牙周病基础治疗前后龈下菌群的动态观察

目的：动态观察龈下细菌治疗前后在口腔内定植的变化，为牙周病病因学研究和治疗方案确定提供依据。方法：选取26例慢性牙周炎患者治疗前、治疗后1周、1个月和3个月时同一位点的龈下菌斑，测量牙周探诊深度，提DNA，洲浓度。扩增全细菌16SrRNA基因片段，变性梯度凝胶电泳分离，选择特异性的DNA条带，回收、测序。结果：测序结果表明治疗后消失的两个DNA条带与牙龈卟啉单胞菌有98％和99％的同源性；治疗后新出现的两个DNA条带与卟啉菌属的一种有99％的同源性。结论：牙龈卟啉单胞菌是慢性牙周炎的重要可疑致病菌。变性梯度凝胶电泳适用于分析大量微生物标本分布和类型。     

The effect of periodontal treatment on the salivary bacterial load and early plaque formation

AIM: The purpose of the present study was to investigate in periodontitis patients the relationship between the number of bacteria in the saliva and the amount of de novo plaque formation before and after treatment. METHODS: At baseline, before any treatment was provided, patients rinsed with 10 ml sterile saline. After professional tooth cleaning the patients were instructed to abolish all tooth cleaning procedures for the subsequent 24 h. After this period, the rinsing procedure was repeated and the amount of de novo plaque was assessed. Three months after the initial periodontal therapy was completed the experiment was repeated. The microbiological evaluation of the rinsing samples was carried out by means of phase contrast microscopy and anaerobic culturing. RESULTS: After treatment the amount of de novo plaque was less compared to before treatment, 0.40 and 0.65 respectively. Both before and after treatment more de novo plaque was present at sites with inflammation than at healthy sites. In order to evaluate the contribution of the numbers of salivary bacteria to the amount of de novo plaque formation an analysis was carried out for healthy sites. This analysis included only healthy sites as determined before treatment and the same sites after treatment. The results showed a significant reduction in the de novo plaque formation after treatment (0.49 before and 0.22 after treatment). Phase contrast microscopic evaluation showed that the number of bacteria in the rinsing samples after treatment was less than before treatment. After treatment also a reduction was found in the prevalence of Prevotella intermedia, Tannerella forsythensis and Peptostreptococcus micros in the rinsing samples. CONCLUSION: The present study confirms the observation in the literature that the periodontal condition is of major importance in the rate of de novo plaque formation. In addition, the results suggest that the number of bacteria in the saliva may play a role.     

变黑普里沃菌在成人牙周炎患者龈下菌斑中的分布

目的：研究变黑普里沃菌与成人牙周炎的关系。方法：选择64例成年人牙周炎患者,取病变部位和健康部位龈下菌斑,厌氧培养,挑取产黑色素菌落,经多聚酶链反应鉴定变黑普里沃菌。结果：在成人牙周炎的病变部位和健康部位,变黑普里沃菌的检出率分别是16．8％和9．4％,无统计学差异。变黑普里沃菌的检出株数分别是25和23,总产黑色素G^-厌氧杆菌检出株数是6．9％和9．7％。结论：变黑普里沃菌作为可疑的牙周致病菌证据尚不充分。     

Systemic doxycycline administration in the treatment of periodontal infections (I). Effect on the subgingival microbiota

