Centipeda periodontii in human periodontitis.

This study assessed the subgingival occurrence of the flagellated, Gram-negative, anaerobic rod Centipeda periodontii in chronic periodontitis and periodontal health/gingivitis with species-specific nucleic acid probes, and evaluated the in vitro resistance of subgingival isolates to therapeutic levels of amoxicillin, metronidazole, and doxycycline. Subgingival plaque biofilm specimens from 307 adults with chronic periodontitis, and 48 adults with periodontal health/localized gingivitis, were evaluated with digoxigenin-labeled, whole-chromosomal, DNA probes to C. periodontii ATCC 35019 possessing a 10(4) cell detection threshold. Fifty-two C. periodontii subgingival culture isolates were assessed on antibiotic-supplemented enriched Brucella blood agar for in vitro resistance to either amoxicillin at 2 µg/ml, metronidazole at 4 µg/ml, or doxycycline at 2 µg/ml. A significantly greater subgingival occurrence of C. periodontii was found in chronic periodontitis subjects as compared to individuals with periodontal health/gingivitis (13.4 vs. 0 %, P < 0.003), although high subgingival counts of the organism (≥ 10(6) cells) were rarely detected (1.3 % of chronic periodontitis subjects). In vitro resistance was not found to amoxicillin or metronidazole, and to doxycycline in only 2 (3.9 %) of the 52 C. periodontii clinical isolates studied. These findings indicate that C. periodontii is not a major constituent of the subgingival microbiome in chronic periodontitis or periodontal health/gingivitis. The potential contribution of C. periodontii to periodontal breakdown in the few chronic periodontitis subjects who yielded high subgingival levels of the organism remains to be delineated. C. periodontii clinical isolates were susceptible in vitro to therapeutic concentrations of three antibiotics frequently used in treatment of human periodontitis.

Insights into the virulence of oral biofilms: discoveries from proteomics.

This review covers developments in the study of polymicrobial communities, biofilms and selected areas of host response relevant to dental plaque and related areas of oral biology. The emphasis is on recent studies in which proteomic methods, particularly those using mass spectrometry as a readout, have played a major role in the investigation. The last 5-10 years have seen a transition of such methods from the periphery of oral biology to the mainstream, as in other areas of biomedical science. For reasons of focus and space, the authors do not discuss biomarker studies relevant to improved diagnostics for oral health, as this literature is rather substantial in its own right and deserves a separate treatment. Here, global gene regulation studies of plaque-component organisms, biofilm formation, multispecies interactions and host-microbe interactions are discussed. Several aspects of proteomics methodology that are relevant to the studies of multispecies systems are commented upon.

Periodontal disease and preterm birth relationship: a review of the literature.

Despite medical care improves consistently, the rate of preterm birth has risen in recent years. In Italy the rate of preterm birth between the XXXIII and the XXXVI week is 13.5%, while it amounts to 1.3% for preterm birth between XXIV and the XXXII week. Consequently, the identification of risk factors for preterm birth that might be modified would have far-reaching and long-lasting effects. A significant number of preterm birth may be attributed to infections of the urogenital tract, such as bacterial vaginosis. In the last decade, great interest has been generated to support the hypothesis that sub-clinical infection at sites that are also distant from the genito-urinary tract may be an important cause of preterm labour, probably through the activation of abnormal inflammatory responses within the uterus and intrauterine tissues. There is emerging evidence of a possible relationship between maternal periodontal diseases as a potential risk factor of adverse pregnancy outcomes, like preterm low birth weight even though not all of the actual data support such hypothesis. Further studies are clearly required to clarify the causes and/or relationships linking pathologic oral conditions and adverse pregnancy outcomes. So far, from a clinical standpoint, it would appear that the assessment of the periodontal status of pregnant women during an early pregnancy might be useful in providing an important indicator of risk for future obstetric complications.

