterial species in 3 groups of C57BL/6 mice: control (n = 12; without any challenge); Y4 (n = 8; received oral inoculation of Aggregatibacter Actinomycetemcomitans strain FDC Y4) and AIA group (n = 12; chronic antigen-induced arthritis). The results showed that AIA and Y4 group exhibited similar patterns of bone loss. The AIA group exhibited higher counts of most bacterial species analyzed with predominance of Gram-negative species similarly to infection-induced PD. Prevotella nigrescens and Treponema denticola were detected only in the Y4 group whereas Campylobacter showae, Streptococcus mitis and Streptococcus oralis were only found in the AIA group. Counts of Parvimonas micra, Selenomonas Noxia and Veillonella parvula were greater in the AIA group whereas Actinomyces viscosus and Neisseira mucosa were in large proportion in Y4 group. In conclusion, AIA is associated with changes in the composition of the oral microbiota, which might account for the alveolar bone loss observed in AIA mice.

Higher abundance of Campylobacter in the oral microbiome of Japanese patients with moyamoya disease.

Genetic factors alone cannot explain the pathophysiology of moyamoya disease (MMD), and environmental factors such as an immune response are thought to be involved. Oral and gut microbiomes have attracted attention as environmental factors in the pathophysiology of some vascular and autoimmune diseases. However, the relationship between MMD and these microbiomes is yet to be thoroughly investigated. This prospective case-control study aimed to compare the microbiomes of Japanese patients with MMD with those of healthy individuals to identify the specific bacteria involved in MMD. Saliva and fecal samples were collected from 16 patients with MMD who had not undergone revascularization surgery. Fifteen healthy individuals were matched for age, sex, and body mass index. The microbiomes were determined using 16S rRNA sequencing and analyzed using QIIME2. Differentially abundant microbes were identified using LEfSE and ANCOM-BC. In the oral microbiome, the two analytical methods showed that Campylobacter was more abundant in patients with MMD than in healthy individuals. Differences in the gut microbiome were smaller than those in the oral microbiome. In conclusion, the oral microbiome profiles of patients with MMD significantly differ from those of healthy individuals. Campylobacter spp. could be a substantial environmental factor in the pathophysiology of MMD.

Molecular epidemiology of Campylobacter jejuni isolates from wild-bird fecal material in children's playgrounds.

In many countries relatively high notification rates of campylobacteriosis are observed in children under 5 years of age. Few studies have considered the role that environmental exposure plays in the epidemiology of these cases. Wild birds inhabit parks and playgrounds and are recognized carriers of Campylobacter, and young children are at greater risk of ingesting infective material due to their frequent hand-mouth contact. We investigated wild-bird fecal contamination in playgrounds in parks in a New Zealand city. A total of 192 samples of fresh and dried fecal material were cultured to determine the presence of Campylobacter spp. Campylobacter jejuni isolates were also characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), and the profiles obtained were compared with those of human isolates. C. jejuni was isolated from 12.5% of the samples. MLST identified members of clonal complexes ST-45, ST-682, and ST-177; all of these complexes have been recovered from wild birds in Europe. PFGE of ST-45 isolates resulted in profiles indistinguishable from those of isolated obtained from human cases in New Zealand. Members of the ST-177 and ST-682 complexes have been found in starlings (Sturnus vulgaris) in the United Kingdom, and these birds were common in playgrounds investigated in New Zealand in this study. We suggest that feces from wild birds in playgrounds could contribute to the occurrence of campylobacteriosis in preschool children. Further, the C. jejuni isolates obtained in this study belonged to clonal complexes associated with wild-bird populations in the northern hemisphere and could have been introduced into New Zealand in imported wild garden birds in the 19th century.

Detection of Helicobacter and Campylobacter spp. from the aquatic environment of marine mammals.

The mechanism by which Helicobacter species are transmitted remains unclear. To examine the possible role of environmental transmission in marine mammals, we sought the presence of Helicobacter spp. and non-Helicobacter bacteria within the order Campylobacterales in water from the aquatic environment of marine mammals, and in fish otoliths regurgitated by dolphins. Water was collected from six pools, two inhabited by dolphins and four inhabited by seals. Regurgitated otoliths were collected from the bottom of dolphins' pools. Samples were evaluated by culture, PCR and DNA sequence analysis. Sequences from dolphins' water and from regurgitated otoliths clustered with 99.8-100% homology with sequences from gastric fluids, dental plaque and saliva from dolphins living in those pools, and with 99.5% homology with H. cetorum. Sequences from seals' water clustered with 99.5% homology with a sequence amplified from a Northern sea lion (AY203900). Control PCR on source water for the pools and from otoliths dissected from feeder fish were negative. The findings of Helicobacter spp. DNA in the aquatic environment suggests that contaminated water from regurgitated fish otoliths and perhaps other tissues may play a role in Helicobacter transmission among marine mammals.

