Actinomycetemcomitans-associated Periodontitis Research Articles

Lactobacillus acidophilus LA-5 Ameliorates Inflammation and Alveolar Bone Loss Promoted by A. actinomycetemcomitans and S. gordonii in Mice and Impacts Oral and Gut Microbiomes.

The benefits of probiotics on dysbiotic microbiomes and inflammation are dependent on the tested strain, host factors, and the resident microbiome. There is limited knowledge on the effects of probiotics in A. actinomycetemcomitans-associated periodontitis. Thus, Lactobacillus acidophilus LA5 (LA5) was orally inoculated for 30 days in C57Bl/6 mice infected with A. actinomycetemcomitans JP2 (Aa) and S. gordonii (Sg). Alveolar bone loss, gingival gene expression, and oral and gut microbiomes were determined. LA5 controlled bone loss in Aa+Sg-infected mice, downregulated the expression of Il-1β and upregulated Il-10 in gingival tissues, and altered the oral and gut microbiomes. LA5 increased the diversity of the oral microbiome of Aa+Sg infected mice, and Aa+Sg and Aa+Sg+LA5 oral or gut microbiomes clustered apart. LA5 induced shifts in Aa+Sg infected mice by increasing the abundance of Muribaculaceae and decreasing Bifidobacteriaceae in the oral cavity and increasing the abundance of Verrucomicrobiae and Eggerthellales in the gut. In conclusion, LA5 oral administration controls experimental Aa-associated periodontitis by altering inflammatory gene expression and the oral and gut microbiomes.

Lactobacilli Attenuate the Effect of Aggregatibacter actinomycetemcomitans Infection in Gingival Epithelial Cells.

Probiotics may be considered as an additional strategy to achieve a balanced microbiome in periodontitis. However, the mechanisms underlying the use of probiotics in the prevention or control of periodontitis are still not fully elucidated. This in vitro study aimed to evaluate the effect of two commercially available strains of lactobacilli on gingival epithelial cells (GECs) challenged by Aggregatibacter actinomycetemcomitans. OBA-9 GECs were infected with A. actinomycetemcomitans strain JP2 at an MOI of 1:100 and/or co-infected with Lactobacillus acidophilus La5 (La5) or Lacticaseibacillus rhamnosus Lr32 (Lr32) at an MOI of 1:10 for 2 and 24 h. The number of adherent/internalized bacteria to GECs was determined by qPCR. Production of inflammatory mediators (CXCL-8, IL-1β, GM-CSF, and IL-10) by GECs was determined by ELISA, and the expression of genes encoding cell receptors and involved in apoptosis was determined by RT-qPCR. Apoptosis was also analyzed by Annexin V staining. There was a slight loss in OBA-9 cell viability after infection with A. actinomycetemcomitans or the tested probiotics after 2 h, which was magnified after 24-h co-infection. Adherence of A. actinomycetemcomitans to GECs was 1.8 × 107 (± 1.2 × 106) cells/well in the mono-infection but reduced to 1.2 × 107 (± 1.5 × 106) in the co-infection with Lr32 and to 6 × 106 (± 1 × 106) in the co-infection with La5 (p < 0.05). GECs mono-infected with A. actinomycetemcomitans produced CXCL-8, GM-CSF, and IL-1β, and the co-infection with both probiotic strains altered this profile. While the co-infection of A. actinomycetemcomitans with La5 resulted in reduced levels of all mediators, the co-infection with Lr32 promoted reduced levels of CXCL-8 and GM-CSF but increased the production of IL-1β. The probiotics upregulated the expression of TLR2 and downregulated TLR4 in cells co-infected with A. actinomycetemcomitans. A. actinomycetemcomitans-induced the upregulation of NRLP3 was attenuated by La5 but increased by Lr32. Furthermore, the transcription of the anti-apoptotic gene BCL-2 was upregulated, whereas the pro-apoptotic BAX was downregulated in cells co-infected with A. actinomycetemcomitans and the probiotics. Infection with A. actinomycetemcomitans induced apoptosis in GECs, whereas the co-infection with lactobacilli attenuated the apoptotic phenotype. Both tested lactobacilli may interfere in A. actinomycetemcomitans colonization of the oral cavity by reducing its ability to interact with gingival epithelial cells and modulating cells response. However, L. acidophilus La5 properties suggest that this strain has a higher potential to control A. actinomycetemcomitans-associated periodontitis than L. rhamnosus Lr32.

