Femoral osteomyelitis caused by oral anaerobic bacteria with mixed bacteremia of Campylobacter rectus and Parvimonas micra in a chronic periodontitis patient: a case report.

BACKGROUND: Campylobacter rectus is a gram-negative rod, and Parvimonas micra is a gram-positive coccus, both of which are oral anaerobes that cause chronic periodontitis. Chronic periodontitis can cause bacteremia and systemic diseases, including osteomyelitis. Hematogenous osteomyelitis caused by anaerobic bacteria is uncommon, and to date, there have been no reports of mixed bacteremia with C. rectus and P. micra. Here, we report the first case of osteomyelitis of the femur caused by anaerobic bacteria with mixed bacteremia of C. rectus and P. micra caused by chronic periodontitis. CASE PRESENTATION: A 75-year-old man with chronic periodontitis, hyperuricemia, and benign prostatic hyperplasia was admitted to the hospital with a fracture of the left femur. The patient had left thigh pain for 4 weeks prior to admission. Left femoral intramedullary nail fixation was performed, and a large amount of abscess and necrotic tissue was found intraoperatively. The cultures of abscess specimens were identified as P. micra, Fusobacterium nucleatum, and C. rectus. C. rectus and P. micra were also isolated from blood cultures. C. rectus was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16 S ribosomal RNA sequencing. Sulbactam-ampicillin was administered for approximately 1 month, after which it was replaced by oral clavulanic acid-amoxicillin for long-term suppressive treatment. CONCLUSIONS: Only five cases of bloodstream infection with C. rectus have been reported, and this is the first report of mixed bacteremia with P. micra. Clinicians should consider that chronic periodontitis caused by rare oral anaerobic bacteria can cause systemic infections, such as osteomyelitis.

Characterization of ecotin homologs from Campylobacter rectus and Campylobacter showae.

Ecotin, first described in Escherichia coli, is a potent inhibitor of a broad range of serine proteases including those typically released by the innate immune system such as neutrophil elastase (NE). Here we describe the identification of ecotin orthologs in various Campylobacter species, including Campylobacter rectus and Campylobacter showae residing in the oral cavity and implicated in the development and progression of periodontal disease in humans. To investigate the function of these ecotins in vitro, the orthologs from C. rectus and C. showae were recombinantly expressed and purified from E. coli. Using CmeA degradation/protection assays, fluorescence resonance energy transfer and NE activity assays, we found that ecotins from C. rectus and C. showae inhibit NE, factor Xa and trypsin, but not the Campylobacter jejuni serine protease HtrA or its ortholog in E. coli, DegP. To further evaluate ecotin function in vivo, an E. coli ecotin-deficient mutant was complemented with the C. rectus and C. showae homologs. Using a neutrophil killing assay, we demonstrate that the low survival rate of the E. coli ecotin-deficient mutant can be rescued upon expression of ecotins from C. rectus and C. showae. In addition, the C. rectus and C. showae ecotins partially compensate for loss of N-glycosylation and increased protease susceptibility in the related pathogen, Campylobacter jejuni, thus implicating a similar role for these proteins in the native host to cope with the protease-rich environment of the oral cavity.

Frequency of putative periodontal pathogens among type 1 diabetes mellitus: a case-control study.

OBJECTIVE: The aim of the present study is to compare and assess the risk of periodontitis due to the presence of four putative periodontopathic bacteria viz., Eikenella corrodens, Campylobacter rectus, Prevotella intermedia and Prevotella nigrescens. To fulfil the above objective, polymerase Chain reaction using the primers targeting 16S rRNA gene of the bacterial species was performed with the subgingival plaque collected from the permanent first molars of type 1 diabetic children and age matched healthy children. RESULTS: The prevalence of periodontal pathogens in diabetic and healthy children was 6% and 16% for E. corrodens, 18% and 36% for C. rectus, 2% and 2% for P. intermedia, 4% and 0%, for P. nigrescens respectively. Statistically, significant difference was not observed for the prevalence of all the four periodontal pathogens between type 1 diabetic and healthy children (P = 1.00). The results of the present study thus reveal a negative correlation of type I diabetes to periodontitis in association to Eikenella corrodens, Campylobacter rectus, Prevotella intermedia and Prevotella nigrescens.