The biofilm forming potential of bacterial species in the genus Campylobacter.

The biofilm forming abilities of 16 strains representative of 14 of the 16 species comprising the genus Campylobacter were determined on glass, stainless steel, and polystyrene plastic. The formation of biofilms has been suggested as a means by which Campylobacter is able to persist within an inhospitable environment. Of the eight microaerophilic Campylobacter species, including two strains each of Campylobacter jejuni and Campylobacter fetus, only C. jejuni strain 81-176 reliably produced a visible biofilm on multiple surfaces. Alternately, all six strains of the anaerobic Campylobacter species reliably produced visible biofilms on multiple surfaces. Electron micrographs of the individual biofilms showed relatively homogeneous biofilms produced by the anaerobic strains, while the microaerophilic C. jejuni strain 81-176 produced a biofilm containing similar quantities of both the spiral and coccoid forms. This survey suggests a difference in the biofilm forming potentials and the morphologies of the bacteria comprising the biofilms between anaerobic and microaerophilic species of Campylobacter. Additionally, differences observed in the biofilm forming ability of two strains of C. jejuni suggest the need for a further investigation of the biofilm forming potential of this species using a larger number of strains.

Subgingival Microbiota of Mexicans with Type 2 Diabetes with Different Periodontal and Metabolic Conditions.

BACKGROUND: Type-2-Diabetes (T2D) and Periodontitis are major inflammatory diseases. However, not much is known about the specific subgingival microbiota in Mexicans with diabetes and metabolic dysbiosis. The aim of this study was to describe the subgingival microbiota of Mexicans with T2D and the different periodontal and metabolic conditions, through "Checkerboard" DNA-DNA hybridization. METHODS: Subjects were divided into two groups-periodontal-health (PH) (PH_non-T2D; n = 59, PH_T2D; n = 14) and generalized-periodontitis (GP) (GP_non-T2D; n = 67, GP_T2D; n = 38). Obesity (BMI ≥ 30 kg/m2) and serum levels of glycated-hemoglobin (HbA1c), total-lipids, triglycerides, total-cholesterol, high-density-lipids, and low-density-lipids were measured for the T2D individuals. Subgingival microbial identification was processed for 40 species through DNA-probes. RESULTS: Subjects with T2D harbored significantly higher mean total levels (PH: p < 0.001, and GP_NS), a lower proportion of "red" complex (GP: p < 0.01), a higher proportion of "yellow" (GP; p < 0.001), and "orange" (GP; p < 0.01) complex than the non-T2D. GP_T2D individuals exhibited a greater proportion of putative-species-Campylobacter gracilis and S. constellatus (p < 0.001), and Parvimonas micra and Prevotella nigrescens (p < 0.01), than GP_non-T2D. T2D individuals with HbA1c > 8% had presented significantly higher mean pocket-depth and higher levels of G. morbillorum (p < 0.05) and those with obesity or dyslipidemia harbored higher levels, prevalence, or proportion of Streptococcus sp., Actinomyces sp., and Capnocytophaga sp. CONCLUSIONS: T2D individuals harbored a particular microbial profile different to non-T2D microbiota. Metabolic control was related to dysbiosis of microbiota-HbA1c>8% related to periodontitis and obesity or dyslipidemia with the predominance of saccharolytic bacteria, irrespective of their periodontal condition.

Comparative genomics and genome biology of Campylobacter showae.

