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The microbiological status of patients with periodontitis in southern Estonia after non-surgical periodontal therapy

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Our objective was to investigate the presence of periodontal pathogens in comparison with the total degree of microorganisms after non-surgical periodontal therapy. The study material consisted of microbiological samples from periodontal pockets originating from 140 consecutive patients with chronic generalized severe periodontitis. The subgingival samples from periodontal pockets were obtained by a sterile curette, placed into 2 ml of the VMGA III medium, homogenized and serially diluted in the Brucella broth. 100 microl aliquots from the dilutions were inoculated onto the Brucella and the TSBV agar. The plates were incubated in an anaerobic chamber and under microaerobic conditions. The isolates were identified according to colonial and cellular morphology, the potency disk pattern, and the biochemical profiles. After instrumentation, no periodontal pathogens were isolated in 46 (33%) patients, while 94 patients (67%) were infected with one to five different periodontal pathogens. However, higher degree of the total microflora was positively correlated with number of isolated pathogens, a putative indicator of their presence. Therefore, due to the occurrence of residual microorganisms after non-surgical mechanical treatment, information about the pattern of these pathogens is needed for application of antimicrobial therapy. The level of microbial load in gingival pockets, including both pathogenic and non-pathogenic species, is one of the determinants of presence of residual pathogens after non-surgical periodontal therapy.
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Stomatologija, Baltic Dental and Maxillofacial Journal, 2005, Vol. 7., N. 2. 45
SUMMARY
Our objective was to investigate the presence of periodontal pathogens in comparison with the total
degree of microorganisms after non-surgical periodontal therapy. The study material consisted of microbio-
logical samples from periodontal pockets originating from 140 consecutive patients with chronic generalized
severe periodontitis. The subgingival samples from periodontal pockets were obtained by a sterile curette,
placed into 2 ml of the VMGA III medium, homogenized and serially diluted in the Brucella broth. 100 µl aliquots
from the dilutions were inoculated onto the Brucella and the TSBV agar. The plates were incubated in an
anaerobic chamber and under microaerobic conditions. The isolates were identified according to colonial and
cellular morphology, the potency disk pattern, and the biochemical profiles. After instrumentation, no peri-
odontal pathogens were isolated in 46 (33%) patients, while 94 patients (67%) were infected with one to five
different periodontal pathogens. However, higher degree of the total microflora was positively correlated with
number of isolated pathogens, a putative indicator of their presence. Therefore, due to the occurrence of
residual microorganisms after non-surgical mechanical treatment, information about the pattern of these patho-
gens is needed for application of antimicrobial therapy. The level of microbial load in gingival pockets, includ-
ing both pathogenic and non-pathogenic species, is one of the determinants of presence of residual patho-
gens after non-surgical periodontal therapy.
Key words: invasive pathogens antimicrobial susceptibility
1
Department of Clinical Microbiology, United Laboratories of Tartu
University Clinics.
2
University of Tartu, Medical Faculty, Department of Stomatology.
Krista Lõivukene
1
- MD, PhD, clinical microbiologist.
Paul Naaber
1
- MD, PhD, Head of Department of Microbiology of
United Laboratories of Tartu.
Mare Saag
2
- MD, PhD, professor; Head of the Department of Stoma-
tology.
Ene-Renate Pähkla
2
- MD, dentist, lecturer.
Taive Koppel
2
- MD, dentist, lecturer.
Address correspondence to Krista Lõivukene, Department of Clinical
Microbiology, United Laboratories of Tartu University Clinics,
Puusepa 1A, Tartu 50406, Estonia.
E-mail: krista.loivuke@kliinikum.ee
The Microbiological Status of Patients with Periodontitis
in Southern Estonia after Non-surgical
Periodontal Therapy
Krista Lõivukene, Ene-Renate Pähkla, Taive Koppel, Mare Saag, Paul Naaber
SCIENTIFIC ARTICLES
Stomatologija, Baltic Dental and Maxillofacial Journal, 7:45-7, 2005
INTRODUCTION
Periodontitis is a chronic infectious disease, which leads
to the destruction of periodontal ligament fibres and alveo-
lar bone until tooth loss [1]. Porphyromonas gingivalis, Ac-
tinobacillus actinomycetemcomitans, Tannerella
forsythensis, Fusobacterium nucleatum, Prevotella
intermedia/nigrescens, Micromonas micros, and
Campylobacter rectus have been suggested as periodontal
pathogens [2,3]. Dominating periodontal pathogens may
vary geographically and regionally [4]. However, in different
populations the presence of the pathogens may reflect rather
the microecology of the mouth; on the other hand, the pres-
ence of suspected pathogens may not be strictly related to
the disease [5,6].
