Purification and characterization of hemolysin from periodontopathogenic bacterium Eikenella corrodens strain 1073.

Eikenella corrodens 1073 was found to show hemolytic activity when grown on sheep blood agar. A high and dose-dependent hemolytic activity was detected in the cell envelope fraction, which was further purified by ion-exchange and gel-filtration chromatography. Consequently, a 65-kDa protein with hemolytic activity was obtained, suggesting that this protein might be a hemolysin. Its N-terminal amino acid sequence was nearly identical to that of X-prolyl aminopeptidase from E. corrodens ATCC 23834. To confirm that X-prolyl aminopeptidase functions as a hemolytic factor, we expressed the hlyA gene, encoding X-prolyl aminopeptidase, in Escherichia coli. After induction with isopropyl ß-D-1-thiogalactopyranoside, a protein of about 65 kDa was purified on a Ni column, and its hemolytic activity was confirmed. Meanwhile, a strain with a disrupted hlyA gene, which was constructed by homologous recombination, did not show any hemolytic activity. These results suggested that X-prolyl aminopeptidase might function as a hemolysin in E. corrodens.

Quantification of five putative periodontal pathogens in female patients with and without chronic periodontitis by real-time polymerase chain reaction.

Chronic periodontitis is a highly prevalent endogenous polymicrobial disease. To better understand the etiology of the disease a quantitative approach is mandatory and real-time PCR is the molecular technique currently preferred to achieve this purpose. Taking into account that such a kind of study is still scarce, we aimed to evaluate the association between periodontal microbiota and chronic periodontitis. A total of 60 low-income age-matched female adults, 30 with chronic periodontitis and 30 without periodontal disease, were enrolled. DNA obtained from subgingival specimens was used for quantification of Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia by real-time PCR. A. actinomycetemcomitans, E. corrodens, and F. nucleatum were detected in all subjects, P. gingivalis was observed in 70.0% and 46.6% and P. intermedia in 90.0% and 80.0% of chronic periodontitis patients and periodontally healthy subjects, respectively. P. gingivalis mean count was significantly higher in patients with chronic periodontitis than in periodontally healthy individuals. Accurate detection and quantification of five putative periodontal pathogens was feasible using a simple and fast real-time PCR protocol. Although P. gingivalis and P. intermedia have been found more commonly in chronic periodontitis patients, no statistical difference was observed between periodontally diseased and healthy groups. Quantitative data indicated association between P. gingivalis and chronic periodontitis. However, because of its uneven distribution, it should not be solely taken as a marker of periodontal status.

Frequency of putative periodontal pathogens among type 1 diabetes mellitus: a case-control study.

OBJECTIVE: The aim of the present study is to compare and assess the risk of periodontitis due to the presence of four putative periodontopathic bacteria viz., Eikenella corrodens, Campylobacter rectus, Prevotella intermedia and Prevotella nigrescens. To fulfil the above objective, polymerase Chain reaction using the primers targeting 16S rRNA gene of the bacterial species was performed with the subgingival plaque collected from the permanent first molars of type 1 diabetic children and age matched healthy children. RESULTS: The prevalence of periodontal pathogens in diabetic and healthy children was 6% and 16% for E. corrodens, 18% and 36% for C. rectus, 2% and 2% for P. intermedia, 4% and 0%, for P. nigrescens respectively. Statistically, significant difference was not observed for the prevalence of all the four periodontal pathogens between type 1 diabetic and healthy children (P = 1.00). The results of the present study thus reveal a negative correlation of type I diabetes to periodontitis in association to Eikenella corrodens, Campylobacter rectus, Prevotella intermedia and Prevotella nigrescens.

Culture-independent detection of Eikenella corrodens and Veillonella parvula in primary endodontic infections.

