Reclassification of Actinobacillus muris as Muribacter muris gen. nov. comb. nov.

To reinvestigate the taxonomy of [Actinobacillus] muris, 474 strains mainly from mice and rats were characterized by phenotype and 130 strains selected for genotypic characterization by 16S rRNA and partial rpoB gene sequencing. The type strain was further investigated by whole genome sequencing. Phylogenetic analysis of the DNA sequences showed one monophyletic group with intra group similarities of 96.7 % and 97.2 % for 16S rRNA and rpoB genes, respectively. The lowest 16S rRNA similarity to the closest related valid named taxon outside the group was 95.9 % to the type strain of [Pasteurella] pneumotropica. The closest related taxon based on rpoB sequence comparison was 'Haemophilus influenzae-murium' with 88.4 %. A new genus, Muribacter is proposed based on a distinct phylogenetic position based on 16S rRNA and rpoB gene sequence comparisons with major divergence to the existing genera of Pasteurellaceae. The new genus includes the characteristics of [Actinobacillus] muris with the emendation that acid formation from (-)-D-mannitol is variable as well the hydrolysis of esculin while the α-glucosidase test is positive. There is no requirement for exogenously supplied nicotinamide adenine dinucleotide (V factor) for the majority of strains investigated, however, one strain was found positive. The major fatty acids of the type strain of Muribacter muris were C 14:0, C 14:0 3OH/C 16:1 ISOI, C 16:1 ω7c and C 16:0 which is in line with most genera of Pasteurellaceae. The type strain of Muribacter muris is CCUG 16938T ( = NCTC 12432T = ATCC 49577T).

Clinical and microbiological findings at sites treated with orthodontic fixed appliances in adolescents

INTRODUCTION: Fixed orthodontic appliances can alter the subgingival microbiota. Our aim was to compare the subgingival microbiota and clinical parameters in adolescent subjects at sites of teeth treated with orthodontic bands with margins at (OBM) or below the gingival margin (OBSM), or with brackets (OBR). METHODS: Microbial samples were collected from 33 subjects (ages, 12-18 years) in treatment more than 6 months. The microbiota was assessed by the DNA-DNA checkerboard hybridization method. RESULTS: Bacterial samples were taken from 83 OBR,103 OBSM, and 54 OBM sites. Probing pocket depths differed by orthodontic type (P <0.001) with mean values of 2.9 mm (SD, 0.6) at OBSM sites, 2.5 mm (SD, 0.6) at OBM sites, and 2.3 mm (SD, 0.5) at OBR sites. Only Actinomyces israelii (P <0.001) and Actinomyces naeslundii (P <0.001) had higher levels at OBR sites, whereas Neisseria mucosa had higher levels at sites treated with OBSM or OBM (P <0.001). Aggregatibacter actinomycetemcomitans was found in 25% of sites independent of the appliance. CONCLUSIONS: Different types of orthodontic appliances cause minor differences in the subgingival microbiota (A israelii and A naeslundii) and higher levels at sites treated with orthodontic brackets. More sites with bleeding on probing and deeper pockets were found around orthodontic bands.

