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PCR methodology as a valuable tool for identification of endodontic pathogens.

OBJECTIVES: This paper reviews the principles of polymerase chain reaction (PCR) methodology, its application in identification of endodontic pathogens and the perspectives regarding the knowledge to be reached with the use of this highly sensitive, specific and accurate methodology as a microbial identification test. DATA SOURCES: Studies published in the medical, dental and biological literature. STUDY SELECTION: Evaluation of published epidemiological studies examining the endodontic microbiota through PCR methodology. CONCLUSIONS: PCR technology has enabled the detection of bacterial species that are difficult or even impossible to culture as well as cultivable bacterial strains showing a phenotypically divergent or convergent behaviour. Moreover, PCR is more rapid, much more sensitive, and more accurate when compared with culture. Its use in endodontics to investigate the microbiota associated with infected root canals has expanded the knowledge on the bacteria involved in the pathogenesis of periradicular diseases. For instance, Tannerella forsythensis (formerly Bacteroides forsythus), Treponema denticola, other Treponema species, Dialister pneumosintes, and Prevotella tannerae were detected in infected root canals for the first time and in high prevalence when using PCR analysis. The diversity of endodontic microbiota has been demonstrated by studies using PCR amplification, cloning and sequencing of the PCR products. Moreover, other fastidious bacterial species, such as Porphyromonas endodontalis, Porphyromonas gingivalis and some Eubacterium spp., have been reported in endodontic infections at a higher prevalence than those reported by culture procedures.

Bacteria↗

Serum antibody responses of cats to soluble whole cell antigens of feline Porphyromonas gingivalis.

The whole cell soluble antigens of two strains (VPB 3457 and VPB 3492) of feline Porphyromonas gingivalis were analysed by Western blotting using serum taken from 40 domestic cats with various grades of periodontal disease. Five strongly immunogenic protein bands (70, 34, 27, 24 and 19kDa) from VPB 3457 and seven from VPB 3492 (58, 44, 34, 27, 25, 24 and 21kDa) were selected for further study. A significant positive correlation was found between the serum antibody response to the 70, 34, 27, 24 and 19kDa bands of VPB 3457 and the 58, 44, 25, 24 and 21kDa bands of VPB 3492 and the overall periodontal grade. A significant positive correlation was also found between the serum antibody response to the 24kDa band of VPB 3457 and the total colony forming units of P. gingivalis. N-terminal sequencing of the 44kDa band of VPB 3492 showed 75% identity with the translated amino acids from the hag A (haemagglutinin) gene of a human strain of P. gingivalis and N-terminal amino acid sequence of the 27kDa band of VPB 3457 showed 88% identity with the amino acid sequences translated from DNA of purported genes coding for variously named proteinases of human strains of P. gingivalis.

Amino Acid Sequence↗

Bacteriological warfare amongst cats: what have we learned about cat bite infections?

Cat bite infections are one of the most common infectious diseases presenting to veterinary practices and to emergency rooms at human hospitals. This review describes the disease in humans and cats, the origin of organisms involved in cat bite abscesses and the importance of selected organisms such as members of the genus Porphyromonas in the disease. It also discusses future directions, the importance of identifying significant organisms and why an understanding of antimicrobial susceptibility patterns is of consequence to the outcome of the disease in humans and cats.

Abscess↗

Comparative phospholipid analogue distributions of Porphyromonas gingivalis isolated from cats in Australia and the USA.

DNA-DNA homology measurements and phospholipid (PL) analogue profiling have shown heterogeneity of Porphyromonas gingivalis. The aim of this study was to determine whether there were differences between cat strains of P. gingivalis from Australia and USA with respect to PL analogue distribution. Lipids were extracted with chloroform-methanol and examined by fast atom bombardment-mass spectrometry (FAB-MS) in negative-ion mode, using published methods. For PL analogues, the major anions included those with mass-to-charge (m/z)=634, 648, 662, 705, 932, 946 and 960, respectively, corresponding to expected presence of PE (28:0), PE (29:0), PE (30:0), PG (32:1), and three unknown homologues of a glycero-phospholipid with a single nitrogen. Analyses were compared to calculate a matrix of Pearson coefficients of linear correlation from which a dendrogram was produced of strains clustered by single linkage. One cluster was comprised solely of Australian cat-to-cat bite isolates and a second cluster included exclusively USA cat- and dog-to-human bite isolates except for one Australian cat-to-cat bite isolate (VPB 5089). The US cluster included three outliers, one of which was the Australian cat isolate VPB 5089. The human type strain (ATCC 33277) was quite remote from all dog and cat strains. It was shown that P. gingivalis human and non-human animal isolates have distinct PL analogue profiles from each other. Furthermore, the cat strains from the USA and those from Australia showed quantitative differences in polar lipid profiles that correlated largely with country of isolation.