Lesiones perirradiculares persistentes: revisión narrativa / Persistent periradicular lesions: a narrative review

La persistencia de lesiones perirradiculares luego del tra- tamiento endodóntico es un problema que requiere del clínico un conocimiento cabal de la histofisiología y de la histopato- logía del sistema de conductos radiculares del tejido pulpar y de los tejidos perirradiculares (periodonto y hueso); además de considerar siempre la posible existencia de enfermedades sistémicas que también pueden actuar como factores de in- fluencia. La presencia de bacterias remanentes a posteriori del tratamiento es considerada como una de las causas principales y más frecuentes para la perpetuación de las lesiones perirra- diculares. Sin embargo, existen otros factores causales, como la existencia de conductos laterales o accesorios infectados y no tratados, la reabsorción dentinaria interna, intercomunica- ciones, cul-de-sacs o istmos; que representan áreas de difícil acceso durante la instrumentación e irrigación. Cuando la cau- sa original se localiza en la zona perirradicular, como en los casos de actinomicosis, reacciones a cuerpo extraño, cristales de colesterol (CRCo) y granulomas o quistes con alto conte- nido de CRCo, la indicación más adecuada es el retratamiento y la cirugía periapical como complemento (AU)

The persistence of periradicular lesions after endodontic treatment is a problem that requires the doctor to have a thor- ough knowledge of the histophysiology and histopathology of the root canal system, the pulp tissue and periradicular tis- sues (periodontium and bone); as well as always considering the possible existence of systemic alterations that can also be influencing factors. Persisting bacteria within the root canal system after treatment is one of the major and most frequent causes for the perpetuation of periradicular lesions. Howev- er, there are other possible causal factors such as the exist- ence of untreated lateral or accessory canals, internal dentin resorption, intercommunications, cul-de-sacs or isthmuses; areas that represent a difficulty in access during instrumen- tation and irrigation. If the original cause is located in the periradicular area, in cases like actinomycosis, foreign-body reactions, cholesterol crystals (CRCo) and granulomas or cysts with high content of CRCo, retreatment coupled with periapical surgery is the best approach to treatment (AU)

Etiology of ventilator-associated pneumonia.

This review focuses on the top ten causes of ventilator-associated pneumonia (VAP), updating an earlier study. These pathogens have specific risk factors, different patterns of clinical resolution, and a wide range of attributable mortality. The discussion herein analyzes these aspects, placing particular emphasis on risk factors, attributable mortality, resistance, and the implications for management.

Physical and genetic map of the chromosome of Dichelobacter nodosus strain A198.

A physical map of the chromosome of Dichelobacter nodosus strain A198 was constructed using the restriction endonucleases EagI and StuI. Mapping data indicated the presence of a single, circular chromosome of 1.54 Mb. The three rRNA operons and the virulence related locus (vrl) were precisely positioned at the junctions of EagI and StuI fragments, and their transcriptional orientations were also determined. Other D. nodosus genes were assigned to specific EagI and StuI fragments. Analysis of the resultant map revealed that the putative virulence genes were not clustered on the chromosome which suggests that the D. nodosus virulence determinants have been acquired gradually and that virulence in D. nodosus is an evolving trait.

A single amino-acid change between the antigenically different extracellular serine proteases V2 and B2 from Dichelobacter nodosus.

Dichelobacter nodosus (Dn), the causative organism of ovine footrot, secrets three distinct types of extracellular serine proteases which have been implicated in virulence. Southern analyses have shown that the proteases are encoded by three separate genes, and the genes encoding an acidic protease V5 and a basic protease have already been characterised from virulent Dn strain 198. The gene encoding the third protease type, as represented by acidic protease V2, was isolated from an EcoRI-BamHI library of strain 198 genomic DNA by probing with a polymerase chain reaction (PCR) fragment generated with oligodeoxyribonucleotides based on protease V2 amino acid (aa) sequences. A further clone from an RsaI library was isolated to complete the 5' region of the gene to yield an ORF of 1803 bp encoding a protein precursor of 601 aa. The acidic protease V2 gene, aprV2, shows the same precursor structure as the bprV and aprV5 genes with 72% and 69% similarity at the nucleotide (nt) level and with 73% and 69% similarity at the aa level, respectively. As monoclonal antibodies consistently distinguish the virulent (V) and benign (B) forms of this protease, the gene encoding the acidic protease B2 from benign Dn strain 305 was isolated using the PCR and characterized to investigate the molecular basis for this difference in antigenicity. A 2-bp substitution in a single codon was identified which appeared to be responsible for a change of epitope.