Short-term effects of systemic antibiotics during periodontal healing

OBJECTIVES: To investigate the short-term effects of nonsurgical therapy (scaling and root planing, SRP) on the subgingival microbiota in chronic (CP) and aggressive (AP) periodontal disease. METHOD AND MATERIALS: Ninety-seven CP and AP subjects underwent full-mouth SRP on 2 consecutive days. AP patients were randomly assigned to either receive systemic metronidazole plus amoxicillin (AP+AB) or were treated mechanically alone (AP). Pathogens were identified with 16S rRNA oligodeoxynucleotide probes and dot-blot hybridization before and at days 2, 3, 4, 7, 10, and 21 of healing. CP subjects were treated by scaling and root planing along with placebo tablets. RESULTS: Initially, AP cell counts were 69.9- (Porphyromonas gingivalis), 10.2- (Aggregatibacter actinomycetemcomitans), 5.7- (Tannerella forsythia), and 3.3-fold (Prevotella intermedia) enhanced compared to CP cell counts. Following SRP, immediate elimination occurred in single individuals of all three treatment groups at day 2. After SRP plus antibiotic therapy (AP+AB), the prevalence scores dropped beyond the levels of AP and CP, beginning at day 7, and remained low until day 21 (P =or< .05). Clinical healing statistically benefited from SRP with no differences among the three treatment groups. CONCLUSION: Nonsurgical therapy resulted in both a suppression and early elimination of single taxa immediately after completion of active treatment. Systemic antibiotics significantly accelerate the suppression of the periodontal microflora, but have limited effect on the elimination of target isolates during healing.

Mechanical non-surgical treatment of peri-implantitis: a single-blinded randomized longitudinal clinical study. II. Microbiological results

BACKGROUND: Peri-implantitis is common in patients with dental implants. We performed a single-blinded longitudinal randomized study to assess the effects of mechanical debridement on the peri-implant microbiota in peri-implantitis lesions. MATERIALS AND METHODS: An expanded checkerboard DNA-DNA hybridization assay encompassing 79 different microorganisms was used to study bacterial counts before and during 6 months following mechanical treatment of peri-implantitis in 17 cases treated with curettes and 14 cases treated with an ultrasonic device. Statistics included non-parametric tests and GLM multivariate analysis with p<0001 indicating significance and 80% power. RESULTS: At selected implant test sites, the most prevalent bacteria were: Fusobacterium nucleatum sp., Staphylococci sp., Aggregatibacter actinomycetemcomitans, Helicobacter pylori, and Tannerella forsythia. 30 min. after treatment with curettes, A. actinomycetemcomitans (serotype a), Lactobacillus acidophilus, Streptococcus anginosus, and Veillonella parvula were found at lower counts (p<0.001). No such differences were found for implants treated with the ultrasonic device. Inconsistent changes occurred following the first week. No microbiological differences between baseline and 6-month samples were found for any species or between treatment study methods in peri-implantitis. CONCLUSIONS: Both methods failed to eliminate or reduce bacterial counts in peri-implantitis. No group differences were found in the ability to reduce the microbiota in peri-implantitis.

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.

Cleavage of IgG1 in gingival crevicular fluid is associated with the presence of Porphyromonas gingivalis

BACKGROUND AND OBJECTIVES: Immunoglobulin (Ig) G1 plays an important role in the adaptive immune response. Kgp, a lysine-specific cysteine protease from Porphyromonas gingivalis, specifically hydrolyses IgG1 heavy chains. The purpose of this study was to examine whether cleavage of IgG1 occurs in gingival crevicular fluid (GCF) in vivo, and whether there is any association with the presence of Porphyromonas gingivalis and other periodontopathogens. MATERIAL AND METHODS: GCF was obtained from nine patients with aggressive periodontitis, nine with chronic periodontitis and five periodontally healthy individuals. The bacterial loads of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Prevotella intermedia and Tannerella forsythia were analysed by real-time polymerase chain reaction, and the presence and cleavage of IgG1 and IgG2 were determined using Western blotting. Kgp levels were measured by ELISA. RESULTS: Cleaved IgG1 was identified in the GCF from 67% of patients with aggressive periodontitis and in 44% of patients with chronic periodontitis. By contrast, no cleaved IgG1 was detectable in healthy controls. No degradation of IgG2 was detected in any of the samples, regardless of health status. Porphyromonas gingivalis was found in high numbers in all samples in which cleavage of IgG1 was detected (P < 0.001 compared with samples with no IgG cleavage). Furthermore, high numbers of Tannerella forsythia and Prevotella intermedia were also present in these samples. The level of Kgp in the GCF correlated with the load of Porphyromonas gingivalis (r = 0.425, P < 0.01). The presence of Kgp (range 0.07-10.98 ng/mL) was associated with proteolytic fragments of IgG1 (P < 0.001). However, cleaved IgG1 was also detected in samples with no detectable Kgp. CONCLUSION: In patients with periodontitis, cleavage of IgG1 occurs in vivo and may suppress antibody-dependent antibacterial activity in subgingival biofilms especially those colonized by Porphyromonas gingivalis.