Oral hygiene instruction, scaling and root planing in
local anaesthesia should constitute the basis for periodon-
tal therapy [7,8]. Non-surgical periodontal therapy alone
may fail to eliminate pathogens because of their location
within the gingival tissues or in tooth structures inacces-
sible to periodontal instruments. Evidence of bacterial
specification in periodontitis patients has led to the use of
periodontal instrumentation combined with systemic anti-
biotic treatment for certain patients [9]. However, the choice
of antibiotics depends on the spectrum of the main patho-
gens and their susceptibility patterns [10-12]. Beginning
from 2001, patients with periodontitis from Southern Esto-
nia have been investigated microbiologically after the
mechanical debridement. Our objective was to investigate
the presence of periodontal pathogens in comparison with
the total level of microorganisms after non-surgical peri-
odontal therapy.
MATERIALS AND METHODS
The study material was collected from January 2001 to
April 2003 from 140 adult patients with chronic generalized
severe periodontitis referred to the Policlinic of the Tartu
University Dental Clinic. The diagnosis was defined by
CPITN score 4 in at least three sextants (periodontal pocket
46 Stomatologija, Baltic Dental and Maxillofacial Journal, 2005, Vol. 7., N. 2.
SCIENTIFIC ARTICLES V.Brukiene et al.
depth 6 mm, clinical attachment loss 5 mm) in at least two
teeth and the minimum radiographic marginal alveolar bone
loss 1/3 of the root length in at least three quadrants. The
patients were systemically healthy and had not received
antibiotics within three months prior to entering the investi-
gation. Non-surgical periodontal treatment (scaling and root
planing under local anaesthesia at 4 up to 6 appointments
during 2-3 weeks) was performed and three weeks after
completion of the treatment periodontal status was clini-
cally evaluated by periodontal specialists. From patients
with clinical signs of inflammation, such as bleeding and/or
suppuration on probing, redness and swelling of gingivae,
the subgingival samples from six deepest periodontal pock-
ets were obtained by a sterile curette [3], placed into 2 ml of
the VMGA III medium [13] and taken to the laboratory within
4 hours. The samples were homogenized with a Vortex mixer.
The bacterial suspension was then serially diluted in 5-fold
steps in the Brucella broth (Oxoid, Basingstoke, Hampshire,
UK), 100 µl aliquots from the dilutions were inoculated onto
the Brucella agar (Oxoid) enriched with 5% horse blood and
1% menadione, and the TSBV (Oxoid) agar. The Brucella
Agar plates were incubated in an anaerobic chamber
(Sheldon Manufacturing Inc., Cornelius, Oregon, USA; gas
mixture: 5% CO2, 5% H2, 90% N2) and TSVB plates under
microaerobic (Oxoid, CampyPak) conditions. After incuba-
tion at 35°C for 5 to 7 days, the isolates were identified
according to colonial and cellular morphology, the potency
disk pattern (Vancomycin, Kanamycin, Colistin, Brilliant
Green, and Oxgall), catalase, oxidase and spot indole reac-
tions, long-wave UV light fluorescence, and MUG assay
[3,14]. All anaerobic microorganisms were tested for absence
of growth under microaerobic conditions. The total level of
microbial load was calculated as the logarithmic value of
colony forming unit per 1mL (log10 CFU/ml).
Ethics Review Committee (ERC) on Human Research
of the University of Tartu approved our study protocol.
Statistical analysis (Descriptive Statistics and
Spearman's correlation) was carried out with the
JandelSigmaStat 2.0 program, and the p-values less than
0.05 were considered statistically significant.
RESULTS AND DISCUSSION
After instrumentation, no periodontal pathogens were
isolated in 46 (33%) patients, while 94 patients (67%) were
infected with one to five different periodontal pathogens
(53 patients harboured one, 27 harboured two, 12 harboured
three and 2 patients harboured five pathogens).
The pathogens isolated from the periodontal pockets
of Estonian patients with chronic generalized severe peri-
odontitis (n=140) are depicted in Figure 1.