Campylobacter showae a bacterium historically linked to gingivitis and periodontitis, has recently been associated with inflammatory bowel disease and colorectal cancer. Our aim was to generate genome sequences for new clinical C. showae strains and identify functional properties explaining their pathogenic potential. Eight C. showae genomes were assessed, four strains isolated from inflamed gut tissues from paediatric Crohn's disease patients, three strains from colonic adenomas, and one from a gastroenteritis patient stool. Genome assemblies were analyzed alongside the only 3 deposited C. showae genomes. The pangenome from these 11 strains consisted of 4686 unique protein families, and the core genome size was estimated at 1050 ± 15 genes with each new genome contributing an additional 206 ± 16 genes. Functional assays indicated that colonic strains segregated into 2 groups: adherent/invasive vs. non-adherent/non-invasive strains. The former possessed Type IV secretion machinery and S-layer proteins, while the latter contained Cas genes and other CRISPR associated proteins. Comparison of gene profiles with strains in Human Microbiome Project metagenomes showed that gut-derived isolates share genes specific to tongue dorsum and supragingival plaque counterparts. Our findings indicate that C. showae strains are phenotypically and genetically diverse and suggest that secretion systems may play an important role in virulence potential.

Comparative genomics of Campylobacter concisus: Analysis of clinical strains reveals genome diversity and pathogenic potential.

In recent years, an increasing number of Campylobacter species have been associated with human gastrointestinal (GI) diseases including gastroenteritis, inflammatory bowel disease, and colorectal cancer. Campylobacter concisus, an oral commensal historically linked to gingivitis and periodontitis, has been increasingly detected in the lower GI tract. In the present study, we generated robust genome sequence data from C. concisus strains and undertook a comprehensive pangenome assessment to identify C. concisus virulence properties and to explain potential adaptations acquired while residing in specific ecological niche(s) of the GI tract. Genomes of 53 new C. concisus strains were sequenced, assembled, and annotated including 36 strains from gastroenteritis patients, 13 strains from Crohn's disease patients and four strains from colitis patients (three collagenous colitis and one lymphocytic colitis). When compared with previous published sequences, strains clustered into two main groups/genomospecies (GS) with phylogenetic clustering explained neither by disease phenotype nor sample location. Paired oral/faecal isolates, from the same patient, indicated that there are few genetic differences between oral and gut isolates which suggests that gut isolates most likely reflect oral strain relocation. Type IV and VI secretion systems genes, genes known to be important for pathogenicity in the Campylobacter genus, were present in the genomes assemblies, with 82% containing Type VI secretion system genes. Our findings indicate that C. concisus strains are genetically diverse, and the variability in bacterial secretion system content may play an important role in their virulence potential.

Molecular epidemiology and comparative genomics of Campylobacter concisus strains from saliva, faeces and gut mucosal biopsies in inflammatory bowel disease.

Campylobacter concisus is an emerging pathogen associated with inflammatory bowel disease (IBD), yet little is known about the genetic diversity of C. concisus in relation to host niches and disease. We isolated 104 C. concisus isolates from saliva, mucosal biopsies and faecal samples from 41 individuals (26 IBD, 3 Gastroenteritis (GE), 12 Healthy controls (HC)). Whole genomes were sequenced and the dataset pan-genome examined, and genomic information was used for typing using multi-locus-sequence typing (MLST). C. concisus isolates clustered into two main groups/genomospecies (GS) with 71 distinct sequence types (STs) represented. Sampling site (p < 0.001), rather than disease phenotype (p = 1.00) was associated with particular GS. We identified 97 candidate genes associated with increase or decrease in prevalence during the anatomical descent from the oral cavity to mucosal biopsies to faeces. Genes related to cell wall/membrane biogenesis were more common in oral isolates, whereas genes involved in cell transport, metabolism and secretory pathways were more prevalent in enteric isolates. Furthermore, there was no correlation between individual genetic diversity and clinical phenotype. This study confirms the genetic heterogeneity of C. concisus and provides evidence that genomic variation is related to the source of isolation, but not clinical phenotype.

Preliminary analysis of salivary microbiome and their potential roles in oral lichen planus.

Several studies have explored the origin and development mechanism of oral lichen planus (OLP) with limited attention to the role of bacteria in the progression of this common oral disease. Here we utilized MiSeq sequencing of 16S rRNA gene amplicons to identify complex oral microbiota associated with OLP from saliva samples of two subtypes (reticular and erosive) of OLP patients and healthy controls. Our analyses indicated that the overall structure of the salivary microbiome was not significantly affected by disease status. However, we did observe evident variations in abundance for several taxonomic groups in OLP. Porphyromonas and Solobacterium showed significantly higher relative abundances, whereas Haemophilus, Corynebacterium, Cellulosimicrobium and Campylobacter showed lower abundances in OLP patients, as compared with healthy controls. In addition, we explored specific microbial co-occurrence patterns in OLP, and revealed significantly fewer linkers of Streptococcus comprising species in erosive OLP. Furthermore, the disease severity and immune dysregulation were also genus-associated, including with Porphyromonas that correlated to disease scores and salivary levels of interleukin (IL)-17 and IL-23. Overall, this study provides a general description of oral microbiome in OLP, and it will be useful for further investigation of their potential roles in the initiation and immune modulation of OLP.