Oral microbiota in Swiss adolescents

OBJECTIVES: The purpose of the study was to determine the prevalence of different oral microbes in gingival plaque samples and in samples from the dorsum of the tongue in a Swiss adolescent population. MATERIALS AND METHODS: Ninety-nine adolescents between 15 and 18 years were enrolled. Plaque index, bleeding on probing (BOP), the periodontal screening index, and decayed missed filled tooth (DMFT) index were recorded. Samples from subgingival plaque and swabs from the tongue were analyzed by the Checkerboard DNA-DNA hybridization method. Additionally, counts of Streptococus mutans and Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola were determined by real-time PCR. RESULTS: Periodontitis was not diagnosed in any of the subjects but all of them presented signs of gingival inflammation displaying a mean BOP of 28%. Ten (10.1%) subjects were tested positive for P. gingivalis, each 22 (22.2%) for A. actinomycetemcomitans and T. forsythia, (47.5%) for T. denticola. T. denticola and S. mutans showed a high affinity to the gingival plaque, whereas T. forsythia was often detected from the dorsum of the tongue. DMFT was associated with S. mutans counts, and BOP correlated with counts of P. gingivalis and T. denticola. CONCLUSIONS: The present data indicate that: (a) gingivitis but not periodontitis is a common finding among Swiss adolescents, and (b) bacteria associated with periodontitis were frequently detected in the subgingival dental plaque and on the dorsum of the tongue in Swiss adolescents with gingivitis. CLINICAL RELEVANCE: Although gingivitis was a frequent finding in Swiss adolescents, periodontitis was not detected in this population. The dorsum of the tongue appears to represent an important reservoir for periodontopathic bacteria.

Periodontal pathogens affect the level of protease inhibitors in gingival crevicular fluid

In periodontitis, an effective host-response is primarily related to neutrophils loaded with serine proteases, including elastase (NE) and protease 3 (PR3), the extracellular activity of which is tightly controlled by endogenous inhibitors. In vitro these inhibitors are degraded by gingipains, cysteine proteases produced by Porphyromonas gingivalis. The purpose of this study was to determine the level of selected protease inhibitors in gingival crevicular fluid (GCF) in relation to periodontal infection. The GCF collected from 31 subjects (nine healthy controls, seven with gingivitis, five with aggressive periodontitis and 10 with chronic periodontitis) was analyzed for the levels of elafin and secretory leukocyte protease inhibitor (SLPI), two main tissue-derived inhibitors of neutrophil serine proteases. In parallel, activity of NE, PR3 and arginine-specific gingipains (Rgps) in GCF was measured. Finally loads of P. gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola were determined. The highest values of elafin were found in aggressive periodontitis and the lowest in controls. The quantity of elafin correlated positively with the load of P. gingivalis, Ta. forsythia and Tr. denticola, as well as with Rgps activity. In addition, NE activity was positively associated with the counts of those bacterial species, but not with the amount of elafin. In contrast, the highest concentrations of SLPI were found in periodontally healthy subjects whereas amounts of this inhibitor were significantly decreased in patients infected with P. gingivalis. Periodontopathogenic bacteria stimulate the release of NE and PR3, which activities escape the control through degradation of locally produced inhibitors (SLPI and elafin) by host-derived and bacteria-derived proteases.