One third of the patients remained pathogen-free, in
concordance with the results of other authors, where pri-
mary therapy reduced the amount of pathogens [7,8]. On
the other hand, we found that 67% of the primarily treated
patients were equally infected with mostly one pathogen,
but also with combinations of different pathogens. How-
ever, scaling and root planing alone do not eliminate peri-
odontal pathogens effectively [15]. Therefore, due to the
occurrence of residual microorganisms after non-surgical
mechanical therapy, information about the pattern of these
pathogens is needed for application of antimicrobial therapy
on patients who do not respond to mechanical treatment.
Periodontal microbes can be divided into different risk
groups according to their association with periodontal dis-
ease, where the ``red`` group includes real pathogens and
the ``green`` group comprises the normal oral microflora [16].
Among the well-known pathogens of periodontitis, some
other potentially pathogenic bacteria, eg. Fusobacterium sp.,
Bacteroides sp., Prevotella sp., enterococci, enterobacteria,
and others can play some role in different populations.
Among the periodontal pathogens, Prevotella intermedia/
nigrescens (37 patients) and Actinobacillus
actinomycetemcomitans (36 patients) were dominating,
Micromonas micros was detected in 12, Porphyromonas
gingivalis in 7, Tannerella forsythensis in 4, and
0 5 10 15 20 25 30 35 40
A. actinomycetemcomitans
P. gingivalis
P. intermedia/nigrescens
C. rectus
M. micros
T. forsythensis
Prevotella sp.
Bactroides sp.
Fusobacterium sp.
Enterococcus sp.
Enterobacter sp.
S. aureus
P. aeruginosa
Number of patients
Potential
pathogens
Periodontal
pathogens
Fig. 1. Specific and potential periodontal pathogens in Estonian patients with periodontitis (n=140) after non-surgical periodontal therapy
Stomatologija, Baltic Dental and Maxillofacial Journal, 2005, Vol. 7., N. 2. 47
V.Brukiene et al. SCIENTIFIC ARTICLES
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Received: 15 04 2005
Accepted for publishing: 20 06 2005
REFERENCES
Campylobacter rectus in 2 patients. Proportional recovery
of Prevotella intermedia/nigrescens varied from 2.3 to 63%
(median 16.9), of Actinobacillus actinomycetemcomitans from
2.4 to 100% (median 23), of Micromonas micros from 3 to
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The total level of microbial load (log10 CFU/ml) of all
isolated microbes varied from zero to 8.4 log, whereas sterile
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found that higher microbial load was positively correlated
(p<0.001) with number of residual pathogens. In the case of
periodontitis with developed deep gingival pockets, the in-
creased amount of the microflora creates good possibilities
for overgrowth of anaerobes, indicating also the presence
of periodontal pathogens.
CONCLUSIONS
Therefore, due to the occurrence of residual microor-
ganisms after non-surgical mechanical treatment, informa-
tion about the pattern of residual pathogens is needed for
application of appropriate antimicrobial therapy for patients
not responding to non-surgical treatment. The higher level
of microbial load in gingival pockets, including both patho-
genic and non-pathogenic species, may be one of the deter-
minants of presence of residual pathogens after non-surgi-
cal mechanical therapy. As periodontitis is a chronic recur-
rent infection, successful diagnosis and therapy should be
based on the individual microbiological examination for rea-
sons including quality control of mechanical therapy as well
as an aid of treatment planning.
ACKNOWLEDGEMENTS
This work was financed by grant No. 5756 from Esto-
nian Science Foundation.
