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.     

Antibiotic resistance in the subgingival microflora in patients with adult periodontitis. A comparative survey between Spain and the Netherlands

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.     

侵袭性牙周炎和慢性牙周炎的龈下优势菌分析

目的 :分析侵袭性牙周炎 (aggressiveperiodontitis ,AgP)与慢性牙周炎 (chronicperiodontitis ,CP)的龈下优势菌群 ,为探讨牙周炎分类、病因和诊断提供实验依据。方法 :将中学生流调筛选 (16例 )及牙周病专科就诊(2 4例 )的AgP和CP患者 ,采集龈下菌斑样本 ,在厌氧菌基础培养基 (CDC)和选择性培养基 (TSBV)上培养分析。结果 :局限型AgP患者的伴放线放线杆菌 (Actinobacillusactinomycetemcomitans ,Aa)及兼性厌氧菌的检出率显著高于中度CP患者 (P <0 .0 5 ,P <0 .0 1) ,而广泛型AgP和重度CP患者的厌氧菌总数较局限型AgP和中度CP患者显著增加 (P <0 .0 5 )。结论 :局限型AgP和中度CP的龈下优势菌有明显差别 ,Aa是一个重要的危险因子。     

The composition of the subgingival microflora of young adults suffering from juvenile periodontitis

The composition of subgingival plaque, from healthy and periodontally diseased regions, as well as the clinical periodontal condition of 6 patients, 17 to 24-years-old suffering from juvenile periodontitis were examined. 6 older patients with rapidly progressive periodontitis served as a control. Samples of subgingival plaque were taken from first molars and central incisors and were analysed morphologically by dark-field microscopy. In the control group in healthy regions the ratio between non-motile and motile bacteria was 27:1 and about 1:1 in deep pockets. In the juvenile periodontitis group in diseased regions, motile bacteria made up only 1/4 of the bacteria present. The results clearly show that our relatively old patients with juvenile periodontitis had a completely different microflora in their periodontally diseased regions than patients with common periodontitis. As regards our observations by dark-field microscopy, one can assume, however, that with increasing age, there might be a shift of the flora associated with juvenile periodontitis lesions from a rather simple composition to a more complex composition very similar to the flora seen in rapidly progressive adult periodontitis lesions.     

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.     

Antibiotic resistance of subgingival species in chronic periodontitis patients

Ardila CM, Granada MI, Guzmán IC. Antibiotic resistance of subgingival species in chronic periodontitis patients. J Periodont Res 2010; 45: 557–563. © 2010 John Wiley & Sons A/S Background and Objective: The increasing rate of resistance of microorganisms to penicillin and other antibiotics has generated concern among health authorities in Latin America. The present investigation determined the in vitro susceptibility of Porphyromonas gingivalis, Fusobacterium nucleatum, black‐pigmented Prevotella spp. and Aggregatibacter actinomycetemcomitans to metronidazole, amoxicillin, amoxicillin/clavulanic acid, clindamycin and moxifloxacin in patients with chronic periodontitis. Material and Methods: Subgingival plaque samples from patients with periodontitis were collected and cultured on selective and nonselective culture media. The antimicrobial susceptibility of periodontopathogenic isolates was studied in chronic periodontitis patients in Colombia. Metronidazole, amoxicillin, amoxicillin/clavulanic acid, clindamycin and moxifloxacin were tested on all bacterial isolates and the percentage of resistant strains was calculated. Results: Of the 150 bacteria identified, 51 were P. gingivalis, 45 were black‐pigmented Prevotella spp., 36 were F. nucleatum and 18 were A. actinomycetemcomitans. All the isolates were sensitive to amoxicillin/clavulanic acid and to moxifloxacin, but exhibited variable susceptibility patterns to the other antimicrobial agents tested. Conclusion: The results of the present study suggest that periodontal microorganisms in patients with chronic periodontitis can be resistant to the antimicrobial agents commonly used in anti‐infective periodontal therapy. We suggest that the indiscriminate use of antimicrobials could result in the appearance of more highly antibiotic‐resistant strains of bacteria associated with periodontal diseases in our population compared with the populations of other countries.     