Systemic doxycycline is one of the more common antimicrobial agents used in the treatment of periodontal infections and yet little is known of its effect on subgingival plaque composition during and after its administration. The purpose of the present investigation was to evaluate changes in subgingival plaque composition during and after 14 days of doxycycline administration. 20 subjects with adult periodontitis were randomly assigned to test (n = 10) and control (n = 10) groups. The subjects received full mouth clinical assessment of pocket depth, attachment level, BOP, gingival redness, suppuration and plaque accumulation at baseline and 90 days. All subjects received full mouth SRP at baseline and, additionally, the test group received 100 mg doxycycline daily for 14 days. Subgingival plaque samples were taken from the mesial surface of up to 28 teeth in each subject at baseline and 90 days. In addition, plaque samples were taken from 2 randomly selected teeth at 3, 7 and 14 days during and after antibiotic administration. Control subjects were sampled at the same time points. Counts of 40 subgingival species were determined using checkerboard DNA-DNA hybridization and fluorescent detection. Significance of differences between test and control groups was determined at each time point using the Mann Whitney test. Significance of changes over time within test and control groups was determined using the Quade test. A modest but significant reduction in mean pocket depth from baseline to 90 days occurred in both test and control groups. A significant decrease in the % of sites with gingival redness occurred in the test group. There were no significant differences in proportions between test and control groups for 33 of the test species at any time point. Test subjects exhibited lower proportions of 4 Actinomyces species and an increase in 3 Streptococcus species during antibiotic administration. After cessation of doxycycline, Actinomyces sp. increased while Streptococcus sp. returned to baseline proportions. The relationship between these 2 genera appeared to be reciprocal; an increase in one was accompanied by a decrease in the other. Periodontal pathogens including B. forsythus, P. gingivalis, T. denticola and A. actinomycetemcomitans were not significantly altered by oral administration of doxycycline using conventional therapeutic dosage.     

Capnocytophaga granulosa and Capnocytophaga haemolytica: novel species in subgingival plaque

BACKGROUND: The oral cavity accommodates one of the most diverse microfloras in the human body. Knowledge of this microflora, and of the periodontal microflora in particular, proves crucial towards an understanding of the bacterial-host interactions which lead to the development of infectious inflammatory periodontal diseases. Capnocytophaga species have been implicated as putative periodontal pathogens. To date, only 3 members of this genus (C. gingivalis, C. ochracea and C. sputigena) have been isolated from subgingival plaque. AIM: This communication reports the isolation of 2 recently-speciated strains, namely C. granulosa and C. haemolytica, from subgingival plaque collected from adult periodontitis patients. MATERIAL AND METHODS: Subgingival plaque was collected from 29 patients with chronic adult periodontitis. Plaque samples were inoculated onto fastidious anaerobe agar and incubated anaerobically for 5 days. Routine identification of clinical isolates was performed by 16S rRNA PCR-RFLP analysis, using Cfo I as restriction enzyme and corroborated by 16S rRNA gene sequencing. RESULTS: 16 of 29 patients (55%) tested positive for either C. granulosa and or C. haemolytica. A total of 70 isolates (63 C. granulosa and 7 C. haemolytica) were cultivated from subgingival plaque. 15 (51%) patients tested positive for C. granulosa, and 3 (10%) patients tested positive for C. haemolytica. CONCLUSION: This is the 1st report which recounts the presence of C. granulosa and C. haemolytica in subgingival plaque. Further research is required to establish the relative proportions of these species subgingivally in health and disease.     

Detection rates of presumptive periodontal pathogens in subgingival plaque samples of untreated periodontitis using either four or six pooled samples

Aim: A comparison of the detection frequency and number of periodontal pathogens in patients with aggressive or generalized, severe chronic periodontitis using a gene‐probe analysis. Methods: In 16 aggressive and 34 generalized, severe chronic periodontitis patients, plaque was sampled from the deepest pockets per quadrant (MT4) and per sextant (MT6). After sampling two paper points simultaneously, one paper point from each pocket was pooled with three paper points of the other pockets (MT4). The remaining four paper points were pooled with two paper points from the deepest pockets from the two remaining sextants (MT6). Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola were detected by 16S rRNA gene probes. Results: Log‐transformed counts for Aggregatibacter actinomycetemcomitans were statistically significantly higher with MT6 (aggressive: 3.21 ± 2.94; generalized, severe chronic: 2.22 ± 2.70) than MT4 (aggressive: 2.04 ± 2.74; generalized, severe chronic: 1.50 ± 2.37) (P < 0.05). The detection frequency and mean counts were high for Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola (>95%/>6.0). Conclusion: Aggregatibacter actinomycetemcomitans was detected in higher numbers for MT6 than MT4. For both MT4 and MT6, Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola were detected in >95% of all patients and with mean log‐transformed numbers >6.0.     