Eikenella corrodens and Veillonella parvula are normal cultivable inhabitants of the oral cavity but their presence in endodontic infections has not been as common as it could be anticipated. This might have been because of shortcomings of culture techniques when it comes to bacterial isolation or identification. The present study intended to survey samples from primary endodontic infections for the presence of E. corrodens and V. parvula using a culture-independent 16S rRNA gene-based nested PCR protocol. Genomic DNA was isolated directly from samples taken from different forms of periradicular lesions, and the presence of E. corrodens and V. parvula was determined by nested PCR. Specificity for each primer pair was confirmed by sequence analysis of PCR products from positive clinical samples. V. parvula and E. corrodens were, respectively, detected in 33% and 14% of the root canals associated with chronic apical periodontitis. Both V. parvula and E. corrodens were found in 10% of the cases diagnosed as acute apical periodontitis. V. parvula and E. corrodens were detected in 21% and 26% of the samples from acute apical abscesses, respectively. In general, species-specific nPCR allowed the detection of V. parvula in 24% and E. corrodens in 18% of the samples taken from primary endodontic infections. Findings confirmed that V. parvula and E. corrodens can take part in the microbiota of primary endodontic infections, but in prevalence values somewhat higher when compared to most of the previous culture studies that had reported recovery of these species.

Isolation and characterization of a plasmid DNA from periodontopathogenic bacterium, Eikenella corrodens 1073, which affects pilus formation and colony morphology.

Eikenella corrodens (Ec) is one of a group of periodontopathogenic bacteria. A plasmid DNA (8.7 kb) isolated from Ec 1073 was designated pMU1. Agarose gel electrophoresis and Southern analysis suggested that pMU1-like plasmids were carried in 2 Ec strains, including 1073, with higher hemagglutination (HA) activity than other strains. We determined the nucleotide sequence of this plasmid and identified 7 ORFs. A homology search revealed that 4 ORFs of pMU1 were homologous to ORFs in pJTPS1, found in a spontaneous avirulent mutant of the phytopathogenic bacterium, Ralstonia solanacearum. pJTPS1 is a putative hypovirulent plasmid, which is thought to control the virulence of R. solanacearum. We also found the ORF to be homologous to the recombinase specific to the type IV pilin gene. We introduced a part of pMU1 into the Ec 23834 strain, which has a pilus structure on its cell surface and forms corroding colonies on solid medium. No pilus structure was observed on the surface of transformants, most of which formed non-corroding colonies. When such transformants (or Ec 1073) were cured of pMU1 with acridine orange, they remained non-foliated and non-corroding. The results suggest that pMU1 might irreversibly affect pilus formation and colony morphology, and might be involved in the pathogenicity and virulence of Ec.

Eikenella corrodens in human oral and non-oral infections: a review.

There is substantial evidence in support of the existence of distinct clinical forms of human periodontal disease. Moreover, these different forms of periodontal disease may be associated with relatively distinct subgingival microflora, often involving microaerophilic or anaerobic Gram-negative bacterial species. Eikenella corrodens is a facultative Gram-negative bacillus which is a common inhabitant of the oral cavity and the intestinal and genital tracts. Its primary ecologic niche within the oral cavity appears to be dental plaque, both in periodontally healthy individuals and in periodontitis patients. However, E. corrodens is recognized as an infrequent human pathogen capable of causing extraoral infections, either as the sole infectious agent or as part of a mixed infection, its potential role in the etiology of periodontal disease is not well understood. E. corrodens is often present in the supra- and subgingival plaque of periodontally healthy subjects. On the basis of cross-sectional and longitudinal studies, E. corrodens appears to be somewhat more prevalent in subgingival plaque samples of periodontitis subjects than periodontally healthy individuals. However, the percentage of E. corrodens in the total cultivable microflora did not vary between the two groups. Microbiologic studies attempting to define the relationship between E. corrodens and periodontal disease assume that this species is essentially homogeneous and that all strains exhibit comparable pathogenic potential. However, E. corrodens exhibits 1) variable colony morphology, biochemical and serologic reactivity; 2) marked phenotypic diversity with respect to outer membrane protein and lipopolysaccharide structure; and 3) marked diversity in the restriction patterns of total genomic DNA. Thus, it is possible that a limited number of clones of E. corrodens may be associated with periodontal disease and/or extraoral infection, while other strains are relatively harmless commensals. Additional studies, possibly employing strain-specific nucleic acid probes, may be required to define the role of E. corrodens as a human periodontal pathogen.

Comparison of DNA probe and ELISA microbial analysis methods and their association with adult periodontitis.