Spiramycin resistance in human periodontitis microbiota

Purpose The occurrence of in vitro resistance to therapeutic concentrations of spiramycin, amoxicillin, and metronidazole was determined for putative periodontal pathogens isolated in the United States. Materials and methods Subgingival plaque specimens from 37 consecutive adults with untreated severe periodontitis were anaerobically cultured, and isolated putative periodontal pathogens were identified to a species level. In vitro resistance to spiramycin at 4 μg/ml, amoxicillin at 8 μg/ml, and/or metronidazole at 16 μg/ml was noted when putative periodontal pathogen growth was noted on the respective antibiotic-supplemented primary culture plates. Results A total of 18 (48.7%) subjects yielded antibiotic-resistant putative periodontal pathogens with spiramycin at 4 μg/ml in drug-supplemented primary isolation plates, as compared to 23 (62.2%) subjects with amoxicillin at 8 μg/ml, and 10 (27.0%) subjects with metronidazole at 16 μg/ml. Spiramycin in vitro resistance occurred among species of Fusobacterium nucleatum (44.4% of organism-positive subjects), Prevotella intermedia/nigrescens (11.1%), Parvimonas micra (10.8%), Streptococcus constellatus (10%), Streptococcus intermedius (10%), Porphyromonas gingivalis (6.7%), and Tannerella forsythia (5.3%). Amoxicillin in vitro resistance was found in P. intermedia/nigrescens (55.5%), T. forsythia (15.8%), S. constellatus (10%), F. nucleatum (5.6%), and P. micra (2.7%). Only S. constellatus (70%) and S. intermedius (40%) exhibited in vitro resistance to metronidazole. When subject-based resistance data for spiramycin and metronidazole were jointly considered, all isolated putative periodontal pathogens were inhibited in vitro by one or the other of the antibiotic concentrations, except for one strain each of S. constellatus and S. intermedius from one study subject. Similarly, either amoxicillin or metronidazole at the drug concentrations tested inhibited in vitro all recovered putative periodontal pathogens, except S. constellatus in one subject. Conclusions In vitro spiramycin resistance among putative periodontal pathogens of United States origin occurred in approximately one-half of severe periodontitis patients evaluated, particularly among subgingival F. nucleatum species. In vitro resistance patterns also suggest that therapeutic concentrations of spiramycin plus metronidazole may have potential antimicrobial efficacy in non- Aggregatibacter actinomycetemcomitans-associated periodontitis similar to amoxicillin plus metronidazole, which may be beneficial, where spiramycin is clinically available, for patients hypersensitive to amoxicillin or other beta-lactam antibiotics.

Rationale for use of antibiotics in periodontics.

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

Oral ecology and person-to-person transmission of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis.