Efficacy of taurolidine against periodontopathic species--an in vitro study

The antimicrobial effect of taurolidine was tested against periodontopathic species in comparison to chlorhexidine digluconate in the presence or absence of serum. Minimal inhibitory concentrations (MIC), microbiocidal concentrations (MBC), as well as killing were determined against 32 different microbial strains including 3 Porphyromonas gingivalis, 3 Aggregatibacter actinomycetemcomitans, and 15 potentially superinfecting species with and without 25% v/v human serum. The MIC(50) of taurolidine against the tested microbial strains was 0.025% and the MIC(90) 0.05%. The respective values for the MBCs were 0.05% and 0.1%. Addition of 25% serum (heat-inactivated) did not change the MIC and MBC values of taurolidine. In contrast, MICs and MBCs of chlorhexidine (CHX) increased by two steps after addition of serum. Taurolidine killed microorganisms in a concentration and time-dependent manner, the killing rate of 1.6% taurolidine was 99.08% ± 2.27% in mean after 2 h. Again, killing activity of taurolidine was not affected if serum was added, whereas addition of inactivated serum clearly reduced the killing rate of all selected bacterial strains by CHX. Therefore, taurolidine possesses antimicrobial properties which are not reduced in the presence of serum as a main component in gingival crevicular fluid and wound fluid. Taurolidine may have potential as an antimicrobial agent in non-surgical and surgical periodontal treatment.

Effect of ozone on periodontopathogenic species--an in vitro study

The in vitro study was aimed to determine the effect of ozone on periodontopathogenic microorganisms. Ozone was generated for 6 s-2 × 24 s (corresponding to 0.56 mg-2 × 2.24 mg of ozone) against 23 mainly anaerobic periodontopathogenic species. Agar diffusion test was used as a screening method. Then, the killing activity was tested in a serum-free environment and with 25% v/v inactivated serum. Further, the effect of ozone on bactericidal activity of native serum was analyzed against Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans. Agar diffusion test showed a high efficacy of ozone against microorganisms, especially against Porphyromonas gingivalis. This result was confirmed by the killing tests; most of the strains in a concentration of 10(5) were completely eliminated after twofold 18-s application of ozone. Only four of the six potentially "superinfecting" species (Staphylococcus aureus, Enterococcus faecalis, Enterobacter cloacae, Candida albicans) survived in part. Addition of heat-inactivated serum reduced the killing rate of ozone by 78% after 6-s and by 47% after twofold 18-s exposures; no strain was completely eradicated after any application of ozone. The bactericidal effect of native serum was enhanced after application of ozone; no effect was visible on the included A. actinomycetemcomitans strain which was found to be completely resistant to the bactericidal action of serum. In conclusion, (a) ozone has a strong antibacterial activity against putative periodontopathogenic microorganisms, and (b) the bactericidal effect is reduced in the presence of serum. Ozone may have potential as an adjunctive application to mechanical treatment in periodontitis patients.

The role of RTX toxins in host specificity of animal pathogenic Pasteurellaceae

RTX toxins are bacterial pore-forming toxins that are particularly abundant among pathogenic species of Pasteurellaceae, in which they play a major role in virulence. RTX toxins of several primary pathogens of the family of Pasteurellaceae are directly involved in causing necrotic lesions in the target organs. Many RTX toxins are known as haemolysins because they lyse erythrocytes in vitro, an effect that is non-specific, but which serves as a useful marker in bacteriological identification and as an easily measurable signal in vitro in experimental studies. More recent studies have shown that the specific targets of most RTX toxins are leukocytes, with RTX toxins binding to the corresponding beta-subunit (CD18) of beta2 integrins and then exerting cytotoxic activity. After uptake by the target cell, at sub-lytic concentrations, some RTX toxins are transported to mitochondria and induce apoptosis. For several RTX toxins the binding to CD18 has been shown to be host specific and this seems to be the basis for the host range specificity of these RTX toxins. Observations on two very closely related species of the Pasteurellaceae family, Actinobacillus suis, a porcine pathogen particularly affecting suckling pigs, and Actinobacillus equuli subsp. haemolytica, which causes pyosepticaemia in new-born foals (sleepy foal disease), have revealed that they express different RTX toxins, named ApxI/II and Aqx, respectively. These RTX toxins are specifically cytotoxic for porcine and equine leukocytes, respectively. Furthermore, the ApxI and Aqx toxins of these species, when expressed in an isogenetic background in Escherichia coli, are specifically cytotoxic for leukocytes of their respective hosts. These data indicate the determinative role of RTX toxins in host specificity of pathogenic species of Pasteurellaceae.