The purposes of this study were two-fold: to compare the DNA probe and enzyme linked immunosorbent assay (ELISA) microbial identification tests and correlate the levels of microorganisms with adult periodontitis. A single plaque sample were taken from each of 2 sites in 52 patients. Twelve of these patients were also sampled during and after treatment. The experimental site had clinical indicators of disease (bleeding on probing, probing and attachment loss of > or = 6 mm) and the contralateral site (control) was clinically healthy. A total of 176 plaque samples were collected, divided, processed, and sent for both types of quantitative microbial analyses. All of these samples were used to compare the DNA probe and ELISA methods while only the initial 104 pretreatment sites were used to correlate microorganisms/method with clinical indicators of adult periodontitis. DNA probes were used to assay for A. actinomycetemcomitans, P. gingivalis, P. intermedia, E. corrodens, F. nucleatum, T. denticola, and C. rectus. An ELISA utilizing monoclonal antibodies was used to assay for P. gingivalis, E. corrodens, T. denticola, and C. rectus. Comparison of the two methods revealed that the ELISA test identified P. gingivalis and C. rectus significantly more often than the DNA probe method and that T. denticola was detected more frequently with the DNA probe. The sensitivities and specificities varied widely among organisms and by test. P. gingivalis, as identified by ELISA, had the highest degree of sensitivity and specificity (0.90 and 0.82 respectively) to clinical indicators of adult periodontitis.

Oral food consumption and subgingival microorganisms: subgingival microbiota of gastrostomy tube-fed children and healthy controls.

This study examined the effect of oral food consumption on the prevalence and levels of subgingival bacteria and yeasts in 20 gastrostomy tube-fed children and 24 healthy controls. Microbial identification was carried out using anaerobic culture and 16S rRNA-based PCR identification methods. Streptococcal and Actinomyces species were recovered from 100% and 76% of all subjects and averaged 66% and 11% of total cultivable organisms, respectively. In decreasing order of prevalence, Fusobacterium, enteric rods, Prevotella intermedia/Prevotella nigrescens, Capnocytophaga, Propionibacterium, yeasts, Actinobacillus actinomycetemcomitans, coagulase-negative Staphylococcus, Campylobacter rectus, Bacteroides forsythus, and Porphyromonas gingivalis were detected in 48% to 2% of the study subjects. The cultivable levels of these species varied widely among subjects. PCR detection showed C. rectus and Eikenella corrodens both to occur in 93% of the study subjects and to be the most prevalent putative periodontal pathogens examined. In decreasing order of prevalence, PCR identified Treponema denticola, A. actinomycetemcomitans, P. nigrescens, P. intermedia, B. forsythus, and P. gingivalis in 38% to 21% of the subjects studied. Tube-fed children and healthy controls exhibited similar subgingival microbial compositions. It appears from this study that oral food consumption is not a major determinant for the establishment of subgingival microbiota in children.

LuxS affects biofilm maturation and detachment of the periodontopathogenic bacterium Eikenella corrodens.

Previously, we reported that biofilm formation of Eikenella corrodens is regulated by autoinducer-2 (AI-2), based on observations that biofilm-forming efficiency of ΔluxS mutant was greater than that of the wild type (Azakami et al., J. Biosci. Bioeng., 102, 110-117, 2006). To determine whether the AI-2 molecule affects biofilm formation directly, we added purified AI-2 to luxS mutant and wild-type E. corrodens and compared biofilm formations by using a static assay. Results indicated that biofilm formation in E. corrodens was enhanced by the addition of AI-2. We also compared the biofilms formed by flow cell system for the luxS mutant and the wild type by using scanning electron microscopy and confocal laser scanning microscopy. The number of viable bacteria in the luxS mutant biofilm was dramatically reduced and more sparsely distributed than that of the wild type, which suggested that AI-2 might enhance the mature biofilm. Conversely, further analysis by modified confocal reflection microscopy indicated that the wild-type biofilm was matured earlier than that of the luxS mutant, and became thinner and more sparsely distributed with time. These data suggest that LuxS may facilitate the maturation and detachment of biofilm in E. corrodens.

A new checkerboard panel for testing bacterial markers in periodontal disease.