Fatal thoracic empyema involving Campylobacter rectus: A case report.

We report the case of a 69-year-old man admitted for septic shock secondary to necrotic pneumoniae complicated by thoracic empyema of fatal issue. Microbiological examination of pleural liquid revealed a mixed anaerobic flora involving Campylobacter rectus and Actinomyces meyeri. Campylobacter rectus is an infrequent anaerobic pathogen of oral origin To our knowledge, this is the first case report of fatal C. rectus - associated thoracic empyema, and only the second reported case in which identification was successfully performed by MALDI-TOF MS.

Salivary Periodontopathic Bacteria in Children and Adolescents with Down Syndrome.

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

First report of severe acute otitis media caused by Campylobacter rectus and review of the literature.

Campylobacter rectus is a member of the human oral flora and is associated with periodontal disease. We report the first case of severe acute otitis media (AOM) due to C. rectus in a previous healthy 15-year-old boy, which was confirmed by 16S ribosomal RNA gene sequencing. C. rectus is a possible causative pathogen of AOM.

Quantitation of biofilm and planktonic life forms of coexisting periodontal species.

BACKGROUND: Complexity of oral polymicrobial communities has prompted a need for developing in vitro models to study behavior of coexisting bacteria. Little knowledge is available of in vitro co-growth of several periodontitis-associated species without early colonizers of dental plaque. THE AIM: was to determine temporal changes in the quantities of six periodontal species in an in vitro biofilm model in comparison with parallel planktonic cultures. MATERIAL AND METHODS: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Parvimonas micra, Campylobacter rectus and Fusobacterium nucleatum were anaerobically grown as multispecies and monospecies biofilms and parallel planktonic cultures using cell culture plates and microfuge tubes, respectively. After incubating 2, 4, 6, 8 days, biofilms and planktonic cultures were harvested, DNA extracted and the target species quantified using qPCR with species-specific 16S rDNA primers. Biofilm growth as monocultures was visualized at day 2 and 8 with confocal microscopy and crystal violet staining. RESULTS: The six species were found throughout the test period in all culture conditions, except that P. gingivalis and F. nucleatum were not detected in multispecies planktonic cultures at day 8. In multispecies biofilm, P. gingivalis qPCR counts (cells/ml) increased (P<0.05) from day 2-8 and were then higher (P<0.05) than those of A. actinomycetemcomitans and C. rectus, whereas in monospecies biofilm, P. gingivalis counts were lower (P<0.05) than those of the other species, except A. actinomycetemcomitans. When multi- and monospecies biofilm cultures were compared, P. gingivalis counts were higher (P<0.05) but those of the other species, except P. intermedia, lower (P<0.05) in multispecies biofilm. Comparison between planktonic and biofilm cultures showed that A. actinomycetemcomitans, P. micra and C. rectus had higher (P<0.05) counts in planktonic cultures no matter whether grown in mono- or multispecies environment. CONCLUSIONS: Six periodontal species were able to form multispecies biofilm up to 8 days in vitro without pioneer plaque bacteria. P. gingivalis seemed to prefer multispecies biofilm environment whereas P. micra and A. actinomycetemcomitans planktonic culture.

Genome-wide association study of periodontal pathogen colonization.

