Comparisons of bacterial patterns present at implant and tooth sites in subjects on supportive periodontal therapy. I. Impact of clinical variables, gender and smoking

OBJECTIVE: (I) To compare the oral microflora at implant and tooth sites in subjects participating in a periodontal recall program, (II) to test whether the microflora at implant and tooth sites differ as an effect of gingival bleeding (bleeding on probing (BOP)), or pocket probing depth (PPD), and (III) to test whether smoking and gender had an impact on the microflora. MATERIAL AND METHODS: Data were collected from 127 implants and all teeth in 56 subjects. Microbiological data were identified by the DNA-DNA checkerboard hybridization. RESULTS: PPD> or =4 mm were found in 16.9% of tooth, and at 26.6% of implant sites (P<0.01). Tooth sites with PPD> or =4 mm had a 3.1-fold higher bacterial load than implant sites (mean difference: 66%, 95% confidence interval (CI): 40.7-91.3, P<0.001). No differences were found for the red, orange, green, and yellow complexes. A higher total bacterial load was found at implant sites with PPD> or =4 mm (mean difference 35.7 x 10(5), 95% CI: 5.2 (10(5)) to 66.1 (10(5)), P<0.02 with equal variance not assumed). At implant sites, BOP had no impact on bacterial load but influenced the load at tooth sites (P<0.01). CONCLUSION: BOP, and smoking had no impact on bacteria at implant sites but influenced the bacterial load at tooth sites. Tooth sites harbored more bacteria than implant sites with comparable PPD. The 4 mm PPD cutoff level influenced the distribution and amounts of bacterial loads. The subject factor is explanatory to bacterial load at both tooth and implant sites.

Bacterial profile and burden of periodontal infection in subjects with a diagnosis of acute coronary syndrome

BACKGROUND: Periodontitis has been identified as a potential risk factor in cardiovascular diseases. It is possible that the stimulation of host responses to oral infections may result in vascular damage and the inducement of blood clotting. The aim of this study was to assess the role of periodontal infection and bacterial burden as an explanatory variable to the activation of the inflammatory process leading to acute coronary syndrome (ACS). METHODS: A total of 161 consecutive surviving cases admitted with a diagnosis of ACS and 161 control subjects, matched with cases according to their gender, socioeconomic level, and smoking status, were studied. Serum white blood cell (WBC) counts, high- and low-density lipoprotein (HDL/LDL) levels, high-sensitivity C-reactive protein (hsC-rp) levels, and clinical periodontal routine parameters were studied. The subgingival pathogens were assayed by the checkerboard DNA-DNA hybridization method. RESULTS: Total oral bacterial load was higher in the subjects with ACS (mean difference: 17.4x10(5); SD: 10.8; 95% confidence interval [CI]: 4.2 to 17.4; P<0.001), and significant for 26 of 40 species including Porphyromonas gingivalis, Tannerella forsythensis, and Treponema denticola. Serum WBC counts, hsC-rp levels, Streptococcus intermedius, and Streptococcus sanguis, were explanatory factors to acute coronary syndrome status (Nagelkerke r2=0.49). CONCLUSION: The oral bacterial load of S. intermedius, S. sanguis, Streptococcus anginosus, T. forsythensis, T. denticola, and P. gingivalis may be concomitant risk factors in the development of ACS.

Comparison of gingival crevicular fluid sampling methods in patients with severe chronic periodontitis

The analysis of samplings from periodontal pockets is important in the diagnosis and therapy of periodontitis. In this study, three different sampling techniques were compared to determine whether one method yielded samples suitable for the reproducible and simultaneous determination of bacterial load, cytokines, neutrophil elastase, and arginine-specific gingipains (Rgps). Rgps are an important virulence factor of Porphyromonas gingivalis, the exact concentration of which in gingival crevicular fluid (GCF) has not been quantified.

Comparison of real-time polymerase chain reaction and DNA-strip technology in microbiological evaluation of periodontitis treatment

The impact of a semiquantitative commercially available test based on DNA-strip technology (microIDent®, Hain Lifescience, Nehren, Germany) on diagnosis and treatment of severe chronic periodontitis of 25 periodontitis patients was evaluated in comparison with a quantitative in-house real-time PCR. Subgingival plaque samples were collected at baseline as well as at 3, 6, and 12 months later. After extracting DNA, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and several other periodontopathogens were determined by both methods. The results obtained by DNA-strip technology were analyzed semiquantitatively and additionally quantitatively by densitometry. The results for the 4 major periodontopathogenic bacterial species correlated significantly between the 2 methods. Samples detecting a high bacterial load by one method and negative by the other were always found in less than 2% of the total samples. Both technologies showed the impact of treatment on microflora. Especially the semiquantitative DNA-strip technology clearly analyzed the different loads of periodontopathogens after therapy and is useful in microbial diagnostics for patients in dental practices.

