Comparison of restriction enzyme analysis and amplified fragment length polymorphism typing of Porphyromonas gingivalis isolated from spouses.

INTRODUCTION: In the past, theories on the transmission of Porphyromonas gingivalis between individuals have been based on, among other techniques, restriction enzyme analysis (REA) of bacterial DNA. Currently, amplified fragment length polymorphism (AFLP) may be a more sophisticated alternative. The possibility of automatic pattern analysis and digital storage of the typing data enables the comparison of patterns from a large number of strains in a broad time frame. The aim of this study was to compare REA profiles with AFLP patterns of P. gingivalis strains isolated from periodontitis patients and their spouses. METHODS: Forty-two P. gingivalis strains were isolated from different sites in the mouth from six adult patients with periodontitis and their spouses. DNA of the bacterial isolates was subjected to REA and AFLP analysis. RESULTS: One single type of P. gingivalis was found in each individual with both methods, regardless of the site of isolation. Indistinguishable types were found in four of the six couples with both techniques. Different types were found in two couples with both the REA and the AFLP method. CONCLUSIONS: The AFLP typing technique confirms earlier observations on the transmission of P. gingivalis between spouses. This new technique can replace REA typing.

[Dissertations 25 years after date 12. Classification and virulence of black-pigmented bacteria in relation to periodontitis]. / Proefschriften 25 jaar na dato 12. Classificatie en virulentie van zwart-gepigmenteerde bacteriën in relatie tot parodontitis.

Bacteria in dental plaque play an essential role in the origin and development of periodontitis. In the seventies of the last century it became clear that black-pigmented bacteria of the genus Bacteroides play a vital role in this process. These bacteria are currently known as Porphyromonas gingivalis and Prevotella intermedia. In a PhD dissertation 25 years ago it was shown by DNA analysis that this group of bacteria is very heterogeneous, and that different species exist, which are associated with different oral infections. Because Porphyromonas gingivalis plays an important role in periodontitis, this bacterium has been investigated extensively during the last decades. The entire genome is now known at the DNA level. In addition, transmission between spouses has been shown to be possible, although it does not always cause periodontal disease. It is not yet possible to conclude if for patients with Porphyromonas gingivalis a different antibiotic policy should be used compared to patients without this bacterium.

Adhesion of Porphyromonas gingivalis strains to cultured epithelial cells from patients with a history of chronic adult periodontitis or from patients less susceptible to periodontitis.

BACKGROUND: The present study aimed to explain the interindividual variation in periodontitis susceptibility by differences in the initial adhesion rate of Porphyromonas gingivalis to the pocket epithelium of these individuals, and/or by inter-P. gingivalis strain differences in association capacity (adhesion and internalization). METHODS: Adhesion assays were performed on epithelial monolayers (cultured in vitro from pocket epithelium belonging to patients who were less or more susceptible to chronic adult periodontitis) using 11 genetically different clinical strains of P. gingivalis. RESULTS: Both the disease category (less susceptible versus susceptible) and the interstrain variation were found to have a significant effect (both P <0.05) on the initial bacterial association. The chronic adult periodontitis group showed significantly more association of P. gingivalis when compared to less susceptible patients (4.2 x 10(6) versus 3.5 x 10(6)). Also, the interstrain variation was significant, with strains Pg 4 and 5 representing the least and best associating bacteria (1.8 x 10(6) colony forming units for Pg 4, 9 x 10(6) for Pg 5). CONCLUSIONS: These results indicate that periodontitis susceptibility is influenced by both the interindividual differences in pocket epithelium (allowing more adhesion of P. gingivalis) or by the strain type by which the patient is infected (intra-species differences in adhesion capacity).

Occurrence of Prevotella intermedia and Prevotella nigrescens in relation to gingivitis and gingival health.