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ABSTRACT The purpose of this study was to evaluate the prevalence of anaerobic bacteria in the sub-gingival plaque in periodontitis in relation to pocket depth. In this procedure ninety seven sub-gingival plaque samples were collected with sterile paper points (size 50), transported and diluted in thioglycolate broth and cultivated on blood agar under anaerobic conditions for (72-96) hours, the total count of anaerobic bacteria were enumerated, the identification of anaerobic bacteria were carried out using morphological and cultural characteristics, biochemical tests and antibiotics susceptibility tests. The ninety seven samples (55 males and 42 females), their ages ranged (18-60) years were distributed according to the clinical parameters and environmental factors; according to pocket depth: 23(< 3mm), 34(3- 5mm), 40(>5mm). According to mean gingival index: 28 (low), 46 (moderate), 23 (sever). According to Plaque index; 27 (low), 52 (moderate), (18 heavy). For smoking 64 were non smoker and 33 smokers, 45 low educated and 52 well educated. The results showed a significant effect of pocket depth on both total count and prevalence of different types of anaerobic bacteria in sub gingival plaque. The total counts were; (3368.7, 23043.53, 120473) CFU/ml for the three groups of pocket depth samples: (<3mm, 3-5mm, > 5mm) respectively. The study also showed a significant effect of both gingival index and plaque index on the total count of anaerobic bacteria, for the (low, moderate, and sever) gingival index the counts were; (7152.57, 55202.61, 127831.30) CFU/ml respectively and for the (low, moderate and heavy) plaque index the counts were; (6868,15, 57360, 139540) CFU/ml respectively. The study showed shifting of anaerobic bacterial isolates from mainly Grampositive facultative anaerobic like Actoinomyces spp. and oral III streptococci in samples of shallow pockets, to mainly Gram-negative strict anaerobic bacteria like Prevotella spp., Porphyromonas spp., Bacteriodes spp. and Fuspbacterium spp. in samples of deep pockets. The environmental factors included in the study (gender, smoking, education, and age) had no effect on the total count of anaerobic bacteria in sub gingival plaque, but smoking caused a significant reduction of Actinomyces spp. in samples of smoker compared with non smoker while Porphyromonas spp., A.actinomycetemcomitans and Fusobacterium spp. were significantly increased in samples of smokers. Sub-gingival plaque samples of low educated had high prevalence of Porhpyromonas spp. compared with educated, while Actinomyces spp. were increased significantly in samples of educated individuals compared with low educated .Age had only a simple correlation r2 = 0.009 with total count of anaerobic bacteria in sub gingival plaque samples.
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The purpose of this study was to evaluate, on a short-term basis, the clinical and microbiological effects of a single course of scaling and root planing as compared with those obtained by flap surgery in patients with moderate to advanced periodontitis. 11 patients participated in the study. Using a split-mouth design, one quadrant of the mouth was treated with reverse bevel flap surgery, whereas the contralateral one was subjected to a single course of scaling and root planing. 2 approximal sites on single-rooted teeth with a pocket depth greater than or equal to 5 mm were monitored clinically and microbiologically for 16 weeks after active treatment. Both techniques resulted in a gain of probable attachment levels, a reduction in bleeding on probing and a reduced mean pocket depth, although 31.2% of the sites in the scaling and root planing group still had 6-7 mm deep pockets at 8 and 16 weeks after treatment. Both techniques reduced median relative proportions and frequencies of detection of black-pigmented Bacteroides species. A highly statistically significant increase (p less than 0.01) in median proportions of oral streptococci was recorded only for surgery within the 1st month post-operatively. No correlation was found between residual pocket depth and any of the microbiological parameters considered in the study, suggesting that residual pocket depth does not exert a significant influence on bacterial subgingival recolonization after therapy. The results from this study suggest that surgery can be as effective as scaling and root planing in favoring the establishment of micro-organisms compatible with periodontal health, although this effect is limited to the 1st month after therapy.
Article
Antibiotic treatment of periodontitis aims at eradicating or controlling specific pathogens. Prime candidates for antibiotic therapy are patients with recently diagnosed active periodontitis or a history of recurrent disease who fail to stabilize following mechanical/surgical therapy. Since a variety of microbes with differing antimicrobial susceptibility profiles may cause periodontitis, selection of antimicrobial agents should be based on proper microbial diagnosis and sensitivity testing, as well as consideration of the patient's medical status. The risk of treating chemotherapeutically solely on the basis of clinical features, radiographic findings or a limited microbiological analysis, is failure to control the pathogens or overgrowth of new pathogens. A review of published papers reveals that appropriate systemic antibiotic therapy may enhance healing in patients with recent or high risk of periodontal breakdown. Systemic antibiotic therapy seems more predictable than topical administration in eradicating periodontal pathogens from deep periodontal pockets. Several promising antimicrobial agents for periodontitis treatment need testing in placebo-controlled, double-blind, randomized clinical trials.
Article
The most common dental diseases, periodontal disease and dental caries, are chronic infections caused by bacteria of normal oral flora. When these bacteria increase in number and irritation exceeds the host defence threshold, disease arises. The human oral flora comprises more than 300 different bacteria. During the last decade approximately 10 species, mainly Gram-negative anaerobes, have been noted as putative pathogens in periodontal disease. The Gram-positive and facultatively anaerobic mutans streptococci are aetiologically the most important bacteria in dental caries. Data have rapidly increased on the association of these bacteria with certain periodontal diseases or caries, on phenotypic and genotypic characteristics, pathogenic mechanisms, antibiotic susceptibility patterns and transmission among family members. Chronic dental infections have been the focus of renewed interest because of recent advances in oral microbiology as well as in medicine. We now know that in addition to oral streptococci, recently classified, fastidious periodontal anaerobes can be detected from various extra-oral infections. Oral bacteria may spread into the blood stream through ulcerated epithelium in diseased periodontal pockets and cause transient bacteraemias, which are regarded as increased risk, especially for immunocompromised patients or persons with endoprotheses. In these patients, routine antibiotic prophylaxis is recommended for invasive dental care procedures. Also the new association between dental infections and myocardial/cerebral infarction have offered new challenges for cooperation between dental and medical researchers.