Acquisition of a multifunctional IgA+ plasma cell phenotype in the gut

The largest mucosal surface in the body is in the gastrointestinal tract, a location that is heavily colonized by microbes that are normally harmless. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the gastrointestinal tract is the production and transepithelial transport of poly-reactive IgA (ref. 1). Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T-cell help, undergo class switch recombination of their immunoglobulin receptor to IgA, and differentiate to become plasma cells. However, IgA-secreting plasma cells probably have additional attributes that are needed for coping with the tremendous bacterial load in the gastrointestinal tract. Here we report that mouse IgA(+) plasma cells also produce the antimicrobial mediators tumour-necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS), and express many molecules that are commonly associated with monocyte/granulocytic cell types. The development of iNOS-producing IgA(+) plasma cells can be recapitulated in vitro in the presence of gut stroma, and the acquisition of this multifunctional phenotype in vivo and in vitro relies on microbial co-stimulation. Deletion of TNF-α and iNOS in B-lineage cells resulted in a reduction in IgA production, altered diversification of the gut microbiota and poor clearance of a gut-tropic pathogen. These findings reveal a novel adaptation to maintaining homeostasis in the gut, and extend the repertoire of protective responses exhibited by some B-lineage cells.

Role of Toll interleukin-1 receptor (IL-1R) 8, a negative regulator of IL-1R/Toll-like receptor signaling, in resistance to acute Pseudomonas aeruginosa lung infection

Toll interleukin-1 receptor (IL-1R) 8 (TIR8), also known as single Ig IL-1 receptor (IL-R)-related molecule, or SIGIRR, is a member of the IL-1R-like family, primarily expressed by epithelial cells. Current evidence suggests that TIR8 plays a nonredundant role as a negative regulator in vivo under different inflammatory conditions that are dependent on IL-R and Toll-like receptor (TLR) activation. In the present study, we examined the role of TIR8 in innate resistance to acute lung infections caused by Pseudomonas aeruginosa, a Gram-negative pathogen responsible for life-threatening infections in immunocompromised individuals and cystic fibrosis patients. We show that Tir8 deficiency in mice was associated with increased susceptibility to acute P. aeruginosa infection, in terms of mortality and bacterial load, and to exacerbated local and systemic production of proinflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], IL-1β, and IL-6) and chemokines (CXCL1, CXCL2, and CCL2). It has been reported that host defense against P. aeruginosa acute lung infection can be improved by blocking IL-1 since exaggerated IL-1β production may be harmful for the host in this infection. In agreement with these data, IL-1RI deficiency rescues the phenotype observed in Tir8-deficient mice: in Tir8-/- IL-1RI-/- double knockout mice we observed higher survival rates, enhanced bacterial clearance, and reduced levels of local and systemic cytokine and chemokine levels than in Tir8-deficient mice. These results suggest that TIR8 has a nonredundant effect in modulating the inflammation caused by P. aeruginosa, in particular, by negatively regulating IL-1RI signaling, which plays a major role in the pathogenesis of this infectious disease.

Neuropathogenesis of naturally occurring encephalitis caused by Listeria monocytogenes in ruminants

Listeriosis is a serious food-borne disease with increasing frequency in humans and ruminants. Despite the facts that in both hosts, listeriosis can occur as rhombencephalitis and ruminants are a reservoir of Listeria monocytogenes (LM) strains pathogenic for humans, little work has been done on the pathogenesis in ruminants. This study investigates the neuropathogenesis of listeric encephalitis in over 200 natural cases in cattle, sheep and goats by analyzing anatomical distribution, severity, bacterial load and temporal evolution of the lesions. Our results suggest that LM gains access to the brainstem of all three species via axonal migration not only along the trigeminal nerve, but also along other nerves. The ensuing encephalitis does not remain restricted to the brainstem. Rather, LM spreads further from the brainstem into rostral brain regions likely by intracerebral axonal migration. Significant differences in severity of the lesions and bacterial load were found between cattle and small ruminants, which may be caused by species-specific properties of antibacterial immune responses. As histopathological lesions of human rhombencephalitis caused by LM strongly resemble those of ruminants, the disease likely has a similar pathogenesis in both hosts.