BACKGROUND/AIMS: Various microbiological methods have been used for testing bacterial markers for periodontitis and periodontal disease progression. Most studies have used only a limited number of well recognized bacterial species. The purpose of the present study was to evaluate the association of 13 more recently identified bacterial species in a new panel in comparison with 12 previously more recognized periodontotopathogens ('old panel') using the 'checkerboard' DNA-DNA hybridization method. METHODS: Fifty individuals were chosen who showed at least one site with a probing pocket depth of 6 mm or more (disease) and bleeding on probing and at least one site with a probing pocket depth of 3 mm and without bleeding on probing (health). One diseased and one healthy site on each individual were sampled with the paperpoint technique and the samples were processed in the checkerboard technique against deoxigenin-labeled whole genomic probes to 25 subgingival species representing 12 well recognized and 13 newly identified periodontitis associated species. RESULTS: Twenty-four (out of 25) species were detected more frequently in the subgingival plaque of diseased than healthy sites both at score 1 (> 10(4)) and score 3 (> 10(5)). A significant difference at the higher score (score 3) was noticed for all species of the old panel except for three (Streptococcus intermedius, Selenomonas noxia, and Eikenella corrodens). Of the species in the new panel only Prevotella tannerae, Filifactor alocis, and Porphyromonas endodontalis showed a statistical significant difference between diseased and healthy sites. CONCLUSION: It was concluded that P. tannerae, F. alocis, and P. endodontalis should be added to the 12 species used for routine diagnostics of periodontitis-associated bacterial flora.

Restriction endonuclease analysis of Eikenella corrodens.

Eikenella corrodens is a gram-negative facultative bacillus commonly found in the oral cavity. Although the role of E. corrodens in periodonititis is not clear, its isolation from extraoral infections attests to its pathogenic potential. Previous studies suggested that this species is phenotypically diverse. In the present study, we used restriction endonuclease analysis (REA) to assess the genetic diversity of this species and to explore the applicability of REA in studying the transmission of E. corrodens. Two groups of E. corrodens isolates were used in this study. Group 1 included 47 epidemiologically independent isolates recovered from dental plaques in periodontally healthy subjects and periodontitis patients and from extraoral infections in different geographic areas. Group 2 E. corrodens included 40 isolates recovered from two periodontitis patients and two periodontally healthy subjects. The results indicated that E. corrodens is genetically heterogeneous, as determined by REA. The majority of the group 1 E. corrodens isolates exhibited strain-specific restriction patterns. Forty restriction patterns were distinguishable among the 47 isolates. Analyses of group 2 isolates revealed that three of four subjects harbored more than one clonal type of E. corrodens. In one instance, a periodontitis patient was found to be colonized by six different clones. Furthermore, two different clonal types of E. corrodens were recovered from a single periodontal pocket in this patient. The results indicated that REA may be a useful tool in the epidemiologic investigation of E. corrodens infections.

DNA homologies shared among E. corrodens isolates and other corroding bacilli from the oral cavity.

In a previous microbiological study of Eikenella corrodens, we noted the presence of E. corrodens strains with variability in colony morphology, as well as other corroding bacilli phenotypically similar to E. corrodens but which were unidentifiable on the basis of biochemical reactions. This raised questions as to whether E. corrodens constitutes a genetically heterogeneous group of organisms, and whether the unidentified corroding bacilli represent atypical E. corrodens or genetically unrelated organisms. In the present study, the genetic relationship among 14 E. corrodens isolates and 6 unidentified corroding bacilli was examined. DNA base compositions were determined from the melting temperatures of DNA samples. DNA homologies among E. corrodens and corroding bacilli were determined by DNA hybridization in solution using S1 nuclease. The % G + C content of E. corrodens strains varied from 56 to 58%, and from 56 to 60% for unidentified corroding bacilli. The DNA homologies among 12 E. corrodens isolates and 2 reference strains varied from 57 to 97%. Although these E. corrodens isolates exhibited variabilities in colony morphology and biochemical profile, no subspecies was identified. The unidentified corroding bacilli shared less than 33% homology with either of the E. corrodens reference strains. These corroding bacilli were further divided into 3 species on the basis of DNA hybridization studies using radiolabeled DNA from 2 representative corroding bacilli. One of the unidentified corroding bacilli appears to be a component of the normal flora in the human oral cavity. Our results indicate that E. corrodens is a genetically homogeneous species containing no recognizable subspecies.(ABSTRACT TRUNCATED AT 250 WORDS)

Clonal diversity and stability of subgingival Eikenella corrodens.