Systemic doxycycline administration in the treatment of periodontal infections (II). Effect on antibiotic resistance of subgingival species

The purpose of this investigation was to determine the proportion and prevalence of doxycycline resistant species in subgingival plaque samples taken during and after doxycycline administration. 20 subjects with adult periodontitis were randomly assigned to test (n = 10) or control groups (n = 10). Saliva samples as well as subgingival plaque samples taken from the distal surface of 6 posterior teeth were collected at baseline. All subjects received full mouth SRP and the test group systemic doxycycline at the dosage of 100 mg/day for 14 days. Saliva samples and plaque samples from the distal surface of 2 randomly selected teeth were taken at 3, 7 and 14 days during and after antibiotic administration. Control subjects were sampled at the same time points. Samples were anaerobically dispersed and serially diluted in PRAS Ringer's solution and plated on enriched Trypticase soy blood agar plates with or without 4 microg/ml doxycycline. After 7 days of anaerobic incubation, colonies were counted on both sets of plates. Microbial growth was washed from the doxycycline-containing media and the species identified using 40 DNA probes and checkerboard DNA-DNA hybridization. Differences in proportions of resistant species between test and control groups were tested for significance at each time point using the Mann Whitney test and over time within each group using the Quade test. The mean % (+/-SEM) of isolates resistant to 4 microg/ml doxycycline in the plaque samples of the test subjects increased from 6+/-2 to 48+/-9% during doxycycline administration, decreasing to 25+/-6% 2 weeks later and 9+/-2% at 90 days. In saliva, the % of resistant isolates rose from 13+/-1% to 81+/-10% during doxycycline administration falling to 46+/-8% 2 weeks later and 22+/-5% at 90 days. The % of resistant isolates did not change significantly in plaque or saliva samples of the control subjects at the same time points. For all subject visits combined, the most prevalent resistant species were: Streptococcus anginosus, Streptococcus oralis, Streptococcus intermedius, Streptococcus sanguis, Streptococcus mitis, Veillonella parvula, Actinomyces gerencseriae, Streptococcus constellatus, Actinomyces naeslundii genospecies 2, Streptococcus gordonii, Eikenella corrodens and Actinomyces naeslundii genospecies 1. Doxycycline resistant strains of these species were detected in both plaque and saliva samples prior to therapy and in the control group. Despite the finding of increased resistance, approximately 50% of the organisms present at periodontal sites at the end of 14 days of doxycycline administration tested sensitive to the agent.     

Combined antibiotic (metronidazole) and mechanical treatment effects on the subgingival bacterial flora of sites with recurrent periodontal disease

5 patients in maintenance, 1-3 years after periodontal therapy who showed sites with reinfected pockets and bleeding despite regular recall visits were selected. Darkfield microscopy from 3 sites in each patient showed an average of 41% spirochetes and 21% motile rods. Probing depths ranged from 7 to 9 mm and loss of clinical attachment from 6 to 13 mm in these sites. The patients were given 3 times 250 mg/day of metronidazole (Flagyl) for 10 days. Darkfield microscopy and microbiological cultures of the subgingival plaque were performed twice prior to the study, at the end of the medication and after 3 weeks, 3 and 6 months. The plaque and gingival indices, probing depth and loss of clinical attachment were recorded. During the medication and at 3 and 6 months, the teeth were scaled and root planed. The samples were obtained with 3 paper points and cultured anaerobically in the glove box on non-selective and selective media and representative bacterial colonies identified by aerobic growth, gram stain and rapid biochemical tests. Presumptive pathogenic micro-organisms including Bacteroides were identified. The % of spirochetes, motile rods and non-motile bacteria were enumerated by darkfield microscopy. The clinical results show that administration of metronidazole and repeated root planing significantly decreased gingival inflammation, probing depth and loss of clinical attachment in reinfected sites. After treatment, these sites harbored significantly less spirochetes and more non-motile bacteria, while motile rods tended to return to baseline levels with time. The combined antibiotic and mechanical therapy resulted in a statistically significant decrease of gram-negative rods, Fusobacteria and Bacteroides gingivalis over 6 months.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of smoking and periodontal treatment on the subgingival microflora