Development and application of a real-time TaqMan((R)) qPCR assay for detection and quantification of 'Candidatus Mycoplasma haemolamae' in South American camelids

Two alpacas from a herd in southwest Switzerland died for unknown reasons. Necropsy revealed chronic weight loss and pale mucous membranes. Infection with hemotropic mycoplasmas was suspected and subsequently confirmed by molecular methods. In order to investigate the epidemiological situation in this herd, a real-time TaqMan((R)) qPCR assay for the specific detection and quantification of hemoplasma infection in South American camelids was developed. This assay was based on the 16S rRNA gene and amplified 'Candidatus Mycoplasma haemolamae' DNA, but not DNA from other hemoplasmas or non-hemotropic mycoplasma species. The lower detection limit was one copy/PCR, and the amplification efficiency was 97.4%. In 11 out of 24 clinically healthy herd mates of the two infected alpacas, 'Candidatus M. haemolamae' infection was confirmed. No correlation was found between bacterial load and clinical signs or anemia. The assay described herein enables to detect and quantify 'Candidatus M. haemolamae' and may be used in future studies to investigate the prevalence, pathogenesis and treatment follow-up of hemoplasma infections in South American camelids.

Antimicrobial therapy using a local drug delivery system (Arestin) in the treatment of peri-implantitis. I: Microbiological outcomes

OBJECTIVES: To assess the microbiological outcome of local administration of minocycline hydrochloride microspheres 1 mg (Arestin) in cases with peri-implantitis and with a follow-up period of 12 months. MATERIAL AND METHODS: After debridement, and local administration of chlorhexidine gel, peri-implantitis cases were treated with local administration of minocycline microspheres (Arestin). The DNA-DNA checkerboard hybridization method was used to detect bacterial presence during the first 360 days of therapy. RESULTS: At Day 10, lower bacterial loads for 6/40 individual bacteria including Actinomyces gerensceriae (P<0.1), Actinomyces israelii (P<0.01), Actinomyces naeslundi type 1 (P<0.01) and type 2 (P<0.03), Actinomyces odontolyticus (P<0.01), Porphyromonas gingivalis (P<0.01) and Treponema socranskii (P<0.01) were found. At Day 360 only the levels of Actinobacillus actinomycetemcomitans were lower than at baseline (mean difference: 1x10(5); SE difference: 0.34x10(5), 95% CI: 0.2x10(5) to 1.2x10(5); P<0.03). Six implants were lost between Days 90 and 270. The microbiota was successfully controlled in 48%, and with definitive failures (implant loss and major increase in bacterial levels) in 32% of subjects. CONCLUSIONS: At study endpoint, the impact of Arestin on A. actinomycetemcomitans was greater than the impact on other pathogens. Up to Day 180 reductions in levels of Tannerella forsythia, P. gingivalis, and Treponema denticola were also found. Failures in treatment could not be associated with the presence of specific pathogens or by the total bacterial load at baseline. Statistical power analysis suggested that a case control study would require approximately 200 subjects.

Microbiological composition associated with interleukin-1 gene polymorphism in subjects undergoing supportive periodontal therapy

BACKGROUND: Interleukin-1 gene polymorphism (IL-1 gene) has been associated with periodontitis. The present study examined the subgingival microbiota by IL-1 gene status in subjects undergoing supportive periodontal therapy (SPT). METHODS: A total of 151 subjects with known IL-1 gene status (IL-1A +4845/IL-1B -3954) (IL-1 gene) were included in this study. Clinical data and subgingival plaque samples (40 taxa) were collected. These taxa were determined by the checkerboard DNA-DNA hybridization method. RESULTS: Gender, smoking habits (n-par tests), age, and clinical periodontal conditions did not differ by IL-1 gene status. IL-1 gene-negative subjects had a higher total bacterial load (mean difference, 480.4 x 10(5); 95% confidence interval [CI], 77 to 884 x 10(5); P <0.02). The levels of Actinobacillus actinomycetemcomitans (mean difference, 30.7 x 10(5); 95% CI, 2.2 to 59.5 x 10(5); P <0.05), Eubacterium nodatum (mean difference, 4.2 x 10(5); 95% CI, 0.6 to 7.8 x 10(5); P <0.02), Porphyromonas gingivalis (mean difference, 17.9 x 10(5); 95% CI, 1.2 to 34.5 x 10(5); P <0.05), and Streptococcus anginosus (mean difference, 4.0 x 10(5); 95% CI, 0.2 to 7.2 x 10(5); P <0.05) were higher in IL-1 gene-negative subjects, an observation specifically found at sites with probing depths <5.0 mm. CONCLUSIONS: Bleeding on probing did not differ by IL gene status, reflecting clinical SPT efficacy. IL-1 gene-negative subjects had higher levels of periodontal pathogens. This may suggest that among subjects undergoing SPT, a lower bacterial load is required in IL-1 gene-positive subjects to develop the same level of periodontitis as in IL-1 gene-negative subjects.