Eikenella corrodens is a commensal subgingival bacterium commonly found in both periodontally nondiseased and diseased subjects. The present study examined the clonal diversity and stability of subgingival E. corrodens over time. Ninety-five subjects were enrolled at the baseline examination, including 44 periodontally nondiseased subjects and 51 subjects with aggressive periodontitis. Twenty-two nondiseased subjects and 11 subjects with aggressive periodontitis were subsequently reexamined after an average interval of 14 months. Two subgingival plaque samples were obtained from each subject to determine the total cultivable bacteria. In addition, multiple E. corrodens isolates from each sample were recovered for clonal analysis by arbitrarily primed PCR. The mean numbers of distinct E. corrodens clones harbored by nondiseased subjects and subjects with aggressive periodontitis were 1.3 and 3.0, respectively. Thirty-nine percent of the nondiseased subjects and 63% of the subjects with aggressive periodontitis harbored multiple clones of E. corrodens. The numbers of distinct E. corrodens clones increased significantly (Mann-Whitney ranking test, P < 0.05) in sites from patients with aggressive periodontitis, in sites with pocket depths of 4 mm or greater, in sites with a clinical attachment loss of 2 mm or greater, and in sites coinfected with Porphyromonas gingivalis. Comparison of E. corrodens clones recovered at the baseline and those recovered at the follow-up examination showed that E. corrodens colonization was not stable. Thirty-eight of the 66 follow-up samples (58%) showed a complete change (including de novo colonization of the sites or complete elimination of the organism from the sites) of the subgingival E. corrodens clonal types between the baseline and the follow-up examinations. Our results suggest a complexity of subgingival microbiota not seen previously.

Clonal diversity of oral Eikenella corrodens within individual subjects by arbitrarily primed PCR.

The genetic diversity of 205 Eikenella corrodens isolates recovered from dental plaque, mucosal surfaces, and saliva of 24 subjects was examined by arbitrarily primed PCR. Twenty-two subjects were colonized by multiple clones (range, two to eight; mean, 3.7). This study demonstrates the utility of arbitrarily primed PCR for clonal analysis of E. corrodens and the multiclonal colonization of E. corrodens in the oral cavity.

Detection of Eikenella corrodens and Actinobacillus actinomycetemcomitans by use of the polymerase chain reaction (PCR) in vitro and in subgingival plaque.

The purpose of the present investigation was to identify 2 putative periodontal pathogens: Eikenella corrodens and Actinobacillus actinomycetemcomitans by polymerase chain reaction (PCR) in vitro and in subgingival plaque. On the basis of published sequences coding for 16S rRNA two primer pairs were designed which amplify a 410 bp sequence from E. corrodens DNA and a 547 bp fragment from A. actinomycetemcomitans DNA, respectively. As few as 50 cells could be detected from pure bacterial cultures. Each of the two primer pairs was found to be specific in that it did not give any amplification product neither with cell lysates from the respective alternative bacterium nor with lysates obtained from other putative periodontal pathogens and other bacteria. The PCR method developed turned out to be a simple, rapid and reliable diagnostic tool for the detection of the target microorganisms in clinical samples.

Variable serum immunoglobulin G immune response to genetically distinct Eikenella corrodens strains coexisting in the human oral cavity.

This study examined the variable serum immunoglobulin G (IgG) levels to genetically distinct autologous Eikenella corrodens strains by enzyme-linked immunosorbent assay (ELISA). Twenty subjects, including 10 adult periodontitis patients, 5 juvenile periodontitis patients and 5 periodontally healthy subjects were examined. Each subject was colonized by 2-8 genetically distinct E. corrodens strains. The serum IgG levels to autologous E. corrodens within individuals were significantly different in 7 adult periodontitis patients, 4 juvenile periodontitis patients and a periodontally healthy subject. Poor correlation was found in diseased subjects between serum IgG levels to autologous strains and to reference strains ATCC 23834 or FDC 373. Four adult periodontitis patients and two juvenile periodontitis patients exhibited significant serum IgG levels to autologous E. corrodens strains (two standard deviations above the mean for periodontally healthy subjects); two of these six diseased subjects exhibited low serum IgG levels to reference strains and would have been classified as low immune responders if only reference strains had been used in ELISA. This study showed the importance of using autologous E. corrodens strains in the assessment of serum IgG immune responses to this organism.

DNA probe detection of periodontopathogens in advanced periodontitis.