Pathological shifts of the human microbiome are characteristic of many diseases, including chronic periodontitis. To date, there is limited evidence on host genetic risk loci associated with periodontal pathogen colonization. We conducted a genome-wide association (GWA) study among 1,020 white participants of the Atherosclerosis Risk in Communities Study, whose periodontal diagnosis ranged from healthy to severe chronic periodontitis, and for whom "checkerboard" DNA-DNA hybridization quantification of 8 periodontal pathogens was performed. We examined 3 traits: "high red" and "high orange" bacterial complexes, and "high" Aggregatibacter actinomycetemcomitans (Aa) colonization. Genotyping was performed on the Affymetrix 6.0 platform. Imputation to 2.5 million markers was based on HapMap II-CEU, and a multiple-test correction was applied (genome-wide threshold of p < 5 × 10(-8)). We detected no genome-wide significant signals. However, 13 loci, including KCNK1, FBXO38, UHRF2, IL33, RUNX2, TRPS1, CAMTA1, and VAMP3, provided suggestive evidence (p < 5 × 10(-6)) of association. All associations reported for "red" and "orange" complex microbiota, but not for Aa, had the same effect direction in a second sample of 123 African-American participants. None of these polymorphisms was associated with periodontitis diagnosis. Investigations replicating these findings may lead to an improved understanding of the complex nature of host-microbiome interactions that characterizes states of health and disease.

Three cases of severe invasive infections caused by Campylobacter rectus and first report of fatal C. rectus infection.

We report the first fatal case of Campylobacter rectus infection due to a subdural empyema and ruptured mycotic intracranial aneurysm and two cases of limb-threatening C. rectus necrotizing soft tissue and bone infection and empyema thoracis that responded to amoxicillin-clavulanate and surgical debridement and drainage. All three strains were identified by 16S rRNA sequencing.

Investigation of subgingival profile of periodontopathic bacteria using polymerase chain reaction.

Periodontopathic bacteria such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Campylobacter rectus and Treponema denticola play an important role in the initiation and progression of periodontitis. The aim of this investigation was to evaluate the relationship between periodontal clinical parameters and the subgingival profile of periodontopathic bacteria. Twenty-six periodontitis patients (23-62 years of age; mean age, 40.2±13.2) with no systemic disease agreed to participate in the study. Periodontal clinical parameters, including probing depth (PD) and bleeding on probing (BOP) were recorded. Subgingival plaque samples were obtained from deep (PD≥4 mm) and shallow (PD≤3 mm) pockets in each patient for detection of P. gingivalis, A. actinomycetemcomitans, T. forsythia, C. rectus and T. denticola by polymerase chain reaction technique. The relationship between the periodontal pathogens and clinical parameters was determined with the Fisher exact test, and a statistically significant association was found between detection of P. gingivalis, T. forsythia, C. rectus and T. denticola and PD or BOP. T. denticola was the most prevalent pathogen in both shallow PD and deep PD sites. No statistically significant association was found between detection of A. actinomycetemcomitans and the clinical parameters examined. A statistically significant association was found between detection of the red complex bacteria and the clinical parameters. These results suggest that the red complex pathogens and C. rectus play an important role in the initiation and progression of periodontitis.

Identification of novel genes in the oral pathogen Campylobacter rectus.

INTRODUCTION: A poorly described bacterium, Campylobacter rectus, has been implicated as an etiological agent of periodontal disease. The aim of this study was to use a comparative genomics approach to identify genes that contribute to the lifestyle of C. rectus as an oral pathogen. METHODS: Suppressive subtractive hybridization was used to identify genes encoded by C. rectus ATCC 33238, but not present in the genome of a related Campylobacter species, Campylobacter jejuni ATCC 11168. RESULTS: Suppressive subtractive hybridization identified 154 unique DNA sequences from the C. rectus genome. Ninety-two of the 154 clones were classified as C. rectus-specific, as they did not show significant sequence homology to genes identified in any strain of C. jejuni (blast E-value >1E-3). blast analysis predicted that the 92 C. rectus-specific gene fragments play a role in a variety of biological processes including signal transduction mechanisms (histidine kinase, response regulators, diguanylate cyclases, chemotaxis receptor) and potentially virulence (S-layer RTX and cysteine desulfhydrase). Further analysis of the C. rectus-specific clones showed that 10 genes had Campylobacter homologues that were only found in species that commonly reside within the oral cavity of humans and 10 other fragments shared homology only with non-campylobacter organisms. CONCLUSIONS: These data provide the first substantial insights into the genomic content of C. rectus, a significant oral pathogen. The genes identified in this study are a valuable resource for initiating new research on the virulence of C. rectus during periodontitis.