The ecological characteristics of the oral cavity are dissimilar for A. actinomycetemcomitans and for P. gingivalis, as judged by differences in their colonization preferences and patterns, associations with periodontal disease parameters, relationships with the subgingival microbiota and the type of periodontitis and their clonal persistence in the oral cavity. These features also suggest that as a periodontal pathogen, A. actinomycetemcomitans is different from P. gingivalis. Probably in most infected individuals, low levels of A. actinomycetemcomitans can persist for years in equilibrium with the host and the resident oral microbiota. However, it is well established that A. actinomycetemcomitans can cause disease in some individuals or in some circumstances when the regulatory mechanisms are unable to maintain homeostasis in the ecosystem. Elevated A. actinomycetemcomitans proportions of the biota can be regarded as a sign of ecological imbalance, leading to increased risk of periodontal destruction. There is also evidence showing elevated pathogenic potential of certain A. actinomycetemcomitans clones. Although A. actinomycetemcomitans seems to be relatively rarely transmitted between cohabiting adults, transmission can occur to periodontally healthy children of A. actinomycetemcomitans-positive parents. Parents and children may share factors that promote successful oral colonization of A. actinomycetemcomitans, or the window of opportunity is in childhood. Therefore, to prevent parent-child transmission of A. actinomycetemcomitans, bacterium-positive parents of young children are optimal targets for enhanced information and treatment. In selected populations, screening for specific clones of A. actinomycetemcomitans has been employed in prevention of peridontitis. Future research aiming at finding the reasons which cause the changes in the oral homeostasis to allow the growth of A. actinomycetemcomitans may give insight into novel prevention strategies for A. actinomycetemcomitans-associated periodontitis. Compared with A. actinomycetemcomitans, P. gingivalis shows a different pattern of coexistence with the host. In periodontal health or in children, P. gingivalis is absent or only rarely detected. When present, P. gingivalis is commonly recovered in high numbers from dentitions exhibiting inflamed periodontitis and poor oral hygiene. Contrary to A. actinomycetemcomitans, the data on the vertical transmission of P. gingivalis are limited. The major infection route of P. gingivalis seems to be between adults, indicating that P. gingivalis commonly colonizes in an established oral microbiota. These characteristics suggest that the degree of tolerance between P. gingivalis and the host is inferior to that between A. actinomycetemcomitans and the host. It appears that the association of P. gingivalis with disease is a rule rather than an accidental incident. On these grounds, it seems that the host-P. gingivalis relationship approaches antibiosis. Since P. gingivalis infection is related to a typical periodontal eco-pathology, the susceptibility to person-to-person transmission of this pathogen may be controlled by periodontal treatment and emphasizing the significance of high standard oral hygiene.

Eradication of Actinobacillus actinomycetemcomitans from the oral cavity in adult periodontitis.

Eradication of Actinobacillus actinomycetemcomitans from the oral cavity seems to be a prerequisite for successful therapeutic outcome in patients periodontally infected with the organism. In view of the limited number of subgingival samples obtained in recent studies one cannot conclude, however, whether eradication has actually been achieved. In the present study clinical and microbiological parameters were monitored in 10 adult patients with A. actinomycetemcomitans-associated periodontitis during successive non-surgical and adjunctive metronidazole plus amoxicillin (or ciprofloxacin) (AB) therapy. In every patient, 13 extracrevicular samples and subgingival samples from the deepest site of every tooth present were selectively cultivated for A. actinomycetemcomitans. The organism was isolated in 47 +/- 29% subgingival and 64 +/- 31% extracrevicular samples. Six weeks following subgingival scaling, A. actinomycetemcomitans was detected in 37 +/- 30% subgingival and 55 +/- 38% extracrevicular samples (n.s.). Three months after antibiotic therapy, the organism was recovered from only 1 patient. At baseline, 7.5 +/- 4.2% sites had a probing pocket depth (PPD) > or = 7 mm. This proportion dropped to 2.3 +/- 2.4% after scaling (p < 0.05) and to 0.3 +/- 0.4% after AB (p < 0.05). The proportion of sites with clinical attachment loss (CAL) > or = 6 mm dropped from 23.3 +/- 13.3% to 17.7 +/- 13.4% (p < 0.05) and to 16.8 +/- 14.6%. Statistical analysis revealed that the organism was strongly related, at baseline, to PPD > or = 7 mm (odds ratio 9.8, p < 0.001). Six weeks after scaling, the organism was associated with CAL > or = 6 mm (odds ratio 1.8, p = 0.02). After scaling, high counts of A. actinomycetemcomitans in excess of 10(4) CFU/ml significantly interfered with attachment gain of > or = 2 mm (odds ratio 0.24, p = 0.001). Based on the present findings, eradication of A. actinomycetemcomitans seems to be possible with adjunctive antibiotic treatment. Elimination of the organism after scaling was only weakly associated with clinical improvement.

Extracellular 37-kDa antigenic protein from Actinobacillus actinomycetemcomitans induces TNF-alpha, IL-1 beta, and IL-6 in murine macrophages.