Microflora in adult periodontitis

The subgingival microflora of adult periodontitis was studied in 8 adults (36–47 years) and compared with that of 10 periodontally healthy individuals (36–43 years). A total of 64 periodontal lesions were examined, and classified according to the attachment level in three categories: attachment loss >6 mm, attachment loss 4–6 mm and attachment loss <4 mm. Also for comparative purposes 20 gingival sulci were evaluated. Samples were taken using three standardized paper points and were incubated anaerobically in selective and non-selective media. The results showed a statistically significant association of Capnocytophaga gingivalis and Capnocytophaga sputigena with moderate periodontal lesions, while Haemophilus segnis has been correlated to severe periodontal lesions. We concluded that C. gingivalis, C. sputigena and H. segnis might be potentially conducive to periodontal deterioration in adult periodontitis.     

The subgingival microflora and gingival crevicular fluid cytokines in refractory periodontitis

Abstract Refractory periodontitis manifests as a rapid, unrelenting, progressive loss of attachment despite the type and frequency of therapy. This study examined possible relationships between cytokine levels in gingival crevicular fluid (GCF), occurrence of specific periodontopathic microflora. and disease activity in patients with refractory periodontitis. Refractory periodontitis patients (7 male and 3 female) were selected on the basis of history and longitudinal clinical observations. In each patient. 2 teeth with pocket depths greater than 6 mm were selected and individual acrylic stents were fabricated with reference grooves for each site. The sites were examined at both baseline and 3 months later. The pattern and amount of alveolar bone resorption were assayed by quantitative digital subtraction radiography. Pocket depth and attachment loss were measured with a Florida Probe. The gingival index was measured at 4 sites around each sample tooth. Sites were divided into active sites (2.1 mm loss of attachment in 3 months) or inactive sites (2.0 mm loss of attachment in 3 months). The distribution and prevalence of the predominant microflora in active and inactive sites were compared using anaerobic culture and indirect immunofluorescence. Interleukin-1β, 2, 4, 6 and tumor necrosis factor-α (TNF-α) levels in gingival crevicular fluid (GCF) were quantified by ELISA. Prevotella intermedia and Eikenella corrodens significantly decreased in inactive sites but remained the same in active sites after 3 months. The active sites revealed significantly higher GCF levels of IL-2 and IL-6 than inactive sites at both baseline and at 3 months. IL-1β was also significantly greater in active sites than in inactive sites at 3 months. Alveolar bone loss in active sites correlated with increased GCF levels of IL-1β and 1L-β. These results suggest that GCF levels of IL-1β, IL-2 and IL-6 and P. intermedia and E. corrodens in subgingival plaque may serve as possible indicators of disease activity in refractory periodontitis.     

The effects of antimicrobial acrylic strips on the subgingival microflora in chronic periodontitis

This study investigated the effects of root planing and/or the placement of acrylic strips containing chlorhexidine, metronidazole or tetracycline on the composition and antimicrobial susceptibility of the subgingival flora in chronic periodontitis. 101 periodontal pockets from 73 patients were entered into 6 treatment groups which were, chlorhexidine, metronidazole or tetracycline strips, root planing, root planing followed by metronidazole strips and a control, no treatment group. Total anaerobic counts and anaerobe/aerobe ratios were estimated from samples taken before treatment and 1, 2, 4, 8 and 12 weeks after treatment. In addition, a more detailed analysis of the effects of the treatments on the subgingival flora was carried out on 12 pockets in 12 patients. Tetracycline strips, metronidazole strips and root planing and metronidazole strips were more effective than chlorhexidine strips in causing reductions in total anaerobic count and anaerobe/aerobe ratio. However, the changes in microbial parameters rebounded to approach baseline levels 4 weeks after treatment. Chlorhexidine caused no detectable changes in the composition of the subgingival microflora, while metronidazole had a variable effect. Tetracycline appeared to effect major shifts in the composition of the microflora of treated pockets but caused a marked selection of tetracycline-resistant organisms.     