Periodontal microbiota in patients with coronary artery disease measured by real-time polymerase chain reaction: a case-control study.

BACKGROUND: Recent data have shown that periodontal disease may increase the risk of occurrence of coronary heart disease in which inflammation initiated by bacteria and their compounds might be a common causal factor. This case-control study aimed at studying the relationship between periodontal disease and coronary artery disease (CAD) based on clinical and periodontal microbiologic parameters. METHODS: A total of 90 male subjects, 48 to 80 years of age, were included in this study. Forty-five men had CAD (CAD+), which was confirmed by coronary angiography. Forty-five age-matched controls showed no history or symptoms of CAD (CAD-). All subjects underwent a clinical periodontal examination including assessment of tooth loss, probing depth, clinical attachment level, and bleeding on probing. In the CAD+ group, this examination took place 1 day before coronary angiography. Subgingival microbial samples were taken and evaluated by means of real-time polymerase chain reaction (RT-PCR) for the total amount of bacteria and the following periodontopathogens: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra (formerly Micromonas micros), Dialister pneumosintes, and Campylobacter rectus. RESULTS: Compared to control subjects, CAD+ subjects had significantly deeper pockets (2.28 mm versus 2.96 mm; P <0.001) and greater attachment loss (2.85 mm versus 3.65 mm; P <0.001), and this difference remained statistically significant after adjusting for smoking. No significant differences were observed between cases and controls with regard to the number of teeth present. P. intermedia was the only periodontal pathogen that showed significantly higher mean counts in CAD+ subjects compared to CAD- subjects. Higher counts of total bacteria, P. micra, D. pneumosintes, and C. rectus were found in the CAD- group. CONCLUSION: The results suggest that a relationship between periodontal disease and coronary heart disease exists, although P. intermedia was the only periodontopathogen related to CAD.

Subgingival distribution of Campylobacter rectus and Tannerella forsythensis in healthy children with primary dentition.

OBJECTIVE: It is important to know how many subgingival plaque samples should be assayed from a child to ascertain infection with a periodontal pathogen. Plaque samples from several sites may fail to detect some important bacteria if only a limited number of gingival sites are sampled. The purpose of this study was to evaluate the detection of periodontal pathogens in a large number of subgingival sites in the same children in order to determine the number of samples necessary. METHODS: Ten children, aged 4-6 years, with complete primary dentition were enrolled in this study. Plaque samples from the mesio-buccal aspect of each erupted tooth were first collected by gently inserting a sterile paper point for 10s. Purified genomic DNA from all plaque samples was prepared for polymerase chain reaction. The primers for species-specific 16S ribosomal RNA sequence were selected as the target sequence. Standard strains of Campylobacter rectus and Tannerella forsythensis (formerly Bacteroides forsythus) were used as control strains. RESULTS: All subjects were found positive for C. rectus and T. forsythensis with the mean of positive sites at 17.6 +/- 2.4 (range: 12-20 sites) for C. rectus and 9.3 +/- 5.0 (range: 1-19) for T. forsythensis. The mean number of positive sites was 1.7 +/- 0.8 for C. rectus and 6.5 +/- 4.9 for T. forsythensis, with a confidence ratio of 95%. CONCLUSIONS: We concluded that two or more random sites for C. rectus and seven or more random sites for T. forsythensis from children to detect those bacteria at 95% probability.