Animals↗

Cloning and expression of the superoxide dismutase gene of the feline strain of Porphyromonas gingivalis: immunological recognition of the protein by cats with periodontal disease.

Recent evidence suggests that feline members of the genus Porphyromonas are of consequence in periodontal disease in cats. Several possible virulence factors from feline strains of Porphyromonas gingivalis have been described that have similarities to those of human P. gingivalis. Both human and feline strains of P. gingivalis produce superoxide dismutase (SOD) which has been proposed as modulator of the inflammatory response during infection. The objective of this study was to clone the superoxide dismutase gene of feline P. gingivalis, to compare the characteristics of its product with that of the native enzyme and to determine its immunoreactivity in cats with periodontal disease. The sod gene of the feline strain Veterinary Pathology and Bacteriology (VPB) 3457 of P. gingivalis was amplified by PCR and cloned in frame with the alpha-peptide of the LacZ gene of E. coli in plasmid pUC19. This construct expressed SOD activity in E. coli with characteristics similar to those of the native SOD enzyme of P. gingivalis human strain 381 and the parent feline strain VPB 3457. The recombinant SOD had an apparent molecular weight of 54,700+/-1300 (S.E.M.) and was inactivated by 5mM hydrogen peroxide but not by 2mM KCN. There was a significant association (P=0.005) between the immunoreactivity of cats to P. gingivalis VPB 3457 soluble whole cell proteins on immunoblots and their responsiveness to the SOD protein. This suggests that cats showing a marked serum responsiveness to P. gingivalis itself, react to the SOD enzyme and further supports the role of feline P. gingivalis in periodontal disease.

Animals↗

The isolation and enumeration of three feline oral Porphyromonas species from subcutaneous abscesses in cats.

Samples were examined from 15 subcutaneous fight wound abscesses from 15 cats. All abscesses were closed at the time of sampling and cats had received no prior treatment. Samples were processed within 20 min and quantitative assessment made of total facultative and obligately anaerobic flora isolated. Digoxigenin labelled whole chromosomal DNA probes directed against three feline members of the genus Porphyromonas (P. gingivalis VPB 3492, P. circumdentaria NCTC 12469T and P. salivosa VPB 3313) were used to identify members of this genus and quantification of these species was made from each cat using colony lifts and southern hybridisation from nitrocellulose membranes taken from replicate plates from each abscess sample. Twelve of the 15 abscesses yielded a variety of facultative and obligately anaerobic (FOA) bacterial species and members of the genus Porphyromounas were enumerated from each of these 12 abscesses. Of the 12 abscesses in which Porphyromonas species were detected, seven contained one species only (five contained only P. gingivalis and two contained only P. salivosa) three abscesses contained two species (both P. gingivalis and P. circumdentaria) and two abscesses contained all three species of Porphyromonas. These results show that members of the genus Porphyromonas are likely to be significant contributors to the purulent disease process in subcutaneous abscesses in cats.

Abscess↗

Associations amongst three feline Porphyromonas species from the gingival margin of cats during periodontal health and disease.