AIM: The occurrence of Prevotella intermedia (Pi) and Prevotella nigrescens (Pn) in relation to natural gingivitis, gingival health and 14-day experimental gingivitis was investigated in 25 non-dental students. MATERIALS AND METHODS: Samples were taken from the dorsum of the tongue, the tonsils (or tonsillar area), and the supra- and subgingival plaque. RESULTS: The microbiological results show that 73% of the samples were positive for the bacterial species presumed to be Pi and/or Pn. In natural gingivitis, gingival health and in experimental gingivitis 25, 23 and 25 subjects were found to be positive for Pi and/or Pn, respectively. The results of the 889 isolates that were successfully purified and differentiated, show that almost all subjects were colonized with Pn whereas approximately half of the study population harboured Pi. These 2 species were isolated from both dental plaque and mucosal sites and were found to colonize the oral cavity simultaneously. CONCLUSION: In natural gingivitis, at the start and after 14 days of experimental gingivitis, Pn was the predominant micro-organism.

Peptostreptococcus micros smooth and rough genotypes in periodontitis and gingivitis.

BACKGROUND: Two genotypes can be distinguished within the species Peptostreptococcus micros: a smooth (Sm) and a rough (Rg) type. To date no systematic study has been performed on the prevalence and proportion of both types in untreated periodontitis patients and subjects without destructive periodontal disease. Therefore, the present study was performed to investigate: 1) the relative importance of the Sm and the Rg genotype of P micros in periodontitis and gingivitis; 2) the correlation between smoking and the 2 genotypes of P micros; and 3) the systemic antibody response against the 2 genotypes in relation to the periodontal condition and smoking. METHODS: A total of 104 untreated periodontitis patients and 41 individuals with gingivitis underwent clinical examination and microbiological sampling. Pocket samples were cultured anaerobically on blood agar plates to determine the prevalence and proportion of the Sm and Rg types of P micros. Serum antibody titers against both types of P micros were determined in all subjects by enzyme-linked immunosorbent assay (ELISA) using whole bacterial cells as antigen. Additionally, in a representative group of subjects, the antigen specificity of the serum antibodies was assessed by immunoblotting experiments. RESULTS: The prevalence of the Sm genotype was higher in subjects with periodontitis (94%) compared to subjects with gingivitis (59%), whereas the prevalence of the Rg type was not significantly different (38% versus 29%). Similar analyses were performed for subgroups of smokers and non-smokers; within the periodontitis group, the prevalence of the Sm type was not different between smokers and non-smokers (96% and 92%, respectively), whereas the prevalence of the Rg type was higher in smokers (48%) compared to non-smokers (19%). No difference in prevalence of both types was observed between smokers and non-smokers within the gingivitis group. The titers and specificity of P micros-specific immunoglobulins in periodontitis patients were not different from those in gingivitis subjects, nor were they related to smoking status or culture-positivity. CONCLUSIONS: The results of this study suggest that both the Sm and the Rg genotypes of P micros are part of the normal oral microbiota. However, the elevated prevalence of the Sm genotype in periodontitis and the elevated prevalence of the Rg type in periodontitis patients who smoke implies that both types can behave as opportunistic pathogens in destructive periodontal disease.

Genetic heterogeneity in Prevotella intermedia, Prevotella nigrescens, Prevotella corporis and related species isolated from oral and nonoral sites.

Prevotella intermedia (43 isolates), Prevotella nigrescens (55) and Prevotella corporis (8) from oral and nonoral sites were distinguished by species-specific DNA fragments, after hybridization of DNA fragments with ribosomal RNA (ribotyping). Eight strains previously identified as P. intermedia did not have these specific fragments. P. nigrescens, P. intermedia and P. corporis formed separate clusters in dendrograms constructed using clustering with an unweighted pair group method with arithmetic averages of similarity values derived from ribotype patterns, with 10 subclusters in P. intermedia isolates and 26 in P. nigrescens. Nine groups of P. intermedia isolates and 6 of P. nigrescens shared identical patterns. Specific ribotypes or species were not associated with particular diseases when all isolates were analyzed. However, results from organisms isolated by one laboratory using consistent clinical reporting indicated that P. intermedia was associated with more severe forms of periodontitis and P. nigrescens with mild to moderate disease.

Peptostreptococcus micros coaggregates with Fusobacterium nucleatum and non-encapsulated Porphyromonas gingivalis.