Clonal analysis of Inquilinus limosus isolates from six cystic fibrosis patients and specific serum antibody response

Inquilinus limosus is a novel Gram-negative bacterium of the subdivision alpha-Proteobacteria recently found in the airways of patients with cystic fibrosis (CF). Here, the authors report on the clinical courses of six CF patients colonized with I. limosus. Five patients suffered from either an acute respiratory exacerbation or a progressive loss of pulmonary function, whereas one patient was in a stable clinical situation. This study focused on two aims: (i) the clonal analysis of I. limosus isolates by random amplified polymorphic DNA (RAPD)-PCR, and (ii) the clarification of whether the presence of I. limosus in the respiratory tract is associated with a specific serum antibody response. Serum IgG was detected by immunoblotting using I. limosus whole-cell-lysate proteins as antigens. Sera from healthy blood donors (n=10) and from CF patients colonized with Pseudomonas aeruginosa (n=10) were found to be immunoblot negative. All six Inquilinus-positive patients raised serum IgG antibodies against various I. limosus antigens. Surprisingly, in one patient, a specific I. limosus serum antibody response was already detected 1 year prior to Inquilinus-positive sputum cultures. Two prominent antigens were characterized by MALDI-MS: a 23 kDa protein revealed homology to the outer membrane lipoprotein OmlA of Actinobacillus pleuropneumoniae, and an 18 kDa protein to a protein-tyrosine phosphatase of Burkholderia cepacia. In conclusion, detection of I. limosus is accompanied by a specific serum antibody response and may reflect the infectious/pathogenic potential of I. limosus. Moreover, IgG immunoblotting may be useful to detect early infection with I. limosus and may support the selective cultivation of this novel emerging pathogen.

Two-year outcome with Nobel Direct implants: a retrospective radiographic and microbiologic study in 10 patients

INTRODUCTION: The Nobel Direct implant (Nobel Biocare AB, Göteborg, Sweden) was developed to minimize marginal bone resorption and to result in "soft tissue integration" for an optimized aesthetic outcome. However, conflicting results have been presented in the literature. The aim of this present study was to evaluate the clinical and microbiologic outcomes of Nobel Direct implants. MATERIALS AND METHODS: Ten partially edentulous subjects without evidence of active periodontitis (mean age 55 years) received 12 Nobel Direct implants. Implants were loaded with single crowns after a healing period of 3 to 6 months. Treatment outcomes were assessed at month 24. Routine clinical assessments, intraoral radiographs, and microbiologic samplings were made. Histologic analysis of one failing implant and chemical spectroscopy around three unused implants was performed. Paired Wilcoxon signed-rank test was used for the evaluation of bone loss; otherwise, descriptive analysis was performed. RESULTS: Implants were functionally loaded after 3 to 6 months. At 2 years, the mean bone loss of remaining implants was 2.0 mm (SD +/- 1.1 mm; range: 0.0-3.4 mm). Three out of 12 implants with an early mean bone loss >3 mm were lost. The surviving implants showed increasing bone loss between 6 and 24 months (p = .028). Only 3 out of the 12 implants were considered successful and showed bone loss of <1.7 mm after 2 years. High rates of pathogens, including Aggregatibacter actinomycetemcomitans, Fusobacterium spp., Porphyromonas gingivalis, Pseudomonas aeruginosa, and Tanerella forsythia, were found. Chemical spectroscopy revealed, despite the normal signals from Ti, O, and C, also peaks of P, F, S, N, and Ca. A normal histologic image of osseointegration was observed in the apical part of the retrieved implant. CONCLUSION: Radiographic evidence and 25% implant failures are indications of a low success rate. High counts and prevalence of significant pathogens were found at surviving implants. Although extensive bone loss had occurred in the coronal part, the apical portion of the implant showed some bone to implant integration.