Microbial colonization patterns predict the outcomes of surgical treatment of intrabony defects

AIM: To explore the impact of bacterial load and microbial colonization patterns on the clinical outcomes of periodontal surgery at deep intrabony defects. MATERIALS AND METHODS: One hundred and twenty-two patients with advanced chronic periodontitis and at least one intrabony defect of >3 mm were recruited in 10 centres. Before recruitment, the infection control phase of periodontal therapy was completed. After surgical access and debridement, the regenerative material was applied in the test subjects, and omitted in the controls. At baseline and 1 year following the interventions, clinical attachment levels (CAL), pocket probing depths (PPD), recession (REC), full-mouth plaque scores and full-mouth bleeding scores were assessed. Microbial colonization of the defect-associated pocket was assessed using a DNA-DNA checkerboard analysis. RESULTS: Total bacterial load and counts of red complex bacteria were negatively associated with CAL gains 1 year following treatment. The probability of achieving above median CAL gains (>3 mm) was significantly decreased by higher total bacterial counts, higher red complex and T. forsythensis counts immediately before surgery. CONCLUSIONS: Presence of high bacterial load and specific periodontal pathogen complexes in deep periodontal pockets associated with intrabony defects had a significant negative impact on the 1 year outcome of surgical/regenerative treatment.

Immunization of Macaca fascicularis against experimental periodontitis using a vaccine containing cysteine proteases purified from Porphyromonas gingivalis

INTRODUCTION: Periodontitis is a common infectious disease to which Porphyromonas gingivalis has been closely linked, in which the attachment tissues of the teeth and their alveolar bone housing are destroyed. We conducted a study to determine if immunization using a purified antigen could alter the onset and progression of the disease. METHODS: Using the ligature-induced model of periodontitis in Macaca fascicularis, we immunized five animals with cysteine protease purified from P. gingivalis and used an additional five animals as controls. Alveolar bone loss was measured by digital subtraction radiography. RESULTS: Immunization induced high titers of specific immunoglobuin G serum antibodies that were opsonic. Total bacterial load, levels of P. gingivalis in subgingival plaque and levels of prostaglandin E(2) in gingival crevicular fluid were significantly reduced. Onset and progression of alveolar bone loss was inhibited by approximately 50%. No manifestations of toxicity were observed. CONCLUSIONS: Immunization using a purified protein antigen from P. gingivalis inhibits alveolar bone destruction in a ligature-induced periodontitis model in M. fascicularis.

Adjuvant TACE inhibitor treatment improves the outcome of TLR2-/- mice with experimental pneumococcal meningitis

BACKGROUND: Streptococcus (S.) pneumoniae meningitis has a high lethality despite antibiotic treatment. Inflammation is a major pathogenetic factor, which is unresponsive to antibiotics. Therefore adjunctive therapies with antiinflammatory compounds have been developed. TNF484 is a TNF-alpha converting enzyme (TACE) inhibitor and has been found efficacious in experimental meningitis. Toll-like receptor 2 (TLR2) contributes to host response in pneumococcal meningitis by enhancing bacterial clearing and downmodulating inflammation. In this study, TNF484 was applied in mice, which lacked TLR2 and exhibited a strong meningeal inflammation. METHODS: 103 CFU S. pneumoniae serotype 3 was inoculated subarachnoidally into C57BL/6 wild type (wt) mice or TLR2-/-, CD14-/- and CD14-/-/TLR2-/- mice. Severity of disease and survival was followed over 9 days. Response to antibiotics (80 mg/kg ceftriaxone i.p. for 5 days) and/or TACE inhibitor treatment (1 mg/kg s.c. twice daily for 4 days) was evaluated. Animals were sacrificed after 12, 24, and 48 h for analysis of bacterial load in cerebrospinal fluid (CSF) and brain and for TNF and leukocyte measurements in CSF. RESULTS: TLR2-/- mice were significantly sicker than the other mouse strains 24 h after infection. All knockout mice showed higher disease severity after 48 h and died earlier than wt mice. TNF release into CSF was significantly more elevated in TLR2-/- than in the other strains after 24 h. Brain bacterial numbers were significantly higher in all knockout than wt mice after 24 h. Modulation of outcome by antibiotic and TACE inhibitor treatment was evaluated. With antibiotic therapy all wt, CD14-/- and TLR2-/-/CD14-/- mice, but only 79% of TLR2-/- mice, were rescued. TACE inhibitor treatment alone did not rescue, but prolonged survival in wt mice, and in TLR2-/- and CD14-/- mice to the values observed in untreated wt mice. By combined antibiotic and TACE inhibitor treatment 95% of TLR2-/- mice were rescued. CONCLUSION: During pneumococcal meningitis strong inflammation in TLR2-deficiency was associated with incomplete responsiveness to antibiotics and complete response to combined antibiotic and TACE inhibitor treatment. TACE inhibitor treatment offers a promising adjuvant therapeutic strategy in pneumococcal meningitis.