Identification of a gene encoding a bacteriophage-related integrase in a vap region of the Dichelobacter nodosus genome.

Dichelobacter nodosus is the principal causative agent of ovine footrot. Nucleotide (nt) sequences from the D. nodosus genome have been isolated and a series of overlapping lambda clones defining vap (virulence-associated protein) regions 1, 2 and 3 have been reported [Katz et al., J. Bacteriol. 176 (1994) 2663-2669]. In the present study, the limits of the virulence-associated (va) DNA around vap regions 1 and 3 were determined by dot-blot hybridization experiments using plasmid subclones to probe genomic DNA from the D. nodosus virulent strain A198 and the benign strain C305. This va region was found to be approx. 11.9 kb in length, and to be interrupted by a short DNA segment which is also found in the benign D. nodosus strain. Sequence analysis of the entire region revealed an ORF, intA, which is very similar to the integrases of bacteriophages phi R73, P4 and Sf6. Bacteriophages phi R73 and P4 integrate into the 3' ends of tRNA genes, with the integrase genes adjacent to the tRNA genes. A similar arrangement was found in the D. nodosus va region. A 19-bp nt sequence was found to be repeated at the ends of the va region, and may represent the bacteriphage attachment site. These findings suggest that D. nodosus may have acquired these DNA sequences by the integration of a bacteriophage, or an integrative plasmid that contains a bacteriophage-related integrase gene. The high similarity of the D. nodosus integrase to integrases from coliphages suggests that these va sequences may be transferred between distantly related bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of anaerobic beta-lactamase-producing bacteria in upper respiratory tract infections.

Bacteroides sp. (Bacteroides melaninogenicus, Bacteroides oralis and Bacteroides fragilis), peptostreptococci and Fusobacterium sp. are important pathogens in upper respiratory tract infections. A recent increase in numbers of beta-lactamase-producing strains of anaerobic Gram-negative bacteria in upper respiratory tract infections has been associated with increased failure rates of penicillins in eradication of these infections. These infections include chronic otitis media, chronic sinusitis and mastoiditis, chronic recurrent tonsillitis and lung abscesses. The indirect pathogenicity of these organisms is apparent through their ability not only to survive penicillin therapy but also to protect penicillin-susceptible pathogens from the drug. These direct and indirect virulence characteristics of anaerobic bacteria require the administration of appropriate antimicrobial therapy directed against all pathogens in mixed infections.

Virulence regions and virulence factors of the ovine footrot pathogen, Dichelobacter nodosus.

Ovine footrot is a debilitating and highly infectious disease that is primarily caused by the Gram-negative, anaerobic bacterium Dichelobacter nodosus. The major antigens implicated in virulence are the type IV fimbriae and extracellular proteases. The fimbriae show sequence and structural similarity to other type IV fimbriae, this similarity extends to genes that are involved in fimbrial biogenesis. Several acidic and basic extracellular serine proteases are produced by both virulent and benign isolates of D. nodosus. Subtle functional differences in these proteases appear to be important in virulence. In addition, there are two chromosomal regions that have a genotypic association with virulence. The partially duplicated and rearranged vap regions appear to have arisen from the insertion of a plasmid into a tRNA gene via an integrase-mediated site-specific insertion event. The 27 kb vrl region has several genes often found on bacteriophages and has inserted into an ssrA gene that may have a regulatory role in the cell. The determination of the precise role that each of these genes and gene regions has in virulence awaits the development of methods for the genetic analysis and manipulation of D. nodosus.

Comparison of gene probe and conventional methods for the differentiation of ovine footrot isolates of Dichelobacter nodosus.

