Mutans streptococci in subgingival plaque of treated and untreated patients with periodontitis.

BACKGROUND: The etiology of root caries is thought to be identical to coronal caries, though root caries seem to be more complicated because of the higher susceptibility of exposed roots (dentin) by periodontal therapy to demineralization than intact enamel. This implies that mutans streptococci are the most likely pathogens in the development of root caries. Although it is known that both the numbers of mutans streptococci and the frequency of isolation in root caries lesions are negatively correlated with the distance from the gingival margin, the subgingival sulcus has not been considered a possible habitat for mutans streptococci. However, subgingival occurrence of mutans streptococci in both untreated and treated periodontal patients has not been documented well in the literature. OBJECTIVE: To investigate the presence and levels of mutans streptococci in the subgingival plaque of patients (n=154) in different stages of periodontal therapy. The subgingival sulcus may be a possible habitat for mutans streptococci. This localisation of mutans streptococci may be of importance in the development of root caries after periodontal surgery. MATERIALS AND METHODS: In this cross-sectional study, subgingival plaque samples from 154 consecutive adult periodontitis patients were tested for presence and levels of mutans streptococci and putative periodontal pathogens by anaerobic cultures. These patients were divided into 4 groups based on their stage of periodontal treatment: (1) untreated patients; (2) patients after initial periodontal therapy only; (3) patients in the maintenance phase who not underwent periodontal surgery; (4) patients after periodontal surgery. RESULTS: The prevalence of mutans streptococci in the 4 study groups varied from 82% in untreated patients to 94% in maintenance patients. The mean proportion of mutans streptococci was 6.65% in maintenance patients versus 1.86% in untreated patients (p=0.005) and 2.51% in patients after scaling and root planing (p=0.041). CONCLUSIONS: The subgingival area is a microbial habitat for mutans streptococci that may be of importance in the development of root caries in periodontitis patients.

Differences in the composition of the subgingival microbiota of two periodontitis populations of different geographical origin. A comparison between Spain and The Netherlands.

The purpose of this study was to compare the subgingival microbiota of two geographically distinct patient populations using identical clinical and bacteriological methods. Adult patients with a diagnosis of periodontitis were consecutively selected according to pre-defined clinical criteria. Microbiological samples were taken from the deepest four sites with bleeding. The samples were plated on blood agar plates, for the determination of the total anaerobic counts and identification of specific bacterial pathogens, and on TSBV and McConkey for isolation of Actinobacillus actinomycetemcomitans and enteric rods, respectively. Thirty-one patients in Spain and 30 patients in The Netherlands were selected. Both patient groups showed similar clinical characteristics, both in terms of age, gender and periodontal clinical variables. A. actinomycetemcomitans was significantly more prevalent (23.3% vs. 3.2%) in the Dutch group, while Porphyromonas gingivalis was significantly more prevalent (64.5% vs. 36.7%) in the Spanish group. Bacteroides forsythus and most commensal periodontal pathogens showed similar prevalences, except Peptostreptococcus micros that was significantly more frequent in the Dutch group (96.7% vs. 74.2%). In summary, the subgingival microbiota from the Spanish group was characterised by a high prevalence of P. gingivalis and low of A. actinomycetemcomitans, while the flora from the Dutch group was characterised by a high prevalence of A. actinomycetemcomitans and P. micros.

Beta-lactamase producing bacteria in the subgingival microflora of adult patients with periodontitis. A comparison between Spain and The Netherlands.