BACKGROUND: The effect of smoking on the prevalence of periodontal pathogens after periodontal treatment is still not clear. Some studies found no effect of the smoking status on the prevalence of periodontal pathogens after therapy, whereas others did. The aim of this retrospective study was to investigate the influence of smoking on the treatment of periodontitis and the composition of the subgingival microflora. METHOD: The study included 59 periodontitis patients (mean age 41.5 years): 30 smokers and 29 nonsmokers. The treatment consisted of initial periodontal therapy and, if necessary, surgery and/or antibiotics. Clinical and microbiological data were obtained before and after treatment at the deepest site in each quadrant. A pooled sample was analysed for the presence of Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotalla intermedia (Pi), Bacteroides forsythus (Bf), Fusobacterium nucleatum (Fn) and Peptostreptococcus micros (Pm). RESULTS: For smokers and nonsmokers a significant improvement of the clinical condition was found after treatment. A decrease could be assessed for bleeding on probing (smokers: 0.46; nonsmokers: 0.52) and probing pocket depth (PPD) (smokers: 1.64 mm; nonsmokers: 2.09 mm). Furthermore, both groups showed gain of attachment (smokers: 0.68 mm; nonsmokers: 1.46 mm). No significant difference in bleeding on probing and PPD reduction was found between smokers and nonsmokers. In contrast, nonsmokers showed significantly more gain of attachment than smokers. The microbiological results revealed no differences in the prevalence of the various bacteria between smokers and nonsmokers before treatment. After treatment in nonsmokers, a significant decrease was found in the prevalence of Aa (11-3), Pg (17-7), Pi (27-11), Bf (27-11), Fn (28-20) and Pm (27-17). In smokers, a significant decrease could be shown only for the prevalence of Pg (15-5). CONCLUSIONS: Nonsmokers showed more gain of attachment and a greater decrease in the prevalence of periodontal bacteria as compared to smokers. The phenomenon that among smokers, more patients remain culture positive for periodontal pathogens after therapy, may contribute to the often observed unfavourable treatment results in smoker periodontitis patients.     

The most frequently isolated gram-negative anaerobes in saliva and subgingival samples taken from young women

Stimulated saliva and a pooled subgingival sample from the first molars were collected from 30 Caucasian women (mean age 30 years) for the presence of gram-negative anaerobic bacteria. The samples were inoculated on nonselective and selective media. The gram-negative anaerobic species occurring in the oral cavity of more than 50% of the subjects were selected for further analysis. Fusobacterium nucleatum and Prevotella melaninogenica were not recovered from subgingival sites without a simultaneous recovery in saliva. When positive subgingivally, Leplotrichia spp., nonpigmented Prevotella spp., Prevotella intermedia, Capnocytopha-ga spp., Bacteroides gracilis and Prevotella loescheii were simultaneously detected from saliva in 76%, 72%), 64%>, 64%, 61% and 55% of the subjects, respectively. Intraindividually, the proportions of F. nucleatum, nonpigmented Prevotella spp., Leptotrichia spp., B. gracilis and P. loescheii of the anaerobic flora were quite similar in the salivary and subgingival samples. Capnocytophaga spp. and P. intermedia were present in higher proportions in subgingival samples than in saliva, whereas P. melaninogenica was present in higher proportions in saliva. Our data indicate that many oral gram-negative anaerobes are frequently recovered from stimulated saliva. Furthermore, when subgingivally positive, most subjects harbored these anaerobes simultaneously in saliva.     

单纯机械治疗对慢性牙周炎患者龈下菌群微生态的影响

目的 采用16S rRNA高通量测序技术,探讨单纯机械治疗对慢性牙周炎龈下菌群微生态的影响.方法 纳入广泛型中重度慢性牙周炎患者,接受口腔卫生宣教,并同时进行龈上洁治.一周后进行超声结合手工龈下刮治及根面平整,在基线、治疗后3个月和治疗后6个月,记录临床指标并采集龈下菌斑.利用Illu-mina MiSeq平台进行16...     