In a collaborative study that involved four Australian veterinary diagnostic laboratories a gene probe test based on the recombinant plasmids pJIR318, pJIR314B, and pJIR313, which contain genomic vap or vrl regions, was compared with conventional tests used for the differential diagnosis of ovine footrot. A total of 771 clinical dichelobacter nodosus isolates were tested and designated as belonging to one of several gene probe categories. The results showed that 87% of the virulent isolates belonged to gene probe category 1, compared to only 6% of the benign isolates. It was concluded that there was good correlation between the gene probe test and the virulence designation of these isolates as well as the results of elastase, gelatin-gel and protease isoenzyme tests. Furthermore, the gene probe test was converted to a polymerase chain reaction (PCR)-based test. It is suggested that diagnostic laboratories consider carrying out both this PCR test and tests based on the extracellular proteases of D. nodosus.

Rationale for use of antibiotics in periodontics.

The purpose of this review is to provide the clinician with some practical rationale for the selection and use of antibiotics in the treatment of destructive periodontal diseases. We have attempted to integrate approximately 20 years of periodontal literature describing antibiotic therapy with personal experience and 21st century ideas. This article addresses antibiotic use during treatment of aggressive periodontitis with emphasis on juvenile disease and adult refractory diseases. The literature review revealed few large, controlled studies that compared efficacy of adjunctive antibiotic use to mechanical therapy alone. Even fewer studies evaluated the efficacy of one antibiotic relative to another. However, based on the evidence available, certain conclusions were drawn. Adjunctive use of an antibiotic along with mechanical debridement is recommended for the treatment of Actinobacillus actinomycetemcomitans-associated periodontitis as an acceptable therapeutic regimen. Due to the emergence of tetracycline-resistant A. actinomycetemcomitans, the combination of metronidazole and amoxicillin may be preferable. In aggressive refractory periodontitis, compelling evidence exists that the use of an appropriate adjunctive antibiotic frequently gives a more favorable clinical response than mechanical therapy alone. Unfortunately, the selection of antibiotic is not as clear and is probably case-dependent. Positive responses have been reported with amoxicillin/clavulanic acid, clindamycin, metronidazole, and the combination therapy metronidazole plus amoxicillin. The introduction of local delivery antibiotics specifically for the treatment of periodontitis offers a novel concept for the treatment of localized disease. The latter, in particular, may prove useful in the treatment of recurrent disease activity or where only a few individual sites are involved.

Effect of initial periodontal therapy on the frequency of detecting Bacteroides forsythus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans.

BACKGROUND: Porphyromonas gingivalis, Bacteroides forsythus, and Actinobacillus actinomycetemcomitans have been described as periodontopathic bacteria, and their presence in subgingival pockets can lead to development of periodontal disease. Until now, clinical parameters have been used to evaluate the effect of conventional periodontal treatment without microbiological parameters. The present study examined the microbiological effects of initial periodontal therapy using DNA probes and the polymerase chain reaction (PCR). METHODS: Twenty-six patients with periodontitis, 10 males and 16 females, were given instructions regarding oral hygiene, then thoroughly treated by conventional scaling and root planing. Bacterial samples were collected on paper points from 4 sites per patient at baseline and after initial therapy (total: 104 sites). Clinical parameters including probing depth, attachment level, and bleeding on probing were also recorded for each site at baseline and after therapy. A DNA probe kit was used to monitor the frequency of B. forsythus, P. gingivalis, and A. actinomycetemcomitans, the last of which was identified by PCR. RESULTS: At baseline, B. forsythus was the bacterium most frequently detected. DNA probe analysis also showed that more than half of the sites were colonized by both B. forsythus and P. gingivalis. Initial therapy resulted in significant clinical improvement such as significant reduction in the frequency of B. forsythus and P. gingivalis detected using the DNA probe. A. actinomycetemcomitans was difficult to detect using the DNA probe, but PCR indicated that levels of A. actinomycetemcomitans did not significantly decrease. CONCLUSIONS: These results indicate that initial conventional therapy can eliminate B. forsythus and P. gingivalis, but not A. actinomycetemcomitans. When levels of these bacteria decreased to below-detectable levels, clinical improvement was significant. These results indicate that monitoring levels of these three periodontopathic bacteria may render periodontal therapy more effective and accurate.