Digoxigenin labelled whole chromosomal DNA probes directed against three feline members of the genus Porphyromonas (P. gingivalis VPB 3492, P. circumdentaria NCTC 12469T and P. salivosa VPB 3313) were used to identify and quantify organisms in samples taken from the gingival margins of 40 domestic cats with different grades of periodontal disease. At the right upper canine tooth, the grade of periodontal disease ranged from 0 to 5 and the cfu of facultative/obligate anaerobes ranged from 5.5 x 10(4) to 2.0 x 10(6)). In 38 of the 40 cats, at least one of the three Porphyromonas species was isolated and regression analysis showed that the cfu of total Porphyromonas sp. was a highly significant indicator of the grade of periodontal disease (p < 0.001, R2 0.510). Feline P. gingivalis was isolated from 37 of the 40 cats and regression analysis showed that it was a highly significant predictor of the grade of periodontal disease (p < 0.001, R2 0.561). The cfu of P. salivosa was a significant predictor of the grade of periodontal disease (p < 0.001, R2 0.286) and regression analysis showed that there was a significant positive relationship between cfu of P. circumdentaria and grade of periodontal disease (p = 0.018, R2 0.116). The periodontal grades at the right upper third premolar tooth ranged from 0 to 6. The cfu of facultative/obligate anaerobes isolated ranged from 1.2 x 10(5) to 7.9 x 10(6), and regression analysis showed that cfu was a significant predictor of periodontal grade (p < 0.001, R2 0.378). The cfu of total Porphyromonas species ranged from 1.2 x 10(4) to 1.7 x 10(6) and regression analysis of the cfu against the grade of periodontal disease showed a highly significant association (p < 0.001, R2 0.633). The cfu of P. gingivalis ranged from 0 to 1.1 x 10(6) and regression analysis of the cfu of P. gingivalis against the grade of periodontal disease showed a highly significant association (p < 0.001, R2 0.439). The cfu of P. salivosa was a significant predictor of the grade of periodontal disease (p < 0.001, R2 0.479) and the same association was found between cfu of P. circumdentaria and grade of periodontal disease (p = 0.002, R2 0.204). This study has established Porphyromonas as anumerically significant and highly prevalent genus in feline periodontal disease.

Animals↗

The association of two recombinant proteinases of a feline strain of Porphyromonas gingivalis with periodontal disease in cats.

Serum from 40 domestic cats with various grades of periodontal disease was used to probe two recombinant functional proteinases from feline strain VPB 3457 of Porphyromonas gingivalis expressed in E. coli. One recombinant proteinase (VPB 2856) was constructed using polymerase chain reaction and had 91% DNA identity with the prtC collagenase gene of the human type strain of P. gingivalis, while the other proteinase (VPB 2814) was isolated from a size selected genomic library and had an amino-terminal sequence with no significant identity with deposited sequences. Thirteen of 40 cats showed a serum antibody response to VPB 2856 using Western immunoblot detection. All the 13 cats had an overall periodontal grade of 3 or greater and greater than 1.68x10(5) cfu P. gingivalis at the canine and premolar periodontium sample sites. Fourteen of 40 cats showed a serum antibody response to VPB 2814. Thirteen of these 14 cats had an overall periodontal grade of 3 or greater. Regression analysis of overall periodontal grade against the serum antibody response showed significant positive relationships for both VPB 2856 (r2 = 0.351; p<0.001) and VPB 2814 (r2 = 0.247; p<0.001). Regression analysis of the total colony forming units of feline strain P. gingivalis against the grade of serum antibody response showed a positive relationship for both VPB 2856 (r2 = 0.662; p<0.001) and VPB 2814 (r2 = 0.531; p<0.001). These data provide strong evidence that the recombinant proteinases of feline P. gingivalis expressed in E. coli clones VPB 2856 and VPB 2814 are associated with periodontal disease in cats.

Amino Acid Sequence↗

Effect of chitosan on a periodontal pathogen Porphyromonas gingivalis.

Local delivery systems of antimicrobial agents for treatment of the periodontal diseases received considerable attention during the past decade due to the disadvantages of the systemic administration. An ideal formulation should exhibit ease of delivery, a good retention at the application site, and a controlled release of the drug. The application of bioadhesive gels provides a long stay in the oral cavity, adequate drug penetration, high efficacy and acceptability. In dentistry and oral medicine, various applications of chitosan, which is a bioadhesive polymer have been proposed due to its favorable properties such as biocompatibility and biodegradability. The aim of this study was to determine the antimicrobial activity of chitosan formulations either in gel or film form against a periodontal pathogen, Porphyromonas gingivalis. The viscosity, bioadhesive properties and antimicrobial activity of chitosans at different molecular weight and deacetylation degree were evaluated in the absence or presence of chlorhexidine gluconate (Chx), incorporated into the formulations at 0.1 and 0.2% concentrations. The flow property of the gels were found to be suitable for topical application on the oral mucosa and to syringe into the periodontal pocket. Bioadhesion of the gels and films examined ex-vivo using fresh porcine buccal mucosa showed that both the film and gel formulations exert bioadhesive properties and was not affected by incorporation of Chx. Chitosan is shown to have an antimicrobial activity against P. gingivalis and this was higher with high molecular weight chitosan. The combination of chitosan with Chx showed a higher activity when compared to that of Chx alone, which would provide Chx application at lower concentrations thus avoiding its unwanted side effects. Chitosan films and gels seem to be promising delivery systems for local therapy of periodontal diseases with its bioadhesive property and antimicrobial activity.