Coaggregation is one of the potential colonization strategies of oral microorganisms, often involving fimbrial structures in the interactions. In this study, the coaggregation characteristics of the rough and smooth genotypes of the periodontal pathogen Peptostreptococcus micros were compared to investigate the role of the fibril-like structures of the rough genotype in coaggregation. Of the 11 oral species tested, only Fusobacterium nucleatum strains and non-encapsulated Porphyromonas gingivalis strains coaggregated with P. micros. No differences in coaggregation between the smooth type (Sm), the rough type (Rg) and the smooth variant of the Rg type (Rg(Sm)) of P. micros were observed. Heat-stable, periodate-sensitive structures on P. micros appeared to interact with heat- and protease-sensitive structures on F. nucleatum and P. gingivalis. These data indicate that these unimodal coaggregations are not mediated by the proteinaceous fibril-like structures of the Rg genotype, but by carbohydrates present on both genotypes of P. micros.

Cloning of fibA, encoding an immunogenic subunit of the fibril-like surface structure of Peptostreptococcus micros.

Although we are currently unaware of its biological function, the fibril-like surface structure is a prominent characteristic of the rough (Rg) genotype of the gram-positive periodontal pathogen Peptostreptococcus micros. The smooth (Sm) type of this species as well as the smooth variant of the Rg type (RgSm) lack these structures on their surface. A fibril-specific serum, as determined by immunogold electron microscopy, was obtained through adsorption of a rabbit anti-Rg type serum with excess bacteria of the RgSm type. This serum recognized a 42-kDa protein, which was subjected to N-terminal sequencing. Both clones of a lambdaTriplEx expression library that were selected by immunoscreening with the fibril-specific serum contained an open reading frame, designated fibA, encoding a 393-amino-acid protein (FibA). The 15-residue N-terminal amino acid sequence of the 42-kDa antigen was present at positions 39 to 53 in FibA; from this we conclude that the mature FibA protein contains 355 amino acids, resulting in a predicted molecular mass of 41,368 Da. The putative 38-residue signal sequence of FibA strongly resembles other gram-positive secretion signal sequences. The C termini of FibA and two open reading frames directly upstream and downstream of fibA exhibited significant sequence homology to the C termini of a group of secreted and surface-located proteins of other gram-positive cocci that are all presumably involved in anchoring of the protein to carbohydrate structures. We conclude that FibA is a secreted and surface-located protein and as such is part of the fibril-like structures.

Adherence of Peptostreptococcus micros morphotypes to epithelial cells in vitro.

Peptostreptococcus micros, which is associated with oral and non-oral mixed anaerobic infections, occurs in three colony morphotypes, the smooth type, the rough type and the smooth variant of the rough type. These types differ in surface structures; the rough type expresses large fibrillar surface appendages, which are absent on the surface of both the smooth and the smooth variant of the rough type. To determine the role of these surface structures in adherence we characterized the adherence of the three morphotypes of P. micros to epithelial cells in vitro. Although all three types adhered well to epithelial cells, adhering numbers of the rough type were significantly lower than those of the smooth and the smooth variant of the rough type. Protease treatment increased the adherence of the rough type of the level of the two other types. The adherence of all three types was reduced more than 85% by treatment with 10 mM sodium periodate. Furthermore, the adherence was pH independent and could not be blocked by incubation with antisera to the bacteria. In addition, we determined the capacity to invade epithelial cells by P. micros. In an acridine orange assay such invasion could not be detected. Our results suggest that the adherence of P. micros to epithelial cells is mediated by periodate-sensitive extracellular polysaccharides and that the protruding fibril-like protein surface structures of the rough type have an obstructive effect on the adherence.

Genetic relatedness between oral and intestinal isolates of Porphyromonas endodontalis by analysis of random amplified polymorphic DNA.

Genomic fingerprints from the DNA of 27 strains of Porphyromonas endodontalis from diverse clinical and geographic origins were generated as random amplified polymorphic DNA (RAPD) using the technique of PCR amplification with a single primer of arbitrary sequence. Cluster analysis of the combined RAPD data obtained with three selected 9- or 10-mer-long primers identified 25 distinct RAPD types which clustered as three main groups identifying three genogroups. Genogroups I and II included exclusively P. endodontalis isolates of oral origin, while 7/9 human intestinal strains of genogroup III which linked at a similarity level of 52% constituted the most homogeneous group in our study. Genotypic diversity within P. endodontalis, as shown by RAPD analysis, suggests that the taxon is composed of two oral genogroups and one intestinal genogroup. This hypothesis remains to be confirmed.