Article
The present clinical trial was performed to study the effect of systemic administration of metronidazole and amoxicillin as an adjunct to mechanical therapy in patients with advanced periodontal disease. 16 individuals, 10 female and 6 male, aged 35-58 years, with advanced periodontal disease were recruited. A baseline examination included assessment of clinical, radiographical, microbiological and histopathological characteristics of periodontal disease. The 16 patients were randomly distributed into 2 different samples of 8 subjects each. One sample of subjects received during the first 2 weeks of active periodontal therapy, antibiotics administered via the systemic route (metronidazole and amoxicillin). During the corresponding period, the 2nd sample of subjects received a placebo drug (placebo sample). In each of the 16 patients, 2 quadrants (1 in the maxilla and 1 in the mandible) were exposed to non-surgical subgingival scaling and root planing. The contralateral quadrants were left without subgingival instrumentation. Thus, 4 different treatment groups were formed; group 1: antibiotic therapy but no scaling, group 2: antibiotic therapy plus scaling, group 3: placebo therapy but no scaling, group 4: placebo therapy plus scaling. Re-examinations regarding the clinical parameters were performed, samples of the subgingival microbiota harvested and 1 soft tissue biopsy from 1 scaled and 1 non-scaled quadrant obtained 2 months and 12 months after the completion of active therapy. The teeth included in groups 1 and 3 were following the 12-month examination exposed to non-surgical periodontal therapy, and subsequently exited from the study. Groups 2 and 4 were also re-examined 24 months after baseline. The findings demonstrated that in patients with advanced periodontal disease, systemic administration of metronidazole plus amoxicillin resulted in (i) an improvement of the periodontal conditions, (ii) elimination/suppression of putative periodontal pathogens such as A. actinomycetemcomitans, P. gingivalis, P. intermedia and (iii) reduction of the size of the inflammatory lesion. The antibiotic regimen alone, however, was less effective than mechanical therapy with respect to reduction of BoP - positive sites, probing pocket depth reduction, probing attachment gain. The combined mechanical and systemic antibiotic therapy (group 2) was more effective than mechanical therapy alone in terms of improvement of clinical and microbiological features of periodontal disease.
Article
Background, aims: The purpose of the present investigation was to determine the effect of weekly professionally administered supragingival plaque removal on the composition of the supra and subgingival microbiota. Methods: 18 adult subjects with periodontitis who had been treated and were in a maintenance phase of therapy were clinically and microbiologically monitored at baseline, 3, 6 and 12 months. After the baseline visit, the subjects received scaling and root planing followed by professional supragingival plaque removal every week for 3 months. Clinical measures of plaque accumulation, bleeding on probing (BOP), gingival redness, suppuration, pocket depth and attachment level were made at 6 sites per tooth at each visit. Separate supra (N = 1804) and subgingival (N = 1804) plaque samples were taken from the mesial aspect of all teeth excluding third molars in each subject at each time point and evaluated for their content of 40 bacterial taxa using checkerboard DNA-DNA hybridization. Significance of changes in mean counts, prevalence and proportions of bacterial species over time in both supra and subgingival samples were determined using the Quade test and adjusted for multiple comparisons. Results: Mean % of sites exhibiting plaque, gingival redness and BOP were significantly reduced during the course of the study. Significant decreases in mean counts were observed in both supra and subgingival samples. Mean total DNA probe counts (x10(5), +/-SEM) at baseline, 3, 6 and 12 months were: 133+/-19, 95+/-25, 66+/-6, 41+/-6 (p<0.001) for supragingival samples and 105+/-22, 40+/-10, 19+/-4, 13+/-3 (p<0.001) for subgingival samples. Mean counts of 22 of 40 and 34 of 40 species tested were significantly reduced in the supra and subgingival samples respectively over the monitoring period. For example, mean counts of Porphyromonas gingivalis x10(5) at baseline, 3, 6 and 12 months in the subgingival plaque samples were 2.0+/-0.4, 0.5+/-0.2, 0.6+/-0.3, 0.3+/-0.1 (p<0.001); Bacteroides forsythus 2.0+/-0.6, 0.4+/-0.1, 0.4+/-0.2, 0.1+/-0.2 (p<0.001); Treponema denticola 3.4+/-1.1, 0.8+/-0.3, 0.4+/-0.2, 0.3+/-0.3 (p<0.01). Similar reductions were seen in supragingival plaque samples. While counts were markedly reduced by professional plaque removal, the proportion and prevalence of the 40 test species were marginally affected. Conclusions: Weekly professional supragingival plaque removal profoundly diminished counts of both supra- and subgingival species creating a microbial profile comparable to that observed in periodontal health. This profile was maintained at the final monitoring visit, 9 months after completion of therapy.