Nested PCR detection of Centipeda periodontii in primary endodontic infections.

In recent years, molecular genetic methodologies have provided significant additional knowledge about components of the microbiota associated with infections of endodontic origin. Following this research line, the purpose of the present study was to investigate the prevalence of Centipeda periodontii in primary endodontic infections using a species-specific nested PCR assay. Samples were collected from fifty teeth having carious lesions, necrotic pulps, and different forms of periradicular diseases. DNA extracted from the samples was initially amplified using universal 16S rDNA primers, and a second round of amplification used the first PCR products to detect a specific fragment of C. periodontii 16S rDNA. This species was detected in 3 (13%) of 23 asymptomatic cases, in 1 (14%) of 7 cases diagnosed as acute apical periodontitis, and in 3 (15%) of 20 pus samples aspirated from acute periradicular abscesses. There was no significant association between C. periodontii and the presence of clinical symptoms. Overall, C. periodontii was detected in 14% of the cases of endodontic infections. This is probably the hitherto first study to detect C. periodontii in primary endodontic infections. The specific role played by this bacterial species in infections of endodontic origin awaits further clarification.

Cytotoxic effect of endodontic bacteria on periapical fibroblasts.

This study was conducted to investigate the effects of sonicated bacterial extracts (SBEs) from anaerobic Gram-negative bacteria on periapical fibroblast obtained from the apical portion of human periodontal ligaments. Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum were chosen from among the endodontic bacteria isolated from root canals having a periapical lesion and compared in terms of their cytotoxicity. The purpose of this study was to examine which bacteria are involved in the development of periapical inflammation. The anaerobes were cultured under strict anaerobic conditions, and the bacterial cells were then harvested by centrifugation after incubation. The concentrated cell suspensions were sonicated and subsequently centrifuged. An SBE was made of each of the filtered supernatants. Each SBE was added to cultures of periapical fibroblasts. The cell growth and proliferation were measured by the MTT method after 3, 5, and 7 days. The SBEs from P. endodontalis, P. gingivalis, and F. nucleatum inhibited the growth of the fibroblasts, whereas the SBE from P. intermedia did not inhibit it. The SBEs from P. gingivalis and F. nucleatum inhibited the fibroblast growth more strongly than did the P. endodontalis, P. gingivalis, and F. nucleatum may participate in the development of periapical lesions.

Influence of five antianaerobic antibiotics on endotoxin liberation by gram-negative anaerobes.

Endotoxin, a lipopolysaccharide (LPS), has for many years been recognized as a key effector molecule in the pathogenesis of gram-negative sepsis and septic shock. Seven strains of the Bacteroides fragilis group were studied for their ability to liberate endotoxin upon exposure to five anti-anaerobic antibiotics, trovafloxacin, cefoxitin, imipenem, meropenem and piperacillin/tazobactam, in an in-vitro experiment. The minimum inhibitory concentrations (MICs) of the antibiotics were determined by using the broth macrodilution technique. Thereafter, endotoxin liberation was detected in the filtered broth cultures of the anaerobic bacteria by the limulus amebocyte lysate (LAL) assay after exposing the organisms to four different concentrations of the antibiotics in supplemented Brucella broth. Aliquots of the broth cultures were also taken at intervals of 0, 6, 24 and 48 h for viable counts. All seven gram-negative anaerobic bacteria investigated liberated induced cell-free endotoxin in filtered broth culture many times higher than the control. There was noticeable variation in the propensity of some antibiotics to induce endotoxin liberation. At four times the MICs, cefoxitin and piperacillin/tazobactam induced negligible quantities of endotoxin after 48 h exposure, whereas the others induced high levels of endotoxin release. After exposure to all concentrations for 48 h, endotoxin activity in the test system was many times higher with the Bacteroides fragilis sensu stricto than with the rest of the species in the Bacteroides group. To varying degrees, all five antibiotics had the capacity to induce endotoxin liberation by gram-negative anaerobic bacteria. This differential endotoxin release by the B. fragilis group may, in part, explain why B. fragilis sensu stricto, more than the other Bacteroides spp., is usually associated with clinical infections and higher morbidity.