The effect of subgingival debridement with hand and ultrasonic instruments on the subgingival microflora

The effect of hand or ultrasonic instrumentation on the subgingival microflora of periodontal pockets was investigated. Pockets with probing depths of 6-9 mm were selected in 12 patients and were randomly assigned per patient to the experimental and control groups. After oral hygiene instruction, instrumentation of the experimental pockets was carried out either by ultrasonic or by hand instruments in a split-mouth design. The treatment effect on the subgingival microbiota was evaluated by microscopic and culture studies of subgingival plaque samples, while in addition, supragingival plaque, bleeding after probing and probing pocket depth were scored. Examinations were carried out before and 7, 21 and 49 days after treatment. The hand and ultrasonic treatments were equally effective in reducing probing pocket depths and bleeding scores. At the end of the experimental period, the probing depths of 54% of the hand-treated pockets and 43% of the ultrasonic-treated pockets were reduced to 4 mm or less while the bleeding scores were reduced to 29% and 22%, respectively. The analysis of microscopical and cultural data did not show any differences between hand and ultrasonic debridement. Both treatments reduced the microscopical counts of rods, spirochetes and motiles and reduced the total colony-forming units and number of black-pigmented Bacteroides and Capnocytophaga, resulting in a subgingival microbiota consistent with periodontal health.     

Smoking and subgingival microflora in periodontal disease

AIM: The present investigation was undertaken to analyze the influence of smoking on the periodontal disease associated subgingival microflora. The population included 33 smokers and 31 non-smokers in the age range 36-86 years. METHODS: Microbial samples were obtained from 4 sites per patient. The checker-board DNA-DNA hybridization technology was used for detection of the bacterial species P. gingivalis, P. intermedia, P. nigrescens, B. forsythus, A. actinomycetemcomitans, F. nucleatum, T. denticola, P. micros, C. rectus, E. corrodens, S. noxia and S. intermedius. RESULTS: Using score 1 as cutoff, contrasting colonized versus non-colonized patients, 8 out of 12 species were detected in > or = 90% of both smokers and non-smokers. Using score 4 as cutoff, contrasting heavily colonized patients versus non-colonized and less heavily colonized patients, the detection rates decreased in both smokers and non-smokers. No significant differences in detection rates were observed between smokers and non-smokers. Logistic regression analysis indicated that neither smoking, probing depth nor gingival bleeding influenced the occurrence of the species analyzed. The lack of a smoking exposure dose-response further supported the indication of a limited influence of smoking. CONCLUSION: Smoking exerts little, if any, influence on the subgingival occurrence of several of the bacteria most commonly associated with periodontal disease.     

The effect of supragingival plaque control on the subgingival microflora

The effect of plaque control on the apical microflora of deep periodontal pockets was studied. 8 subjects exhibiting signs of chronic periodontitis were chosen for the study, each subject having at least one pocket greater than 6 mm. These subjects were placed on a plaque control programme consisting of 3 visits, during which oral hygiene instructions were given. On two visits, the teeth of these subjects were scaled and polished. Bacteriological samples from the apex of a deep pockets from each subject were collected before the commencement of the plaque control programme and again at 8 and 16 weeks after the last scale and polish. No significant difference in the microbial flora was observed before and after plaque control, but marked fluctuation in bacterial composition was noted at the 3 samplings. It was concluded that supragingival plaque reduction was not sufficient to produce significant changes in the subgingival plaque composition of deep periodontal pockets.     

Ultrastructure of the subgingival microflora in juvenile periodontitis

abstract — The ultrastructure of the subgingival deposits on the root surfaces of teeth affected by juvenile periodontitis was studied on 12 teeth from nine individuals, 15–30 years of age. The deposits consisted of either microbial masses associated with a pellicle, or of a cuticular material almost free of bacteria. Gram-negative rods and filaments were the predominant microorganisms. "Corncob" configurations consisting of filamentous bacteria surrounded by Gram-positive cocci, and "bristle brush" formations comprising corncobs surrounded by long rods were observed in the superficial layer of the plaque. Spirochetes and flagellated rods constituted a major segment of the microflora. The present data indicate that the deep pockets in juvenile periodontitis harbor a sparse but relatively characteristic microbial population.     