Porphyromonas gingivalis in an edentulous proband. A case-report.

In a previous study, an edentulous subject showed the presence of P. gingivalis, only in saliva. The present case report is a longitudinal follow-up on this proband (female, 63 years) and her spouse with periodontitis (67 years). Samples were taken from the proband and the spouse for microbiological analysis at several occasions during a 3-year period and a 1-year period respectively. In the edentulous proband, P. gingivalis was present in saliva only, at all times. In the spouse, P. gingivalis was present in the periodontal pocket and on the mucous membranes, but the saliva was culture-negative for this species at both sampling times. The restriction enzyme analysis (REA) patterns of isolates (n=7) of P. gingivalis from the proband recovered over 3 years showed no differences. This indicated that the same clonal type of P. gingivalis persisted for 3 years in the proband. The P. gingivalis isolates (n=13) from the spouse showed no differences when REA patterns were compared. Comparison of REA patterns from the proband and the spouse also showed no differences. Thus the same clonal type of P. gingivalis can persist over several years in an edentulous subject and at least for 1 year in a subject with periodontal disease.

Cystatin and cystatin-derived peptides have antibacterial activity against the pathogen Porphyromonas gingivalis.

We investigated whether cystatins and cystatin-derived peptides, encompassing sequences of secondary structures of cystatin S and papain binding domains of cystatin C, display antimicrobial properties. Of the different microorganisms tested, only the growth of P. gingivalis was inhibited by chicken cystatin and cystatin C. Cystatin S, cystatin S:1-14, cystatin S:61-73 and cystatin S:108-121 also inhibited its growth, whereas cystatin S:21-38, cystatin S:39-55, cystatin S:81-95, cystatin S:94-109, and cystatin C: 9-12/55-60/106-107 did not. No inhibition of the cysteine proteinase activity of P. gingivalis was observed for all cystatin-derived peptides. On the other hand, leupeptin and antipain inhibited P. gingivalis proteinase activity, but had no effect on the growth. These data suggest that cystatins contain antibacterial sequences active against P. gingivalis and that the growth inhibition does not depend on the inhibition of P. gingivalis cysteine proteinases.

Oral microbiota in smokers and non-smokers in natural and experimentally-induced gingivitis.

The present study primarily aimed at investigating the oral microbiota in smokers and non-smokers with established gingivitis and monitoring its composition during experimental gingivitis. Secondly, it aimed at examining whether the composition of the microbiota is associated with different levels of gingival inflammation during this experimental gingivitis trial. For this purpose, 25 non-dental university students with gingivitis were recruited. 11 subjects were smokers and 14 were non-smokers. After achieving gingival health, they entered a 14-day experimental gingivitis trial. Plaque and bleeding were assessed before entering into the study (intake), at day 0, day 5 and at day 14 of the experiment. Microbiological samples from mucosal sites and dental plaque (taken at intake, day 0, and day 14) were analysed for the presence of Actinomyces species, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Fusobacterium nucleatum, Peptostreptococcus micros, Porphyromonas gingivalis, Prevotella intermedia and Streptococcus species. At day 14 of the experimental period, the level of plaque formation was not different between smokers and non-smokers, but bleeding scores were lower in smokers than in non-smokers (15% and 30% respectively, p=0.01). The change from natural gingivitis to a state of gingival health and a subsequent change from gingival health to experimentally induced gingivitis was accompanied by quantitative alterations in the cultivable microbiota in both groups. Changes were most prominent in the transition from gingival health to experimental gingivitis and were found in dental plaque for Actinomyces species, C. rectus, F. nucleatum, and P. intermedia. Within the group of non-smokers, a distinction was made between subjects with a 'weak' or 'strong' inflammatory response. No relationship with a single bacterial species could be established which would likely explain the differences in levels of inflammation. It is concluded that differences in response to experimental gingivitis are not caused by major differences in the composition of the oral microbiota.