Use of elastase test, gelatin gel test and electrophoretic zymogram to determine virulence of Dichelobacter nodosus isolated from ovine foot rot.

A comparative study using the elastase test, gelatin gel test and electrophoretic zymogram was conducted for virulence determination of 96 Dichelobacter nodosus isolates from clinical cases of ovine foot rot. Despite being time consuming, the elastase test provided a classification of D nodosus bacteria in general agreement with the severity of clinical characteristics. The gelatin gel test showed some correlation with the elastase test. While isolates showing elastase activity at 14 days or less were likely to produce more proteases which were of higher thermostability, those showing elastase activity at 21 to 28 days, or elastase negative up to 28 days tended to produce less proteases which were also of lower thermostability. The electrophoretic zymogram based on the virulent pattern 1 (V1), virulent pattern 2 (V2) and benign pattern (B) did not cover all isolates from the field as nine (9.4 per cent) of the 96 isolates formed specific isoenzyme patterns that were different from the V1, V2 and B patterns of classification. However, the patterns formed by these nine isolates could be fitted into another previously reported isoenzyme classification. Nevertheless, the electrophoretic zymogram by itself did not provide adequate information in regard to intermediate foot rot. It appeared that a combination of two tests such as elastase test and zymogram or gelatin gel test and zymogram would offer a useful complementary definition of the virulence of D nodosus bacteria.

Polyclonal B cell activation, endotoxin tolerance, and limulus tests of endotoxin preparations of some periodontopathogens.

Potencies of polyclonal B-cell activation in C3H/HeN mice of Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis endotoxins were 0.36, 0.13 and 0.04, taking Salmonella abortusequi as 1.0. F. nucleatum and P. gingivalis endotoxins showed positive reactions in C3H/HeJ mice. Most activities in C3H/HeN other than that of F. nucleatum were suppressed by polymyxin B. In C3H/HeJ mice, similar inhibitions were only 60% for P. gingivalis and hardly observed with F. nucleatum. The resistances to polymyxin B could be due to protein in the endotoxins. A promoting effect of T cells added to B cells was observed only in the activity of F. nucleatum endotoxin in C3H/HeJ mice; there was no influence in other groups. Test endotoxins had nearly the same ability to produce colony stimulating factor as did references and could not produce the factor in tolerant mice. The clinical significance of tolerance is discussed. Regression lines of endotoxin doses and limulus activities of test endotoxins and Salmonella were parallel, either in specific or non-specific tests. The lines of two test groups were also parallel; values obtained by two tests were very close. These data indicate that the test endotoxins did not contain (1-3)-beta-D-glucan and elicited qualitatively similar limulus reactions to that of the reference, despite their different chemical natures. In conclusion, these test preparations had an endotoxicity similar to that of the reference and contribute to produce periodontitis through polyclonal B cell activation.

Bacteriological diagnosis of periodontal disease.

Dental plaque bacteria cause virtually all the forms of inflammatory periodontal disease. Periodontitis is caused by the specific periodontopathic bacteria, which induce destruction of connective tissue attachment and adjacent alveolar bone. Examinations to identify the infections by Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, Campylobacter rectus, Bacteroides forsythus, Fusobacterium nucleatum, Eikenella corrodens and Treponema denticola have recently become essential in diagnosis of periodontal disease. Bacterial examination permits (1) identification of the causative bacteria, (2) assessment of disease activity and (3) monitoring of the effective of periodontal treatments. The author describes details of accurate and rapid methods for detecting periodontopathogens. Transmission of periodontopathic bacteria as clarified by bacterial examination is also discussed in this review. The pathogenic potential of specific bacteria varies among patients and periodontally healthy individuals and can be controlled by host defense mechanisms such as immune responses. The roles of immune responses against periodontopathic bacteria in balance shifts of periodontal disease processes are therefore also discussed in this review.