Clinical and microbiologic effects of single-dose metronidazole or scaling and root planing in treatment of adult periodontitis

Sites affected with adult periodontitis were observed for 3 months to compare their clinical and microbiologic responses to a single 2 g dose of metronidazole, scaling and root planing, or no treatment. 2 sites with probing depths greater than or equal to 5 mm in each of 18 female subjects (6 in each treatment group) were evaluated clinically (plaque and bleeding indices, probing depth, attachment loss) and microbiologically (%s of cocci, motile rods, non-motile rods and spirochetes, and of obligate anaerobic colony-forming units, black-pigmented Bacteroides, Fusobacterium and Actinobacillus actinomycetemcomitans in subgingival plaque). No significant differences in these variables existed between the 3 groups at baseline. The no-treatment (control) group showed no substantial clinical or microbiologic changes during the study. After 1 month, scaling and root planing had effected significant clinical improvement and significant shifts in the subgingival flora to a pattern more consistent with periodontal health; these changes were still evident at 3 months. In contrast, 1 month after metronidazole, there was some clinical improvement and a significant increase in cocci and a decrease in motile rods, but at 3 months these changes were no longer evident. The results show that the benefits of scaling and root planing are sustained for at least 3 months. However, the benefits of a single 2 g dose of metronidazole are both few and transient, indicating that this regimen, while effective against anaerobic infections in other organ systems, is not clinically or microbiologically effective in the treatment of adult periodontitis.     

Prevalence of 6 putative periodontal pathogens in subgingival plaque samples from Romanian adult periodontitis patients

Abstract The aim of the present study was to determine by standard cultivation procedures the detection frequencies of Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum. Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Capnocytophaga species as well as various enteric rods in subgingival plaque samples form Romanian adult periodontitis patients. DNA probe analysis (Affirm^TM DP Microbial Identification Test) was also used, parallel to cultivation, to identify P. gingivalis. A. actinomycetemcomitans, and B. for- sythus, in deep (≥6 mm) and intermediate (4–5 mm) pockets in some of the subjects investigated. Paper points were used to sample 86 deep pockets in 36 patients and 27 intermediate pockets in 9 of the 36 patients. The x² test was used to test for significance of differences between results obtained by cultivation and DMA analysis in both intermediate and deep pockets. P. gingivalis was recovered in a high percentage of the patients (75,8%) and sites (63.6%) examined, followed by P. intermedia, F. nucleatum, and A. actinomycetemcomitans, respectively. Capnocytophaga species were present in almost all subjects. Enteric rods were recovered in 61.1% of the patients and 55.8% of the sites. Except for this high prevalence of enteric rods, the present group of patients had the periodontal species monitored in %s similar to those commonly perceived in the West. The Affirm M DP Test and cultivation showed poor correlation in detecting P. gingivalis. A. actinomycetemcomitans, and B. forsythus. The cultivation prevalence of P. gingivalis and P. intermedia in deep pockets was similar to their prevalence in intermediate ones. Overall, the prevalence of the periodontal pathogens investigated in the present Romanian periodontitis patients is similar to what has been revealed in matching Norwegian and other Western periodontitis patient populations. The high prevalence of enteric rods in the Romanian patients may have been an artifact resulting from prolonged transport of the samples in VMGA III.     

68例慢性牙周炎患者龈下菌斑致病菌的分布及耐药性分析

目的：了解慢性牙周炎患者龈下菌斑可疑致病菌的分布及抗生素耐药情况,为临床治疗提供参考。方法对口腔门诊诊断为慢性牙周炎患者龈下菌斑中的致病菌进行分离培养,采用琼脂稀释法测定分离菌对甲硝唑、阿莫西林等10种常用抗生素的敏感性。结果从68例慢性牙周炎患者龈下菌斑中共分离到262株可疑致病菌,所有病例均检出有致病菌感染,其中以牙龈卟啉单胞菌和产黑色素普雷沃菌分离率较高,分别检出66.2%和60.3%,其次为伴放线嗜血杆菌（55.9%）、具核梭杆菌（51.5%）和消化链球菌（45.6%）等。药敏结果显示：所测菌株对硝基咪唑类药物敏感性较高,对甲硝唑、替硝唑、奥硝唑的敏感率均在90%以上,对阿莫西林/克拉维酸全部敏感,对阿奇霉素、米诺环素、克林霉素的敏感率在51%~78%之间,但对青霉素、红霉素耐药性高,敏感率仅分别为2.3%和17.9%。结论牙龈卟啉单胞菌、产黑色素普雷沃菌等菌为宁夏青铜峡市小坝地区慢性牙周炎患者龈下菌斑中主要的可疑致病菌,对阿莫西林/克拉维酸和硝基咪唑类药物普遍敏感。     

Detection of Prevotella intermedia in subgingival plaque of adult periodontitis patients by polymerase chain reaction