Evaluation of the whole genome DNA-DNA hybridization technique to identify bacteria in histological sections of periradicular lesions.

Whole genome DNA-DNA hybridization has been used to identify bacteria in periradicular lesions partly because there is no amplification of the bacteria, therefore, minor contaminants are not detected. There are, however, potential pitfalls with this technique, including inability to distinguish dead bacteria, cross-reactions of species within a genus, and inability to detect species present in low numbers because of loss of DNA during extraction and purification. Alternatively, inadequate extraction and purification of DNA could result in false positives. Therefore, controls are required to monitor DNA loss, DNA cross-reactions, and DNA of pure cultures mixed with bacteria-free tissue to monitor for false positives. We determined that the quality of DNA extracted from histological sections of periradicular lesions is excellent for DNA-DNA hybridization. Although lesions contain large numbers of bacteria, histological sections through lesions barely contain sufficient quantity of bacteria for such analysis. This was confirmed by histological observation of sparsely distributed bacteria within lesions. Furthermore, we found that the bacteria are not distributed evenly throughout periradicular lesions, in numbers or species.

Gene expression signatures in chronic and aggressive periodontitis: a pilot study.

This pilot study examined gene expression signatures in pathological gingival tissues of subjects with chronic or aggressive periodontitis, and explored whether new subclasses of periodontitis can be identified based on gene expression profiles. A total of 14 patients, seven with chronic and seven with aggressive periodontitis, were examined with respect to clinical periodontal status, composition of subgingival bacterial plaque assessed by checkerboard hybridizations, and levels of serum IgG antibodies to periodontal bacteria assayed by checkerboard immunoblotting. In addition, at least two pathological pockets/patient were biopsied, processed for RNA extraction, amplification and labeling, and used to study gene expression using Affymetrix U-133 A arrays. Based on a total of 35 microarrays, no significantly different gene expression profiles appeared to emerge between chronic and aggressive periodontitis. However, a de novo grouping of the 14 subjects into two fairly robust clusters was possible based on similarities in gene expression. These two groups had similar clinical periodontal status and subgingival bacterial profiles, but differed significantly with respect to serum IgG levels against the important periodontal pathogens Porphyromonas gingivalis, Tannerella forsythensis and Campylobacter rectus. These early data point to the usefulness of gene expression profiling techniques in the identification of subclasses of periodontitis with common pathobiology.

Campylobacter surface-layers (S-layers) and immune evasion.

Many pathogenic bacteria have evolved mechanisms for evading host immune systems. One evasion mechanism is manifest by the surface layer (S-layer), a paracrystalline protein structure composed of S-layer proteins (SLPs). The S-layer, possessed by 2 Campylobacter species (C. fetus and C. rectus), is external to the bacterial outer membrane and can have multiple functions in immune avoidance. C. fetus is a pathogen of ungulates and immunocompromised humans, in whom it causes disseminated bloodstream disease. In C. fetus, the S-layer is required for dissemination and is involved in 2 mechanisms of evasion. First, the S-layer confers resistance to complement-mediated killing in non-immune serum by preventing the binding of complement factor C3b to the C. fetus cell surface. S-layer expressing C. fetus strains remain susceptible to complement-independent killing, utilizing opsonic antibodies directed against the S-layer. However, C. fetus has also evolved a mechanism for avoiding antibody-mediated killing by high-frequency antigenic variation of SLPs. Antigenic variation is accomplished by complex DNA inversion events involving a family of multiple SLP-encoding genes and a single SLP promoter. Inversion events result in the expression of antigenically variant S-layers, which require distinct antibody responses for killing. C. rectus is implicated in the pathogenesis of periodontal disease and also possesses an S-layer that appears to be involved in evading the human system. Although studied less extensively than its C. fetus counterpart, the C. rectus S-layer appears to confer resistance to complement-mediated killing and to cause the down-regulation of proinflammatory cytokines.