Anti-Bacterial Agents↗

Genetic relationships within the genus Prevotella analyzed by multilocus enzyme electrophoresis and DNA-DNA hybridization.

The genetic diversity and relationships within the genus Prevotella were studied by analyzing twenty-five strains by multilocus enzyme electrophoresis (MLEE) at nine metabolic enzyme loci and DNA-DNA hybridization. MLEE revealed a high genetic diversity with 25 electrophoretic types (ETs) for the 25 strains studied, a mean number of alleles per enzyme locus of 6.8 and a mean genetic diversity per locus of 0.786. The index of association described by Maynard Smith et al. (1993) revealed a clonal structure within the genus Prevotella. A dendrogram generated by cluster analysis of a matrix of ETs showed that species like P. bivia, P. buccae, P. oris, P. oralis, P. nigrescens, and P. denticola form clusters that are consistent with DNA homologies. However, strains identified as P. melaninogenica or P. loescheii by DNA-DNA hybridization did not constitute distinct subpopulations in MLEE. MLEE analysis demonstrated its high power in differentiating closely related strains. It provides an alternative to 16S rRNA analysis for the study of phylogenetic relationships within the genus Prevotella, especially for differentiating strains with high DNA homology or high rRNA homology.

Alleles↗

Cross-reaction between the genus-specific lipopolysaccharide antigen of Chlamydia spp. and the lipopolysaccharides of Porphyromonas gingivalis, Escherichia coli O119 and Salmonella newington: implications for diagnosis.

Seven hybridoma clones, secreting monoclonal antibodies (MAbs) against the genus-specific chlamydial lipopolysaccharide (LPS) antigen were obtained after immunization of BALB/c mice with formalin killed Chlamydia psittaci. The antigen-binding properties of the MAbs were characterized in different immunologic reactions with purified chlamydial elementary bodies and LPS antigens from S- and R-forms of Gram-negative bacteria. Four MAbs reacted with the heterologous LPS antigens of Salmonella R-mutants, Escherichia coli Re chemotype and Acinetobacter calcoaceticus. Two MAbs demonstrated in addition a significant reactivity with Porphyromonas gingivalis, E. coli O119 and Salmonella newington LPS in ELISA, dot-ELISA and passive hemolysis assay (for clone 204G9). One MAb cross-reacted only with Salmonella minnesota Re LPS in ELISA. In indirect immunofluorescent assay six MAbs produced bright green fluorescence with all tested chlamydial strains and five of them reacted with the Re and Rb2 chemotypes of S. minnesota. The results demonstrate a wide cross-reactivity of the produced MAbs with LPS antigens of various Gram-negative bacteria, posing the question for careful consideration and interpretation of serology results for Chlamydia spp.

Animals↗

Toothbrushing and transient bacteremia in patients undergoing orthodontic treatment.

This study was made to determine whether and to what extent bacteremia occurred after toothbrushing in patients undergoing orthodontic treatment with fixed appliances. Twenty patients were selected, all with negative history of heart or hematologic disorders. These patients had not taken antibiotics or had a history of a cold in the previous 30 days. Blood samples of 20 ml were drawn before and 5 minutes after brushing. The immune status of the patients was tested by measurement of isohemagglutins and immunoglobulin levels. Blood samples were incubated in paired culture bottles containing trypticase soy broth (TSB) with an agar paddle and Columbia broth. All samples taken before brushing were negative for bacteria. Five of the 20 patients (25% of the sample) had positive blood tests after brushing. Both anaerobic and aerobic bacteria were identified from the blood samples. Those patients who were found to have a bacteremia did not display poor oral hygiene.

Bacteriological Techniques↗

Beta-lactamase production in Prevotella and in vitro susceptibilities to selected beta-lactam antibiotics [corrected].