A PCR assay was developed that could specifically amplify DNA from the periodontal pathogen Prevotella intermedia. A pair of primers was selected from regions of the 16S rRNA gene of P. intermedia that were both divergent in sequence at their 3' ends with respect to the corresponding regions of the 16S rRNA gene of P. nigreseens , its most closely related species, and used in the PCR assay. Positivity was indicated by amplification of an 855 bp product. Using purified genomic DNA from these 2 species, assay conditions were determined under which only P. intermedia DNA and not P. nigrescens DNA was amplifiable. Absolute specificity of the assay was confirmed by the fact that no amplification products were obtained when using DNA from several other important periodontal organisms. The optimized PCR assay was used to identify P. intermedia in subgingival plaque samples of patients with adult periodontitis. Confirmation of amplification of P. intermedia DNA was achieved by digestion of PCR products with the restriction endonuclease R sal, which gives different restriction patterns for P. intermedia and P. nigrescens. Of the 97 samples analysed, 38 (39%) were positive for P. intermedia. The results obtained confirm P. intermedia as a possible aetiological agent of adult periodontitis. Additionally, PCR primers targeting the corresponding region of the 16S rRNA gene of P. nigrescens were shown to be specific for the organism when used in a PCR assay, although P. nigrescens was not detectable in any of the subgingival plaques analysed.     

Detection of Prevotella intermedia in subgingival plaque of adult periodontitis patients by polymerase chain reaction

A PCR assay was developed that could specifically amplify DNA from the periodontal pathogen Prevotella intermedia. A pair of primers was selected from regions of the 16S rRNA gene of P. intermedia that were both divergent in sequence at their 3′ ends with respect to the corresponding regions of the 16S rRNA gene of P. nigrescens, its most closely related species, and used in the PCR assay. Positivity was indicated by amplification of an 855 bp product. Using purified genomic DNA from these 2 species, assay conditions were determined under which only P. intermedia DNA and not P. nigrescens DNA was amplifiable. Absolute specificity of the assay was confirmed by the fact that no amplification products were obtained when using DNA from several other important periodontal organisms. The optimized PCR assay was used to identify P. intermedia in subgingival plaque samples of patients with adult periodontitis. Confirmation of amplification of P. intermedia DNA was achieved by digestion of PCR products with the restriction endonuclease Rsal, which gives different restriction patterns for P. intermedia and P. nigrescens. Of the 97 samples analysed, 38 (39%) were positive for P. intermedia. The results obtained confirm P. intermedia as a possible aetiological agent of adult periodontitis. Additionally, PCR primers targeting the corresponding region of the 16S rRNA gene of P. nigrescens were shown to be specific for the organism when used in a PCR assay, although P. nigrescens was not detectable in any of the subgingival plaques analysed.     

Beta-lactamase production and antimicrobial susceptibility of subgingival bacteria from refractory periodontitis

This study assessed the extent of beta-lactamase-producing bacteria in subgingival plaque samples obtained from 25 patients with refractory marginal periodontitis in the USA. beta-Lactamase-positive isolates were characterized using commercial diagnostic kits and partial sequencing of the 16S rRNA gene. The susceptibilities to different antimicrobial agents were tested and, in addition, the isolates were screened for the presence of extended spectrum beta-lactamases (ESBLs). beta-lactamase-producing bacteria were detected in 18 (72%) patients. The most prominent beta-lactamase-producing organisms belonged to the anaerobic genus Prevotella. Other enzyme-producing anaerobic strains were Fusobacterium nucleatum, Propionibacterium acnes and Peptostreptococcus sp. Facultative bacteria, such as Burkholderia spp., Ralstonia pickettii, Capnocytophaga spp., Bacillus spp., Staphylococcus spp. and Neisseria sp., were also detected among the enzyme-producers. Minimum inhibitory concentrations (MICs) of ampicillin and amoxicillin were in the range 1.5-256 micrograms/ml and 4-256 micrograms/ml, respectively, for the isolates of the Prevotella species. All Prevotella isolates were susceptible to amoxicillin/clavulanate and metronidazole, but they showed variable resistance to tetracyclines. Two of the Prevotella isolates had high MICs of cefotaxime and ceftazidime. ESBL activity was not detected in any of the beta-lactamase-producing isolates by the Etest method. Thus, our study demonstrated a wide variety of beta-lactamase-producing bacteria that may play a role in refractory periodontal disease.