Routine phenotypic identification of bacterial species of the family Pasteurellaceae isolated from animals

Pasteurellaceae are bacteria with an important role as primary or opportunistic, mainly respiratory, pathogens in domestic and wild animals. Some species of Pasteurellaceae cause severe diseases with high economic losses in commercial animal husbandry and are of great diagnostic concern. Because of new data on the phylogeny of Pasteurellaceae, their taxonomy has recently been revised profoundly, thus requiring an improved phenotypic differentiation procedure to identify the individual species of this family. A new and simplified procedure to identify species of Actinobacillus, Avibacterium, Gallibacterium, Haemophilus, Mannheimia, Nicoletella, and Pasteurella, which are most commonly isolated from clinical samples of diseased animals in veterinary diagnostic laboratories, is presented in the current study. The identification procedure was evaluated with 40 type and reference strains and with 267 strains from routine diagnostic analysis of various animal species, including 28 different bacterial species. Type, reference, and field strains were analyzed by 16S ribosomal RNA (rrs) and rpoB gene sequencing for unambiguous species determination as a basis to evaluate the phenotypic differentiation schema. Primary phenotypic differentiation is based on beta-nicotinamide adenine dinucleotide (beta-NAD) dependence and hemolysis, which are readily determined on the isolation medium. The procedure divides the 28 species into 4 groups for which particular biochemical reactions were chosen to identify the bacterial species. The phenotypic identification procedure allowed researchers to determine the species of 240 out of 267 field strains. The procedure is an easy and cost-effective system for the rapid identification of species of the Pasteurellaceae family isolated from clinical specimens of animals.

Comparative phylogenies of the housekeeping genes atpD, infB and rpoB and the 16S rRNA gene within the Pasteurellaceae

Phylogenies of housekeeping gene and 16S rRNA gene sequences were compared to improve the classification of the bacterial family Pasteurellaceae and knowledge of the evolutionary relationships of its members. Deduced partial protein sequences of the housekeeping genes atpD, infB and rpoB were compared in 28, 36 and 28 representative taxa of the Pasteurellaceae, respectively. The monophyly of representatives of the genus Gallibacterium was recognized by analysis of all housekeeping genes, while members of Mannheimia, Actinobacillus sensu stricto and the core group of Pasteurella sensu stricto formed monophyletic groups with two out of three housekeeping genes. Representatives of Mannheimia, Actinobacillus sensu stricto, [Haemophilus] ducreyi and [Pasteurella] trehalosi formed a monophyletic unit by analysis of all three housekeeping genes, which was in contrast to the 16S rRNA gene-derived phylogeny, where these taxa occurred at separate positions in the phylogenetic tree. Representatives of the Rodent, Avian and Aphrophilus-Haemophilus 16S rRNA gene groups were weakly supported by phylogenetic analysis of housekeeping genes. Phylogenies derived by comparison of the housekeeping genes diverged significantly from the 16S rRNA gene-derived phylogeny as evaluated by the likelihood ratio test. A low degree of congruence was also observed between the individual housekeeping gene-derived phylogenies. Estimates on speciation derived from 16S rRNA and housekeeping gene sequence comparisons resulted in quite different evolutionary scenarios for members of the Pasteurellaceae. The phylogeny based on the housekeeping genes supported observed host associations between Mannheimia, Actinobacillus sensu stricto and [Pasteurella] trehalosi and animals with paired hooves.