Species-specific DNA probes were used to determine the presence of Actinobacillus actinomycetemcomitans (A.a.), Porphyromonas (Bacteroides) gingivalis, Prevotella intermedia, Treponema denticola, Eikenella corrodens, Fusobacterium nucleatum, and Wolinella recta in subgingival plaque from deep pockets/sites of patients with advanced periodontitis. The subjects were 20 patients with severe adult periodontitis, 13 men and 7 women (mean age 45.6 +/- 6.7 yr). For each subject, 9-10 subgingival sites with the deepest probing depths from each quadrant were sampled by the paper point method, a total of 198 sites, with mean probing depth 7.2 +/- 1.6 mm and clinical attachment level 9.5 +/- 2.7 mm. A.a. was present in at least one site in 75% of the subjects; P. gingivalis was found in 95%; P. intermedia and W. recta were found in 90%, respectively; and T. denticola, E. corrodens, and F. nucleatum were found in all subjects. In the 198 samples, A.a. was detected in 25.8%, P. gingivalis in 51.5%, P. intermedia in 64.1%, T. denticola in 60.6%, E. corrodens in 72.9%, F. nucleatum in 74.7%, and W. recta in 65.7%. The predominant combination was the simultaneous presence of P. intermedia, T. denticola, E. corrodens, F. nucleatum, and W. recta in 89.5% of the subjects and 46.8% of the sites. Of these sites, 51.1% showed the combined presence of P. gingivalis and 28.4% that of both A.a. and P. gingivalis. None of the seven bacteria could be detected in 14.4% of the total sites sampled. The present study indicates that severe destructive adult periodontitis is a multibacterial infection and that certain combinations of periodontopathogens seem to be important in the pathogenesis of the disease.

"Checkerboard" versus culture: a comparison between two methods for identification of subgingival microbiota.

The present study compared the "checkerboard" DNA-DNA hybridization methodology with culture techniques for the analysis of the composition of the subgingival microbiota. 70 subjects, presenting with a variety of periodontal conditions, contributed with a total of 283 subgingival plaque samples analyzed with respect to the following species: Porphyromonas gingivalis, Prevotella intermedia/Prevotella nigrescens, Fusobacterium nucleatum, Campylobacter rectus, Eikenella corrodens, Bacteroides forsythus, Actinobacillus actinomycetemcomitans, Streptococcus sanguis and Streptococcus mutans. Species identification and quantification was performed by (i) the checkerboard method, using whole genomic, digoxigenin labeled DNA probes; and (ii) culture, including non-selective and selective media in combination with routine biochemical testing using commercial test panels. We found that the checkerboard technology resulted in higher prevalence figures for half of the species tested when compared to culture data. If the latter were used as the reference, checkerboard detection sensitivities ranged from 0.17 to 0.86, specificities from 0.17 to 1.0, and diagnostic accuracies from 0.51 to 0.81, depending on bacterial species. The use of the checkerboard data as the reference resulted in detection sensitivities for the culture procedures between 0.24 and 1.0 and specificities between 0.21 and 0.87. The checkerboard methodology resulted in statistically significant higher bacterial counts for the majority of the species. It was further observed that, for most species, the higher the total number colony-forming units in the sample, the higher the discrepancy between the results obtained by the two techniques.

Subgingival microbial profile of Papillon-Lefèvre patients assessed by DNA-probes.

The prevalence of 18 selected bacterial species was assessed by means of "checkerboard" DNA-DNA hybridisation in a group of 12 Saudi-Arabian adolescents with Papillon-Lefèvre syndrome. A total of 36 tooth sites were investigated. The patients exhibited severe periodontal disease with deep pockets. All 12 patients harboured the putative bacterial pathogens P. intermedia, F. nucleatum, P. micros and S. intermedius while T. denticola, B. forsythus, P. nigrescens, E. corrodens, S. noxia and C. rectus were recovered from 11 patients. P. gingivalis was recovered from 9 patients and 18 sites while corresponding figures for A. actinomycetemcomitans were 8 and 19, respectively. A number of the investigated species (B. forsythus, T. denticola, P. intermedia, C rectus) reached high levels (> or =10(6) cells) in more than 1/2 of the patients. On the other hand, bacteria such as A. actinomycetemcomitans and P. gingivalis were infrequently encountered at high levels in these subgingival samples. In conclusion, the analysis failed to demonstrate a PLS-specific profile of the subgingival infection, since the bacterial composition of the sampled sites closely resembled that characterising deep pockets in adult periodontitis patients.