Prevalence of Peptostreptococcus micros morphotypes in patients with adult periodontitis.

The prevalence of the smooth and rough colonial morphotypes of Peptostreptococcus micros was examined with culture technique in 123 patients with adult periodontitis (age 24-68 years). Of all subgingival samples, 91% contained the smooth morphotype of P. micros. The smooth morphotype constituted a mean percentage of the total anaerobic viable biota of 6.0%, with a range of 0.02-35.7%. Of these samples, 49% contained colonies of the rough morphotype as well, with a mean percentage of the total anaerobic viable biota of 2.3% (range 0.01-16.2%). None of the samples contained only the rough morphotype. The total percentage of P. micros varied from 0.02-35.71% with a mean of 7.2%. No correlation was found between the prevalence of both morphotypes of P. micros and the age of the subjects or with loss of attachment or pocket depth.

Pathogenicity of Peptostreptococcus micros morphotypes and Prevotella species in pure and mixed culture.

Recently, an atypical rough colony morphotype of Peptostreptococcus micros, a species which is found in ulcerating infections, including periodontitis, was isolated. The virulence of morphotypes alone and in combination with Prevotella intermedia and P. nigrescens was investigated both in vivo and in vitro. All strains tested induced abscesses containing fluid pus in a mouse skin model, and lesions caused by monocultures of the rough morphotype strains of P. micros were statistically significantly larger than those induced by the smooth morphotype strains. Inocula containing both morphotypes produced similar sized abscesses compared to mono-inocula containing the same bacterial load. Both Prevotella species induced small abscesses when inoculated alone, and when Pr. nigrescens was inoculated with one of the other strains, the abscesses were not significantly different from the abscesses induced by the mono-infections of this strain. Synergy, in terms of higher numbers of colony forming units (cfu) in the mixed inocula, was found for all combinations of the rough morphotypes of P. micros and both Prevotella spp. Pus from abscesses caused by combinations of Peptostreptococcus and Prevotella spp. transmitted the infection to other mice, but no abscesses were formed in mice inoculated with pus induced by mono-inocula. These results demonstrated synergic activity between both rough and smooth P. micros strains and oral Prevotella strains. The in-vitro co-culture experiments produced no evidence of growth stimulation. The effect of P. micros strains on the immune system was investigated by testing their ability to initiate luminol-dependent chemiluminescence of polymorphonuclear leucocytes in the presence and absence of human serum. In the latter, the rough morphotype strains initiated higher counts than the smooth morphotype strains. Further work is needed to elucidate the difference in virulence between the smooth and the rough morphotype cells of P. micros and the nature of the interaction with the Prevotella spp.

[Transmission of infectious oral diseases from mother to child]. / Overdracht van infectieuze mondziekten van moeder naar kind.

The relation between oral microbes of mother and child encompasses several aspects. It has been shown that a bad periodontal condition is a risk factor for low birth weight. The bacterium that is most specifically associated with dental caries, Streptococcus mutans, can be transmitted between mother and child; it has been suggested that the age of colonization with this bacterium is a risk factor for caries in the child. Also periodontal bacteria can be transmitted between mother and child. It remains to be elucidated whether this is an additional risk factor for the development of periodontal disease.

Intra-familial transmission and distribution of Prevotella intermedia and Prevotella nigrescens.