The GroEL-like protein from Campylobacter rectus: immunological characterization and interleukin-6 and -8 induction in human gingival fibroblast.

The native GroEL-like protein was purified from Campylobacter rectus, a putative periodontal pathogen, by affinity chromatography on ATP-agarose followed by high performance liquid chromatography on Superose 6. The purified 64-kDa protein (denatured form of GroEL-like protein) was immunoreactive by SDS-PAGE and Western immunoblotting with the monoclonal antibody directed against heat shock protein 60 of human origin. The native GroEL-like protein stimulated both interleukin-6 (IL-6) and IL-8 secretion by a confluent monolayer of human gingival fibroblast in their culture supernatant. During the 22-h incubation, the amounts of IL-6 and IL-8 were increased by 5.4- and 3.5-fold, respectively. These data suggested that the GroEL-like protein might be considered to be a virulence factor of C. rectus in periodontal disease.

Detection of Campylobacter pylori DNA by hybridisation with non-radioactive probes in comparison with a 32P-labelled probe.

A dot-blot hybridisation assay for the detection of Campylobacter pylori was used to compare a 32P-labelled probe with two biotinylated probes and a sulphonated probe. The minimum amount of pure C. pylori DNA that could be detected by the 32P-labelled probe was 100 pg, which corresponded to 5 x 10(4) bacteria. A biotin-labelled DNA (biotin-DNA) probe together with the BluGeneTM detection system produced by Bethesda Research Laboratories (BRL), and a sulphonated probe and ChemiprobeTM detection system (Orgenics) gave similar levels of sensitivity; nylon membranes could be used with both these non-radioactive detection systems. However, a photobiotin-labelled DNA (photobiotin-DNA) probe and detection system produced by Biotechnology Research Enterprises S.A. (BRESA) gave optimum results only with nitrocellulose membranes, and was quantitatively 100 times less sensitive than the other types of probe. The detection systems for the biotin-DNA and photobiotin-DNA probes produced non-specific reactions with crude bacterial blots of heterologous organisms; these non-specific reactions could be removed by treating the dot blots with proteinase K, but not by treatment with RNAase. The sulphonated probe and detection system did not give any reaction with heterologous organism blots.

Comparison of DNA probe and ELISA microbial analysis methods and their association with adult periodontitis.

The purposes of this study were two-fold: to compare the DNA probe and enzyme linked immunosorbent assay (ELISA) microbial identification tests and correlate the levels of microorganisms with adult periodontitis. A single plaque sample were taken from each of 2 sites in 52 patients. Twelve of these patients were also sampled during and after treatment. The experimental site had clinical indicators of disease (bleeding on probing, probing and attachment loss of > or = 6 mm) and the contralateral site (control) was clinically healthy. A total of 176 plaque samples were collected, divided, processed, and sent for both types of quantitative microbial analyses. All of these samples were used to compare the DNA probe and ELISA methods while only the initial 104 pretreatment sites were used to correlate microorganisms/method with clinical indicators of adult periodontitis. DNA probes were used to assay for A. actinomycetemcomitans, P. gingivalis, P. intermedia, E. corrodens, F. nucleatum, T. denticola, and C. rectus. An ELISA utilizing monoclonal antibodies was used to assay for P. gingivalis, E. corrodens, T. denticola, and C. rectus. Comparison of the two methods revealed that the ELISA test identified P. gingivalis and C. rectus significantly more often than the DNA probe method and that T. denticola was detected more frequently with the DNA probe. The sensitivities and specificities varied widely among organisms and by test. P. gingivalis, as identified by ELISA, had the highest degree of sensitivity and specificity (0.90 and 0.82 respectively) to clinical indicators of adult periodontitis.

Oral food consumption and subgingival microorganisms: subgingival microbiota of gastrostomy tube-fed children and healthy controls.