BACKGROUND/AIMS: Countries with a high per capita antibiotic use frequently demonstrate a high level of drug resistance. The aim of this study was to compare the prevalence and levels of beta-lactamase producing bacteria in the subgingival microflora in adult patients with periodontitis in Spain and The Netherlands, and to characterise beta-lactamase producing bacteria in both patient samples. METHOD: Patients with moderate to severe periodontitis were consecutively selected and asked to report on: current systemic disorders and medications, history of use of antibiotics, and smoking habits. Clinical variables included probing pocket depth, clinical attachment level, plaque, bleeding on probing, and suppuration. Pooled subgingival samples of 4 selected sites were anaerobically cultured in blood agar plates with and without amoxicillin, and amoxicillin/clavulanate. Bacterial colonies growing on amoxicillin plates but not on amoxicillin/clavulanate plates were tested for beta-lactamase production. beta-lactamase producing bacteria were isolated and identified. RESULTS: 31 patients were studied in the Spanish group and 30 in the Dutch group. Comparable mean gender and ages were found. Evaluation of previous antibiotic use revealed that, in the previous 12 months, 54.8% of patients in the Spanish group and 10% in the Dutch group reported antibiotic use (p<0.001). The prevalence of beta-lactamase producing bacteria was 87.1% in the Spanish group and 73.3% in the Dutch group. Total counts of beta-lactamase producing bacteria on amoxicillin plates (p<0.01), the mean number of different beta-lactamase producing colonies per patient (p<0.001), and the number of amoxicillin resistant colonies (p<0.001) were significantly higher in the Spanish group. 74 beta-lactamase producing strains in the Spanish group and 33 in the Dutch group were isolated for identification. 23 out of 35 identified strains in the Spanish group, and 32 out of 33 in the Dutch group belonged to Prevotella genus. CONCLUSIONS: A high prevalence of beta-lactamase producing bacteria has been evaluated in two distinct populations, belonging to two European countries with clear differences in antibiotic usage policy. A higher prevalence and a more complex beta-lactamase producing microflora, were found in the Spanish group, associated with a higher antibiotic consumption. This study shows that a higher use of beta-lactam antibiotics is reflected in the % of beta-lactamase producing bacteria in the subgingival microflora of patients with periodontitis. This information may be important in the treatment of severe periodontitis.

Antimicrobial resistance in the subgingival microflora in patients with adult periodontitis. A comparison between The Netherlands and Spain.

BACKGROUND: The widespread use of antibiotics for prophylaxis and treatment of bacterial infections has lead to the emergence of resistant human pathogens. Great differences have been documented between European countries in the use of systemic antibiotics. In parallel, significant differences in levels of resistant pathogens have been documented. AIM: To investigate whether differences in antibiotic use influence the level of antimicrobial resistance of the subgingival microflora of untreated patients with adult periodontitis in The Netherlands and Spain. METHOD: Blood agar plates containing breakpoint concentrations of penicillin, amoxicillin, amoxicillin and clavunalate, metronidazole, erythromycin, azithromycin, clindamycin and tetracycline were used to determine the proportion of bacteria from the subgingival plaque that was resistant to these antibiotics. In the Spanish patients, statistically significant higher mean levels of resistance were found for penicillin, amoxicillin, metronidazole, clindamycin and tetracycline. The mean number of different bacterial species growing on the selective plates was higher in the Spanish patients, as was the % of resistant strains of most periodontal pathogens. A striking difference was observed in the frequency of occurrence of tetracycline-resistant periodontal pathogens. In Spain, 5 patients had > or =3 tetracycline resistant periodontal pathogens, whereas this was not observed in any of the Dutch patients. CONCLUSIONS: The widespread use of antibiotics in Spain is reflected in the level of resistance of the subgingival microflora of adult patients with periodontitis.

[Antibiotic resistance in the subgingival microflora in patients with adult periodontitis. A comparative survey between Spain and the Netherlands]. / Antibioticumresistentie in de subgingivale microflora in patiënten met adulte parodontitis. Een vergelijkend onderzoek tussen Spanje en Nederland.

The widespread use of antibiotics for treatment of bacterial infections has lead to the emergence of resistant human pathogens. Great differences have been documented between European countries in the use of systemic antibiotics. In parallel, significant differences in levels of resistant pathogens have been documented. In order to investigate whether differences in antibiotic use influence the level of antimicrobial resistance of the subgingival microflora, microorganisms from the subgingival plaque of untreated patients with adult periodontitis in The Netherlands (n = 30) and Spain (n = 31) were compared. Blood agar plates containing breakpoint concentrations of penicillin, amoxicillin, amoxicillin and clavulanate, metronidazole, erythromycin, azithromycin, clindamycin and tetracycline were used to determine the proportion of bacteria from the subgingival plaque that was resistant to these antibiotics. In the Spanish patients, statistically significant higher mean levels of resistance were found for penicillin, amoxicillin, metronidazole, clindamycin and tetracycline. The mean number of different bacterial species growing on the selective plates was higher in the Spanish patients, as was the percentage of resistant strains of most periodontal pathogens. A striking difference was observed in the frequency of occurrence of tetracycline-resistant periodontal pathogens. In Spain, 5 patients had > 3 tetracycline resistant periodontal pathogens, whereas this was not observed in any of the Dutch patients. It is concluded that the widespread use of antibiotics in Spain is reflected in the level of resistance of the subgingival microflora of adult patients with periodontitis.

beta-Lactamase producing bacteria in adult periodontitis.