Article
To define the subgingival microbial profiles of adult subjects from a previously identified rural community of indigenous Indians in Guatemala, Central America. A full-mouth periodontal examination was performed in 114 adult subjects from 45 families. Plaque samples were collected from both deep and shallow periodontal pockets and checkerboard DNA-DNA hybridization was employed to identify 17 species previously associated with periodontitis or health. Plaque deposits and gingivitis were universal and widespread, and periodontal pocketing > or =5 mm was highly prevalent (84% of subjects). Streptococcus sanguis, Actinomyces naeslundii genospecies 2 and Fusobacterium nucleatum were significantly more prevalent in shallow sites. At the subject level, Actinomyces naeslundii and Peptostreptococcus micros were significantly more prevalent in periodontally-healthy subjects. Actinobacillus actinomycetemcomitans was not detected in any sample. There was no association between periodontal disease status and presence of suspected periodontal pathogens. These latter results conflict somewhat with those from treated populations. However, in this population where extensive plaque deposits and gingivitis are universal, the presence of putative pathogens may be more reflective of the local environment.
Article
The purpose of the present investigation was to examine subgingival microbial profiles associated with refractory periodontitis and to seek such profiles in periodontally healthy, periodontally well-maintained elder and untreated periodontitis subjects. 36 subjects were defined as refractory on the basis of further attachment loss after scaling and root planing, surgery and systemically administered antibiotics. A total of 890 subgingival plaque samples (mean/subject=24.7) were taken from the mesial aspect of each tooth in each subject at baseline and individually processed for their content of 40 subgingival taxa using checkerboard DNA-DNA hybridization. Cluster analysis was performed on mean within subject species counts using the chord coefficient and an average unweighted linkage sort. Significant differences among clusters for individual and complexes of species were sought using the Kruskal Wallis test. The microbial profiles of the refractory subjects were compared with those of 27 periodontally healthy subjects (n plaque samples=708), 35 periodontally well-maintained elder subjects (n plaque samples=801) and 115 untreated adult periodontitis subjects (n plaque samples=2871). 28 of 36 refractory subjects fell into 4 clusters with >29% similarity. 10 of 40 species and 4 of 7 complexes differed significantly among clusters. Profile (Cluster) I (n=4) was characterized by high proportions of "yellow" and "green" complex species, profile II (n=3) by low total counts and high proportions of "orange" and "purple" complex species, profile III (n=9) by high total counts and counts of Actinomyces and "purple" complex species, profile IV (n=12) by high proportions of "red" and "orange" complex species. The mean profiles of each cluster were subjected to cluster analysis with microbial data from 4380 (mean 24.7) baseline subgingival plaque samples from 27 periodontally healthy, 35 treated, well-maintained elders and 115 untreated adult periodontitis subjects. 12 clusters were formed with >41% similarity. 3 of the refractory profiles were detected in 3 cluster groups. Profile II in a cluster of 1 healthy, 1 elder and 4 untreated periodontitis subjects; profile III in a cluster of 1 healthy, 2 elder and 12 periodontitis subjects; Profile IV, with 1 healthy and 5 untreated periodontitis subjects. The profile not detected in non refractory subjects was dominated by Streptococcus species. 9 clusters did not harbor refractory profiles. 11.1% of healthy, 8.6% of elder and 18.3% of periodontitis subjects were in clusters exhibiting refractory microbial profiles. 4 subgingival microbial profiles were detected among refractory subjects. "Refractory microbial profiles" could be detected in subjects who had not yet exhibited refractory disease.