The extracellular antigens of Actinobacillus actinomycetemcomitans Y4 (serotype b) contain a 37-kDa protein which is a major target for IgGs from patients suffering from severe alveolar bone loss. Since the 37-kDa protein has not been studied sufficiently, our investigation focused on its characteristics, e.g., its localization, specificity, and whether it directly stimulates macrophages to produce cytokines. The 37-kDa protein was purified from the culture supernatant of the Y4 strain by means of chromatofocusing and gel filtration. The 37-kDa protein is a unique glycoprotein which forms immune complexes with monoclonal antibodies against rhamnose-fucose polysaccharide. Patients with A. actinomycetemcomitans-associated periodontitis had higher antibody titers to the purified 37-kDa protein than healthy subjects (p < 0.001). Anti-37-kDa protein antibodies recognized a 37-kDa band in the cytosolic, ribosomal, and total membrane fractions from Y4 cells. Extracellular substances from other strains of A. actinomycetemcomitans (serotypes a and c) also reacted in the Western blots, but Haemophilus spp. or several periodontopathic bacteria did not. These results suggested that the 37-kDa protein is a cytosolic protein that is passed through the cell membrane, and its protein portion is specific for A. actinomycetemcomitans but common to serotypes. This protein induced Il-1 beta, Il-6, and TNF-alpha release from murine macrophages. The Il-6-inducing activity of the 37-kDa protein was higher than that of LPS. These findings suggested that the 37-kDa protein which is released from live cells plays a role in A. actinomycetemcomitans-associated periodontitis, as antigen inducing the release of inflammatory cytokines which are associated with alveolar bone loss.

Microbial ecology of Actinobacillus actinomycetemcomitans, Eikenella corrodens and Capnocytophaga spp. in adult periodontitis.

Information on intraoral distribution of putative periodontal pathogens might be essential for controlling different forms of periodontal disease. Colonization may be either promoted or impeded by other bacteria competing in the subgingival ecosystem. In recent investigations microbial associations between dental organisms have been determined in a multitude of subgingival plaque samples within multiple patients and described by odds ratios, in most circumstances without taking into account the correlated structure of the observations within a single individual. The present investigation had 3 major objectives: (i) to describe the intraoral distribution of some facultatively anaerobic, Gram-negative rods, i.e. Actinobacillus actinomycetemcomitans, Eikenella corrodens-like organisms and Capnocytophaga spp., in a multitude of subgingival and extracrevicular samples of 10 adult subjects with A. actinomycetemcomitans-associated periodontitis; (ii) to analyse possible inconsistencies of microbial associations between these periodontal organisms; and (iii) to determine factors increasing the likelihood of isolating these bacteria in a given subgingival site by employing Generalized Estimation Equation (GEE) methods. Clinical examinations were carried out at 6 sites of every tooth present. In each subject, 13 extracrevicular (2 cheek mucosa, 3 tongue, 4 gingival, 2 tonsillar samples, 1 palatinal, 1 saliva sample) and between 22 and 44 subgingival samples from deepest sites of every tooth present (n = 296) were selectively cultivated for A. actinomycetemcomitans, E. corrodens and Capnocytophaga spp. In extracrevicular material, A. actinomycetemcomitans, Capnocytophaga spp. and E. corrodens were isolated in 9, 10 and 6 patients, and from 65, 82 and 15% samples, respectively. The organisms were recovered from 51, 62 and 27% subgingival plaque samples, respectively. Heterogeneity tests did not reveal significant inconsistencies of microbial associations between bacteria in subgingival plaque. Mantel-Haenszel's odds ratios ranged between 2.0 for A. actinomycetemcomitans and Capnocytophaga spp. and 18.7 for Capnocytophaga spp. and E. corrodens. An exchangeable working dependence structure was employed in the GEE approach. The odds of isolating A. actinomycetemcomitans was increased by factor 3.7 in 4-6 mm deep pockets, and 9.5 in > or = 7 mm deep pockets. The odds of presence of E. corrodens was increased by factor 10.8 in the case of presence of Capnocytophaga spp. and 2.1 in the case of presence of A. actinomycetemcomitans. Capnocytophaga spp. were associated with bleeding on probing and molar sites. Presence of E. corrodens was associated with clinical attachment loss but not periodontal probing depth. Results of the present study indicated an association of A. actinomycetemcomitans with periodontal pathology. Whereas this organism and Capnocytophagae were widely distributed in extracrevicular ecosystems of the mouth, E. corrodens only occasionally appeared in saliva or on mucous membranes of the oral cavity. In general, GEE methods seem to allow to determine factors associated with the presence of periodontal organisms in a multivariate approach and considering the correlated structure of the data.