This study looked for beta-lactamase production in 100 Prevotella isolates. MICs were determined for amoxycillin, ticarcillin, amoxycillin+clavulanate, cephalothin, cefuroxime, cefixime, cefpodoxime and cefotaxime using the reference agar dilution method (standard M11 A4, NCCLS). Beta-lactamase activity was detected in 58 of the 100 isolates, 24 of 46 black-pigmented Provotella and 34 of 54 non-pigmented Prevotella. All beta-lactamase-negative strains were susceptible to all beta-lactam antibiotics with the exception of cefuroxime and cefixime. Overall, resistance rates of Prevotella strains were lower for ticarcillin (8%) and celefotaxime (12%) than for the other cephalosporins. All Prevotella isolates were susceptible to amoxycillin and were all inhibited by 2 mg/l or less amoxycillin [corrected].

Anti-Bacterial Agents↗

Survey of anaerobic susceptibility patterns: a French multicentre study.

In 1996, the in vitro antibiotic susceptibility of 463 anaerobes was measured in five hospitals using the reference agar dilution method. None of the 209 B. fragilis group strains showed resistance to imipenem or ticarcillin-clavulanic acid. High resistance rates (29%) were observed for cefotetan and clindamycin. beta-Lactamase production was detected respectively in 64% of the Prevotella and 7% of the Fusobacterium strains. Because the same standardized methods were used for many years, the authors were able to evaluate the evolution of antibiotic resistance. Clindamycin resistance had increased within the B. fragilis group (from 14% in 1992 to 29% in 1996) and also among strains of clostridia (32%), P. acnes (18%) and Peptostreptococcus (28%). In the B. fragilis group multidrug resistance was unlikely to occur.

Anti-Bacterial Agents↗

Isolation of Mobiluncus species from the human vagina.

We report the results of a study concerning the characteristics of 52 strains of Mobiluncus spp. isolated from 982 vaginal secretions from patients with suspected bacterial vaginosis. 158 of these women presented the features of this bacterial infection. Of the strains isolated, 39 belonged to the species Mobiluncus curtisii, (25 of these which to M. curtisii subsp. curtisii and 14, to M. curtisii subsp. holmesii), and 13, to Mobiluncus mulieris. The vaginal isolates of Mobiluncus spp. were identified by comparing their biochemical profiles with those of the type strains M. curtisii subsp. holmesii (ATCC 35,242), M. curtisii subsp. curtisii (ATCC 35,241) and M. mulieris (ATCC 35,243). All strains of M. mulieris proved to be sensitive to the antimicrobial agents assayed, while strains of M. curtisii were seen to be resistant only to metronidazole.

Adult↗

Fibronectin and laminin binding of urogenital and oral prevotella species.

88 strains of five Prevotella species--P. bivia, P. buccae, P. disiens, P. oralis, and P. oris--were examined for their fibronectin and laminin binding properties with the aid of latex particle agglutination assays. Beside single protein binding activities, all species showed strains that adhered to both fibronectin and laminin. The oral species, P. buccae, P. oralis, and P. oris were found to interact with laminin to a pronouncedly higher extent than with fibronectin. The urogenital species, P. bivia and P. disiens showed comparable activities of binding to fibronectin and laminin, with P. bivia exhibiting higher matrix protein binding rates than P. disiens. Within the oral species group, P. oralis showed a higher percentage of fibronectin and laminin reactive strains than did P. buccae and P. oris. The finding of species-related different binding properties may throw some light on the known differences in clinical relevance and pathogenicity of the urogenital species, P. bivia and P. disiens, but does so only in part concerning the oral species, P. buccae, P. oralis, and P. oris. Moreover, the observed differences in matrix protein binding of Prevotella species may have implications in chemotaxis and opsonization on the one hand and maintenance of colonization activities under antibiotic therapy on the other.

Bacteroidaceae Infections↗

Pathogenic strategies of the oral anaerobe, Porphyromonas gingivalis.

Adult periodontitis is a chronic inflammatory disease that affects over 49 million people in the USA alone. Porphyromonas (formerly Bacteroides) gingivalis, a Gram-negative anaerobe, has a diverse repertoire of virulence factors that may be involved in the induction or progression of periodontitis.

Bacteroidaceae Infections↗