This study examined the effect of oral food consumption on the prevalence and levels of subgingival bacteria and yeasts in 20 gastrostomy tube-fed children and 24 healthy controls. Microbial identification was carried out using anaerobic culture and 16S rRNA-based PCR identification methods. Streptococcal and Actinomyces species were recovered from 100% and 76% of all subjects and averaged 66% and 11% of total cultivable organisms, respectively. In decreasing order of prevalence, Fusobacterium, enteric rods, Prevotella intermedia/Prevotella nigrescens, Capnocytophaga, Propionibacterium, yeasts, Actinobacillus actinomycetemcomitans, coagulase-negative Staphylococcus, Campylobacter rectus, Bacteroides forsythus, and Porphyromonas gingivalis were detected in 48% to 2% of the study subjects. The cultivable levels of these species varied widely among subjects. PCR detection showed C. rectus and Eikenella corrodens both to occur in 93% of the study subjects and to be the most prevalent putative periodontal pathogens examined. In decreasing order of prevalence, PCR identified Treponema denticola, A. actinomycetemcomitans, P. nigrescens, P. intermedia, B. forsythus, and P. gingivalis in 38% to 21% of the subjects studied. Tube-fed children and healthy controls exhibited similar subgingival microbial compositions. It appears from this study that oral food consumption is not a major determinant for the establishment of subgingival microbiota in children.

A 16S rDNA-based nested PCR protocol to detect Campylobacter gracilis in oral infections.

The aim of this study was to describe a 16S rDNA-based nested polymerase chain reaction (nPCR) assay to investigate the occurrence of Campylobacter gracilis in oral infections. Samples were collected from ten infected root canals, ten cases of acute periradicular abscesses and eight cases of adult marginal periodontitis. DNA extracted from the samples was initially amplified using universal 16S rDNA primers. A second round of amplification used the first PCR products to detect C. gracilis using oligonucleotide primers designed from species-specific 16S rDNA signature sequences. The nPCR assay used in this study showed a detection limit of 10 C. gracilis cells and no cross-reactivity was observed with nontarget bacteria. C. gracilis was detected in the three types of oral infections investigated - 4/10 infected root canals; 2/10 acute periradicular abscesses; and 1/8 subgingival specimens from adult periodontitis. The method proposed in this study showed both high sensitivity and high specificity to directly detect C. gracilis in samples from root canal infections, abscesses, and subgingival plaque. Our findings confirmed that C. gracilis may be a member of the microbiota associated with distinct oral infections, and its specific role in such diseases requires further clarification.

The prevalence and polymorphisms of zonula occluden toxin gene in multiple Campylobacter concisus strains isolated from saliva of patients with inflammatory bowel disease and controls.

Campylobacterconcisus is an oral bacterium. A number of studies detected a significantly higher prevalence of C. concisus in the intestinal tract of patients with inflammatory bowel disease (IBD) as compared to controls. The prevalence of zonula occluden toxin (zot) gene, which encodes a toxin known to increase intestinal permeability, in oral C. concisus strains is unknown. Increased intestinal permeability is a feature of IBD. A total of 56 oral C. concisus strains isolated from 19 patients with IBD and 20 controls were examined (some individuals were colonized with multiple strains). A filtration method was used for isolation of C. concisus from saliva samples. SDS-PAGE was used to define strains. PCR was used to amplify zot from C. concisus strains. Positive PCR products were sequenced and the nucleotides and amino acids were compared. Of the 56 oral C. concisus strains examined, 17 strains (30.4%) were positive for zot. The prevalence of zot-positive oral C. concisus strains was 54.5% in patients with active IBD, which was not significantly different from that in healthy controls (40%). Polymorphisms of C. concisus zot were revealed. zot (808T) , zot (350-351AC) and zot (Multiple) were detected only in patients with IBD, but not in healthy controls. Both zot (808T) and zot (Multiple) alleles resulted in substitution of valine at position 270, which occurred in 36.4% of patients with active IBD but not in healthy controls (P = 0.011). Furthermore, the prevalence of multiple oral C. concisus strains in patients with active IBD was significantly higher than that in healthy controls (P = 0.013). This is the first study reporting the prevalence of zot in human oral C. concisus strains and the polymorphisms of C. concisus zot gene. The data suggest that the possible role of C. concisus strains containing specific polymorphic forms of zot gene in human IBD should be investigated.

Campylobacter concisus - A new player in intestinal disease.