Microbiological and clinical effects of metronidazole and amoxicillin in Actinobacillus actinomycetemcomitans associated periodontitis

In this study, we evaluated the microbiological and clinical effects of mechanical debridement in combination with metronidazole and amoxicillin therapy in 48 patients with Actinobacillus actinomycetemcomitans-associated periodontitis, 3 months and at least 24 months after active treatment. The results of this study showed that 47 out of 48 patients were still negative for A. actinomycetemcomitans subgingivally, at the mucous membranes, the tonsillar area and in the saliva, 2 years after therapy. The clinical results showed that a reduction of probing pocket depth, probing attachment level, bleeding index and plaque index was not only seen in the time between baseline and 3 months after therapy, but further clinical improvement was observed between 3 and 24 months after active treatment. We conclude that combined mechanical debridement and metronidazole plus amoxicillin therapy is very effective in suppressing A. actinomycetemcomitans below cultivable levels over a long period of time, suggesting elimination of this organism, and that recolonization of A. actinomycetemcomitans seems to be a rare event. The elimination of A. actinomycetemcomitans is paralleled by a further improvement of the periodontal status of the patients, even up to 24 months after active treatment.

Postoperative bleeding tendency as a risk factor in Actinobacillus actinomycetemcomitans‐associated periodontitis

Frequent bleeding on probing (BOP) has been considered a risk factor for recurrence of periodontitis. In the present study, 29 patients with Actinobacillus actinomycetemcomitans-associated periodontitis were enrolled in a carefully performed recall system. At 6 sites per tooth, periodontal probing depth (PPD), gingival index (GI), plaque index (PlI) and BOP was assessed 6 weeks, 6 months, 1 and 2 years after comprehensive therapy. Professional toothcleaning and subgingival scaling at sites with PPD > or = 5 mm and BOP was carried out every 2nd or 3rd month. Subgingival samples from 2 sites, a pooled subgingival sample, cheek mucosa, saliva and tongue samples were selectively cultivated for A. actinomycetemcomitans after 2 years. Following active therapy, 8% sites had a PPD of > or = 4 mm, whereas 21% sites bled on probing. After 2 years, respective figures were 12 and 27%. During maintenance, frequent BOP (> or = 3 times at 4 visits) had a predictive value of 0.133 to indicate an increase in PPD of > or = 2 mm and a negative predictive value of 0.947. The predictive value of no bleeding to indicate a stable site was 0.972, the negative predictive value 0.078. There was evidence for heterogeneity of associations between increase in PPD of > or = 2 mm and > or = 3 times BOP among patients (chi 2(28) = 41.45, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Failure of adjunctive minocycline-HCl to eliminate oral Actinobacillus actinomycetemcomitans.

Considerable problems have been reported in the eradication of Actinobacillus actinomycetemcomitans from periodontal sites. The present communication describes the 2-year results of a comprehensive combined mechanical/surgical and adjunctive minocycline (200 mg/day for 3 and another 2 weeks) treatment regimen in 28 patients with A. actinomycetemcomitans-associated periodontitis. Elimination of A. actinomycetemcomitans at periodontal sites was a prerequisite for gain of clinical attachment of > or = 2 mm or decrease of probing depth to < or = 4 mm after subgingival scaling plus minocycline (p < 0.01). Whereas 2 years after active treatment A. actinomycetemcomitans could not be detected at monitored sites in 23 patients, the organism was found on buccal mucosa and in saliva in 17 and 12 cases, respectively. One or 2 years after periodontal surgery, there was a significant association between log10-numbers of A. actinomycetemcomitans in buccal samples and numbers of residual pockets of > or = 7 mm as well as gingival sites with overt gingivitis (R2 = 0.687, p < 0.001). Present results indicate failure of an even prolonged administration of adjunctive minocycline to eliminate oral A. actinomycetemcomitans in most cases of A. actinomycetemcomitans-associated periodontitis.