In 23 untreated adult periodontitis patients, the occurrence of beta-lactamase producing periodontal bacteria was determined. In addition to non-selective isolation media, selective isolation and growth of beta-lactamase positive subgingival bacterial species was carried out on blood agar plates supplemented with amoxicillin and plates with amoxicillin+clavulanic acid. Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, Peptostreptococcus micros, Fusobacterium nucleatum, Bacteroides forsythus and Campylobacter rectus isolates from the non-selective medium were tested for beta-lactamase activity by a nitrocefin disk method (DrySlide) and by a laboratory chromogenic nitrocefin-based test. Isolates from the amoxicillin plates that were absent on the amoxicillin/clavulanic acid plates were identified and tested for beta-lactamase production. Based on the non-selective plates, six of 23 P. intermedia isolates, 2 of 19 B. forsythus isolates and 3 of 23 F. nucleatum isolates were beta-lactamase positive. The beta-lactamase positive species Prevotella loescheii, Prevotella buccae, Prevotella buccalis and Actinomyces spp were recovered from the selective amoxicillin plates. beta-Lactamase positive subgingival species were recovered from 17 of 23 patients (74%) but usually comprised low proportions of the subgingival microbiota (range < 0.01-15%). Comparison of the DrySlide test and the nitrocefin-based laboratory test revealed full agreement of test results. beta-Lactamase activity in whole subgingival plaque was detected in 12 patient samples (52%). It was concluded that beta-lactamase activity in subgingival bacteria in adult periodontitis is a common feature. However, since the majority of the samples showed only low-level enzymatic activity, the clinical relevance of this observation with regard to therapy with unprotected enzyme-susceptible beta-lactams is uncertain, though failure on the other hand, is difficult to rule out when a mechanism of resistance is present. The majority of beta-lactamase positive strains was found among species of the Prevotella genus.

Plaque composition, fluoride tolerance and acid production of mutans streptococci before and after the suspension of the use of fluoride toothpastes.

Although fluoride toothpastes are widely used for caries prevention, little is known about the impact of fluoride dentifrices on plaque composition. Also the issue of adaptation of mutans streptococci to grow in vivo in a fluoride environment has received little attention. Such an adaptation may be of interest as it has been suggested that adapted mutans streptococci may show reduced glycolytic activity thereby being less cariogenic. In the present experiments the impact of the suspension of the use of fluoride toothpastes on plaque composition, fluoride tolerance and acid production of mutans streptococci was studied. Pooled plaque samples from the lingual surfaces of the lower incisors were collected from individuals (n = 13) just before and 7 weeks after they had replaced their fluoride toothpastes (0.1-0.15% F) with a non-fluoride one. The samples were analysed for fluoride and the numbers and proportions of streptococci, Streptococcus mutans, Streptococcus sobrinus, Actinomyces species, and lactobacilli, respectively. The fluoride tolerance of the mutans streptococci was estimated by culture of the plaque samples on TYCSB agar supplemented with of 0, 1, 2, 3, 4, and 5 mmol/l fluoride (NaF) at pH 7.2. From each plaque sample six S. mutans strains were isolated for the measurement of the rate of acid production (Vap) at pH 7 in the presence of 0, 5, and 10 mmol/l F. The overnight final pH was measured in cultures of the S. mutans strains with excess of glucose and 0, 5, and 10 mmol/l F. The results showed that the removal of the fluoride pressure from plaque did not affect the numbers or proportions of the various species and genera of bacteria. The fluoride tolerance of the mutans streptococci, and the Vap or the overnight final pH of the isolated strains had not changed. These results suggest that the use of fluoride toothpaste had not affected plaque composition, nor fluoride tolerance or acidogenicity of mutans streptococci. Probably the amount of fluoride delivered by fluoride dentifrices to dental plaque is too low to induce such adaptations.

K-antigens in Porphyromonas gingivalis are associated with virulence.

We investigated antigens in spreading and non-spreading Porphyromonas gingivalis strains. On the basis of differences in virulence in the mouse model, 8 strains were selected for antiserum production in rabbits. Hyperimmune sera were tested by double immunoprecipitation and immunoelectrophoresis. Besides a common antigen, differences in antigenic composition were observed in the thermolabile antigens between all strains tested. Two different heat-stable antigens were found after heating at 120 degrees C. One such antigen was detected after sonication of the pellet fraction of autoclaved P. gingivalis cells. This antigen cross-reacted with 6 of the 8 immune sera. This somatic antigen was almost neutrally charged and sensitive to sodium periodate treatment, suggestive of lipopolysaccharide. A second heat-stable antigen was detected in the supernatant of autoclaved strains of W83, W50, HG184 and A7A1-28. These non-somatic antigens were strain-specific, i.e., no cross-reactivity was found with heterologous hyperimmune sera. An exception was strain W50, which had a non-somatic heat-stable antigen which was recognized by W83 antiserum. These antigens were resistant to DNAse, RNAse and proteinase-K treatment but were degraded by sodium periodate. In immunoelectrophoresis, these antigens appeared to be negatively charged. These properties are characteristics of a K-antigen, which likely represent a thermostable carbohydrate capsule. The presence of K-antigen correlates very well with the serum resistance, the low chemiluminescence, the resistance to phagocytosis and the need for opsonization with specific antibodies for complement-mediated killing of virulent P. gingivalis strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial involvement in denture-induced stomatitis.