Bacterial reduction and persistence after endodontic treatment procedures

Bacteria that persist after endodontic disinfection procedures may lead to treatment failure. Over 50% of the bacteria found in endodontic infections are as-yet-uncultivated so investigations of bacteria that endure treatment procedures should include techniques that side-step cultivation. This culture-independent study evaluated the bacterial reduction promoted by intracanal disinfection procedures and identified the taxa persisting after treatment. Samples taken from the infected canals of teeth with apical periodontitis before treatment (S1), after instrumentation using NaOCl as irrigant (S2) and after interappointment medication with a calcium hydroxide paste (S3) were subjected to 16S rRNA gene clone library and real-time polymerase chain reaction analyses. The S2 and S3 samples from five of the 15 canals showed negative results. In the other cases, instrumentation and instrumentation/medication promoted a significant reduction (99.67% and 99.85%, respectively) in the number of bacteria when compared to S1 samples. Forty-three distinct bacterial taxa were identified, of which 24 (56%) were as-yet-uncultivated phylotypes. Nineteen of these 43 taxa (including eight as-yet-uncultivated phylotypes) were disclosed in post-treatment samples, with streptococci being the most prevalent taxa. Findings demonstrated that culture-independent methods provide a detailed insight into the effects of intracanal disinfection protocols, helping to define more effective strategies to deal with endodontic bacteria, including as-yet-uncultivated phylotypes.     

Plaque development in relation to the periodontal condition and bacterial load of the saliva

AIM: To investigate the influence of the oral bacterial load on plaque development in various groups of periodontitis patients and in healthy subjects. MATERIAL AND METHODS: This study included subjects with a healthy periodontium, a healthy reduced periodontium after treatment, an inflamed reduced periodontium after treatment and untreated periodontitis. At the start of the study, subjects were instructed to rinse with 10 ml reduced transport fluid (RTF) for 10 s in order to evaluate the oral bacterial load. The microbiological evaluation included anaerobe culture and phase-contrast microscopy. Next, the amount of plaque and the clinical condition were evaluated. Thereafter, all supragingival plaque was removed and patients were instructed to refrain from all oral hygiene procedures for 19 h. Subsequently, the rinsing procedure and the evaluation of the amount of plaque were repeated. RESULTS: The amount of plaque that developed in 19 h was significantly higher in the untreated periodontitis group as compared with the two healthy groups. In case of an inflamed reduced periodontium, sites with deep pockets developed more plaque in 19 h than sites with shallow pockets. The number of bacteria present in the rinsing samples of the two inflamed groups was considerably higher than of the two periodontally healthy groups. A significant correlation was found between the bleeding index at intake and the plaque index at 19 h. No correlations were found between gingival recession and the bacterial counts at intake, and the plaque index at 19 h. CONCLUSION: The present findings support the concept that the periodontal condition is the dominating factor in relation to the rate of plaque formation. The number of bacteria present in the oral cavity as ascertained by means of a rinsing sample does not seem to play a role.     

Detection frequency of periodontitis-associated bacteria by polymerase chain reaction in subgingival and supragingival plaque of periodontitis and healthy subjects

The aim of this study was to compare the detection frequencies of 25 bacterial species in subgingival and supragingival plaque of 18 untreated periodontitis subjects and 12 periodontally healthy subjects. Genomic DNA was extracted from subgingival and supragingival plaque samples, and bacterial detection was performed by polymerase chain reaction of the 16S rRNA genes. Fourteen bacteria showed no relationship with periodontitis, and 11 of these 14 species were frequently detected (≥50%) in subgingival plaque in both periodontitis and healthy subjects. Nine bacteria such as Eubacterium saphenum, Prevotella intermedia, and Treponema denticola seemed to be related to periodontitis; their detection frequencies in subgingival plaque samples were higher in periodontitis than in healthy subjects, but these differences were not statistically significant by multiple comparisons (0.002≤ P< 0.05). Two species ( Mogibacterium timidum and Porphyromonas gingivalis ) were detected significantly more frequently in subgingival plaque of periodontitis subjects than of healthy subjects ( P< 0.002), with P. gingivalis being detected only in periodontitis subjects, suggesting that these two species are closely related to periodontitis. There were no significant differences in the detection frequencies of the 25 bacteria between subgingival and supragingival plaque, suggesting that the bacterial flora of supragingival plaque reflects that of subgingival plaque.