In vitro susceptibilities of Actinobacillus actinomycetemcomitans to a number of antimicrobial combinations.

The in vitro susceptibilities of Actinobacillus actinomycetemcomitans to 14 antimicrobial combinations were studied by using the checkerboard titration technique. The results, expressed as the range of the fractional inhibitory concentration indices, were as follows: for metronidazole or its hydroxymetabolite combined with cefixime, 0.2 to 0.6; for moxalactam, 0.2 to 0.6; for penicillin G, 0.3 to 0.6; for tobramycin, 0.8 to 2.0; for erythromycin, 0.8 to 1.7; for ciprofloxacin, 0.2 to 0.6; for tetracycline, 0.8 to 1.2. Our observations indicated that the beta-lactam antibiotics as well as ciprofloxacin act synergistically with both metronidazole and its hydroxymetabolite against A. actinomycetemcomitans. Synergistic interactions were independent of the individual MICs of the antibiotics tested. Erythromycin, tobramycin, and tetracycline combined with either metronidazole or its hydroxymetabolite showed additive to indifferent effects against the five strains of A. actinomycetemcomitans, with the fractional inhibitory concentration indices ranging from 0.8 to 2.0. A. actinomycetemcomitans was found to be highly susceptible to ciprofloxacin (MIC of ciprofloxacin for 90% of strains tested, 0.010 micrograms/ml) and cefixime (MIC of cefixime for 90% of strains tested, 0.8 micrograms/ml). The results indicate that in patients who are allergic to penicillin, cefixime and ciprofloxacin may be useful alternative antibiotics in combination with metronidazole for the treatment of A. actinomycetemcomitans-associated periodontitis.

Microbiological and clinical results of metronidazole plus amoxicillin therapy in Actinobacillus actinomycetemcomitans-associated periodontitis.

We report on the microbiological and clinical effects of mechanical debridement in combination with metronidazole plus amoxicillin therapy in 118 patients with Actinobacillus actinomycetemcomitans-associated periodontitis. Patients were categorized into 3 groups: 28 had localized periodontitis; 50 had generalized periodontitis, and 40 had refractory periodontitis. After initial treatment and metronidazole plus amoxicillin therapy 114 of 118 (96.6%) patients had no detectable A. actinomycetemcomitans. Significant reduction in pocket probing depth and gain of clinical attachment were achieved in almost all patients. Four patients were still positive for A. actinomycetemcomitans after therapy. Metronidazole resistance (MIC greater than 25 micrograms/ml) was observed in 2 of 4 strains from these patients. Patients still positive for A. actinomycetemcomitans or Porphyromonas gingivalis showed a significant higher bleeding tendency after therapy. It was concluded that mechanical periodontal treatment in combination with the metronidazole plus amoxicillin therapy is effective for subgingival suppression of A. actinomycetemcomitans in patients with severe periodontitis.

Microbiology in Diagnosis and Treatment of Severe Periodontitis. A Report of Four Cases

The purpose of this report is to illustrate the use of microbiology in the diagnosis and treatment in four cases of severe adult periodontitis. All four patients were subgingivally infected with Actinobacillus actinomycetemcomitans which was the basis for an adjunct minocycline therapy. Scaling and root planing plus minocycline were not able to establish a clinically stable periodontal condition. A. actinomycetemcomitans was not eliminated from the pockets in any of the patients. Good clinical and microbiological results were obtained after continuous conventional treatment with an adjunct therapy of metronidazole plus amoxycillin. With a 7 day course of this antibiotic therapy, the pockets of all four patients became free of A. actinomycetemcomitans and were still negative on repeated sampling on recall visits. Microbiological monitoring of severe periodontitis in adults seems to be a valuable adjunct to clinical examination. Furthermore, effective antibiotic therapy in A. actinomycetemcomitans-associated periodontitis appears of paramount importance in obtaining a stable periodontal situation.