Denture and mucosal plaque samples were collected from eight full-denture wearers of whom four suffered from denture-induced stomatitis (DIS). Cultures were made, and a proportional identification to species level was carried out of bacteria and yeasts. An inventory was made of the predominant flora. Results showed that the predominant microflora of both groups, both on dentures and the denture-bearing mucosa, consisted mainly of Gram-positive bacteria. Differences in the proportions of cocci were found between the predominant bacterial flora on the dentures. In the control group, 69% of the denture flora consisted of cocci, while on the dentures of the DIS group a mean of 33% cocci was found. Neither group of palates revealed any differences in the proportions of cocci. On these locations a mean of 69% was found. The plaque of dentures and the palates of the healthy group showed means of 35% and 31%, respectively, of obligate anaerobic bacteria, while in the DIS group these percentages were 56% and 43%, respectively. No obligately aerobic bacteria were found. Candida species was detected in both groups, both on dentures and palates in very low numbers (DIS group, median palates 0.02%, median dentures 0.25%). The predominant organisms were Streptococcus species, of which S. salivarius was mostly present on the palates of both groups. Other species which were regularly found at different locations were Veillonella parvula and species of Lactobacillus, Bacteroides, and Actinomyces.

Cross-inhibition between black-pigmented Bacteroides species.

Cross-inhibition within the group of black-pigmented Bacteroides, including both oral and non-oral strains, was studied by means of a membrane filter technique. It was found that B. gingivalis possessed the most extended inhibitory capacity among all species tested. B. gingivalis showed inhibitory activity against B. intermedius, B. endodontalis, B. loescheii, and B. melaninogenicus. B. endodontalis was active against some B. intermedius strains. Among the saccharolytic species, some B. melaninogenicus strains were inhibitory for some B. endodontalis strains, some B. gingivalis strains, and some B. intermedius strains. These inhibitory activities observed in vitro may play a role in the colonization of the periodontal pocket.

Serological characterization of black-pigmented Bacteroides endodontalis.

Serological studies on the black-pigmented Bacteroides species B. endodontalis revealed three serotypes based on capsular determinants. A common antigen (O-antigen) could be demonstrated after decapsulation. Weak cross-reactivity was found with B. asaccharolyticus, but not with B. gingivalis. Similarity between the serology of Enterobacteriaceae and black-pigmented Bacteroides spp. is discussed.

Enzymatic characterization of oral and non-oral black-pigmented Bacteroides species.

The purpose of this study was to investigate the production of various enzymes by oral and non-oral black-pigmented Bacteroides species using chromogenic substrates. The 19 substrates present in the API ZYM system did not differentiate between B. melaninogenicus, B. denticola, B. loescheii and B. levii. The asaccharolytic black-pigmented Bacteroides species showed each species specific enzyme activity, however, differences were based on one enzyme only as far as B. asaccharolyticus and B. endodontalis are concerned. An extended number of 40 chromogenic substrates were tested in order to find more species specific enzyme. With a set of 20 substrates it appeared to be possible to discriminate between all species tested. The possibility to use enzymes for the identification of black-pigmented Bacteroides is discussed.

Further characterization of Bacteroides endodontalis, an asaccharolytic black-pigmented Bacteroides species from the oral S cavity.

In this study, the isolation, characterization, and identification of Bacteroides endodontalis is described. It was found that this asaccharolytic black-pigmented Bacteroides species is associated with infected dental root canals and oral submucous abscesses. B. endodontalis could be differentiated from B. gingivalis by a negative direct hemagglutination test and the absence of trypsin and N-acetyl-beta-glucosamidase. B. endodontalis could be differentiated from B. asaccharolyticus by the absence of alpha-fucosidase, its inability to grow in an atmosphere of 95% N2-5% H2, and a growth requirement for menadione. Immune serum raised against B. endodontalis strain HG 370T agglutinated only B. endodontalis cells. Precautions for the isolation of B. endodontalis are discussed.