The periodontal bacteria Prevotella intermedia and Prevotella nigrescens have been recently separated from each other. The purpose of this study was to investigate the distribution and routes of transmission of these bacteria among family members. Seven patients with moderate to severe periodontitis were selected. These probands, their spouses and 14 of their children were investigated. The presence of Pr. intermedia and Pr. nigrescens was determined by culture techniques in pooled subgingival plaque samples, in the saliva, on the tongue, tonsils and buccal mucosa. Differentiation of Pr. intermedia and Pr. nigrescens was performed by enzyme electrophoretic mobility. From all 7 patients, as well as 4 spouses and 3 of the children, Pr. intermedia could be isolated. Pr. nigrescens was found in 2 of the 7 patients, in 5 of the spouses and in 5 of the 6 children aged 5-10 yr. In the 8 children aged 0-4 yr both species were seldom isolated. These data are in accordance with earlier findings that Pr. intermedia is associated with periodontitis and Pr. nigrescens with a relatively healthy periodontal condition. Ribotyping of bacteria was performed by hybridization of HindIII restriction endonuclease digests of chromosomal DNA with ribosomal DNA. Isolates from unrelated individuals always had distinct ribotypes. Indistinguishable ribotypes of Pr. intermedia and Pr. nigrescens were found both among married couples and among parents and children. This indicates that intrafamilial transmission of Pr. intermedia and Pr. nigrescens is possible both between adults and between parents and children.

Characterization of smooth and rough morphotypes of Peptostreptococcus micros.

Isolation of the smooth (Sm) morphotype of Peptostreptococcus micros, a suspected oral pathogen, is sometimes accompanied by isolation of a rough (Rg) morphotype of P. micros. The Rg type readily changes to a Sm-like variant (RgSm) in broth culture. Sm and Rg isolates and RgSm variants were compared to determine whether these three types are the result of phase variation. The RgSm variants resembled the Sm morphotype in colony morphology; furthermore, the Sm type and the RgSm type did not have the fibrillar surface structures characteristic of the Rg type, and the Sm and RgSm types were more hydrophobic than the Rg type. However, when we compared the sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of whole-cell proteins, serotyping data, pyrolysis mass spectrometry data, 16S ribosomal DNA sequences, and hemolytic activities, the RgSm variants and the Rg isolates were very similar and were clearly distinct from the Sm isolates. These results suggest that the Rg and RgSm types form a cluster distinct from the Sm type and thus provide evidence that P. micros can be differentiated into two groups, one consisting of the Sm type and the other consisting of the Rg and RgSm types.

Effects of chlorhexidine on the bacterial colonization and degradation of dentin and completely demineralized dentin in situ.

The effects of 0.2% chlorhexidine on selected plaque microorganisms were studied in an intraoral dentin caries model. In 8 individuals wearing partial dentures, sound and completely demineralized dentin specimens were placed consecutively in 2 periods of 4 weeks, respectively. Throughout the experimental period, the specimens were treated 2 x daily with 0.2% chlorhexidine; control specimens were treated with water. Plaque accumulation on the specimens was left undisturbed. No protection against demineralization of the dentin or degradation of the dentin collagen by the chlorhexidine treatment was observed. The chlorhexidine treatment did not result in a reduction of the total cultivable flora when compared with the control specimens. A significant reduction of mutans streptococci and total streptococci recovered from completely demineralized dentin treated with chlorhexidine was observed, but the proportions of Actinomyces and lactobacilli were not affected significantly. It is speculated that areas of exposed roots, which are difficult to reach by oral hygiene measurements, such as approximal surfaces, will not be protected by a 0.2% chlorhexidine mouthrinse against the caries process.

Bacterial colonization of mineralized and completely demineralized dentine in situ.

The changing environment in a developing root lesion may result in a succession of the microbial flora in the dentine. As demineralization proceeds, the collagenous matrix is exposed, which could be conducive to the growth of specific microorganisms. In this study both sound and completely demineralized dentine were placed together in the partial prothesis of 8 individuals to test whether the type of substrate influenced the composition of the bacterial flora. After 6 weeks the degradation of the collagenous matrix, the demineralization of the dentine and the microbial composition were assessed. The collagen loss varied between 0 and 69 wt%. Mineral loss from the originally sound dentine specimens ranged from virtually none to complete demineralization. Percentages of total streptococci, mutans streptococci, Actinomyces and lactobacilli isolated from both dentinal substrates did not differ significantly. The percentage of lactobacilli in the dentine specimens was positively correlated to the lesion depth. The percentage of Actinomyces species was significantly higher in both the dentine specimens that had been demineralized in vitro and those that were found to be completely demineralized in situ compared to the partially demineralized dentine specimens. In vitro, no collagenolytic activity of the predominant flora isolated from both dentinal substrates could be shown.