Similarities in the Subgingival Microbiota Assessed by a Curet Sampling Method at Sites With Chronic Periodontitis

Background: The goal of this study was to determine whether site-specific differences in the subgingival microbiota could be detected by the checkerboard method in subjects with periodontitis. Methods: Subjects with at least six periodontal pockets with a probing depth (PD) between 5 and 7 mm were enrolled in the study. Subgingival plaque samples were collected with sterile curets by a single-stroke procedure at six selected periodontal sites from 161 subjects (966 subgingival sites). Subgingival bacterial samples were assayed with the checkerboard DNA-DNA hybridization method identifying 37 species. Results: Probing depths of 5, 6, and 7 mm were found at 50% (n = 483), 34% (n = 328), and 16% (n = 155) of sites, respectively. Statistical analysis failed to demonstrate differences in the sum of bacterial counts by tooth type (P = 0.18) or specific location of the sample (P = 0.78). With the exceptions of Campylobacter gracilis (P <0.001) and Actinomyces naeslundii (P <0.001), analysis by general linear model multivariate regression failed to identify subject or sample location factors as explanatory to microbiologic results. A trend of difference in bacterial load by tooth type was found for Prevotella nigrescens (P <0.01). At a cutoff level of >/=1.0 x 10(5), Porphyromonas gingivalis and Tannerella forsythia (previously T. forsythensis) were present at 48.0% to 56.3% and 46.0% to 51.2% of sampled sites, respectively. Conclusions: Given the similarities in the clinical evidence of periodontitis, the presence and levels of 37 species commonly studied in periodontitis are similar, with no differences between molar, premolar, and incisor/cuspid subgingival sites. This may facilitate microbiologic sampling strategies in subjects during periodontal therapy.

Clinical and microbiological analysis of subjects treated with Brånemark or AstraTech implants: a 7-year follow-up study

AIMS: To assess the impact of different implant systems on the clinical conditions and the microbiota at implants, and whether the presence of bacteria at tooth sites was predictive of the presence at implant sites. MATERIALS AND METHODS: Subjects with either AstraTech or Brånemark in function for 7 years were enrolled. Sub-gingival bacterial samples at tooth and implant sites were collected with sterile endodontic paper points, and analyzed by the checkerboard DNA-DNA hybridization method (40 species). RESULTS: Fifty-four subjects, 27 supplied with AstraTech (n=132 implants) and 27 with Brånemark (n=102) implants, were studied. Test tooth sites had significantly less evidence of bleeding on probing (P<0.001) and presence of plaque (P<0.001) than implant test sites. Implant sites presented with deeper probing pocket depth than tooth sites (mean difference: 1.1 mm, standard error of differences: 0.08, 95% confidence intervals (CI): 0.9-1.3, P<0.001). Tannerella forsythia (P<0.05), Capnocytophaga sputigena (P<0.05), Actinomyces israelii (P<0.05) and Lactobacillus acidophilus (P<0.05) were found at higher levels at tooth surfaces. No differences in bacterial load for any species were found between the two implant systems. The odds of being present/absent at tooth and implants sites were only significant for Staphylococcus aureus [odds ratio (OR): 5.2 : 1, 95% CI: 1.4-18.9, P<0.01]. CONCLUSIONS: After 7 years in function, implants presented with deeper probing depths than teeth. S. aureus was commonly present at both teeth and implants sites. S. aureus at tooth sites was predictive of also being present at implant sites.