Over the last decade Campylobacter concisus, a highly fastidious member of the Campylobacter genus has been described as an emergent pathogen of the human intestinal tract. Historically, C. concisus was associated with the human oral cavity and has been linked with periodontal lesions, including gingivitis and periodontitis, although currently its role as an oral pathogen remains contentious. Evidence to support the role of C. concisus in acute intestinal disease has come from studies that have detected or isolated C. concisus as sole pathogen in fecal samples from diarrheic patients. C. concisus has also been associated with chronic intestinal disease, its prevalence being significantly higher in children with newly diagnosed Crohn's disease (CD) and adults with ulcerative colitis than in controls. Further C. concisus has been isolated from biopsy specimens of patients with CD. While such studies support the role of C. concisus as an intestinal pathogen, its isolation from healthy individuals, and failure of some studies to show a significant difference in C. concisus prevalence in subjects with diarrhea and healthy controls has raised contention as to its role in intestinal disease. Such findings could argue against the role of C. concisus in intestinal disease, however, the fact that C. concisus strains are genetically diverse raises the possibility that differences exist in their pathogenic potential. Evidence to support this view comes from studies showing strain specific differences in the ability of C. concisus to attach to and invade cells and produce virulence factors, including toxins and hemolytic phospholipase A. Further, sequencing of the genome of a C. concisus strain isolated from a child with CD (UNSWCD) and comparison of this with the only other fully sequenced strain (BAA-1457) would suggest that major differences exist in the genetic make-up of this species which could explain different outcomes of C. concisus infection.

Novel gastric helicobacters and oral campylobacters are present in captive and wild cetaceans.

The mammalian gastric and oral mucosa may be colonized by mixed Helicobacter and Campylobacter species, respectively, in individual animals. To better characterize the presence and distribution of Helicobacter and Campylobacter among marine mammals, we used PCR and 16S rDNA sequence analysis to examine gastric and oral samples from ten dolphins (Tursiops gephyreus), one killer whale (Orcinus orca), one false killer whale (Pseudorca crassidens), and three wild La Plata river dolphins (Pontoporia blainvillei). Helicobacter spp. DNA was widely distributed in gastric and oral samples from both captive and wild cetaceans. Phylogenetic analysis demonstrated two Helicobacter sequence clusters, one closely related to H. cetorum, a species isolated from dolphins and whales in North America. The second related cluster was to sequences obtained from dolphins in Australia and to gastric non-H. pylori helicobacters, and may represent a novel taxonomic group. Dental plaque sequences from four dolphins formed a third cluster within the Campylobacter genus that likely represents a novel species isolated from marine mammals. Identification of identical Helicobacter spp. DNA sequences from dental plaque, saliva and gastric fluids from the same hosts, suggests that the oral cavity may be involved in transmission. These results demonstrate that Helicobacter and Campylobacter species are commonly distributed in marine mammals, and identify taxonomic clusters that may represent novel species.

A PCR-DGGE method for detection and identification of Campylobacter, Helicobacter, Arcobacter and related Epsilobacteria and its application to saliva samples from humans and domestic pets.

AIMS: To develop a PCR-denaturing gradient gel electrophoresis (PCR-DGGE) method for the detection and identification of Campylobacter, Helicobacter and Arcobacter species (Epsilobacteria) in clinical samples and evaluate its efficacy on saliva samples from humans and domestic pets. METHODS AND RESULTS: A semi-nested PCR was developed to allow sensitive detection of all Epsilobacteria, with species separation undertaken by DGGE. A database was constructed in BioNumerics using 145 strains covering 51 Campylobacter, Arcobacter and Helicobacter taxa; Nineteen distinct DGGE profile-groups were distinguished. This approach detected Epsilobacteria in all saliva samples collected from humans, cats and dogs, and identified Campylobacter concisus and/or Campylobacter gracilis in the human samples. The pet animal samples were taken from individuals with oral/dental diseases; PCR-DGGE identified up to four different species in each sample. The most common species detected included Wolinella succinogenes, Arcobacter butzleri and two hitherto uncultured campylobacters. The enteropathogen Campylobacter lari was also found. CONCLUSIONS: PCR combined with DGGE is a useful tool for direct detection and preliminary identification of Epsilobacteria in the oral cavity of humans and small animals. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR-DGGE method should allow determination of the true prevalence and diversity of Epsilobacteria in clinical and other samples. Contact with the oral cavity of domestic pets may represent a route of transmission for epsilobacterial enteric diseases.