应用16S rDNA检测固定矫治患者牙周可疑病原菌变化研究

应用16S rDNA检测固定矫治患者龈下菌斑中牙周可疑病原,探讨戴用固定矫治器对牙周组织健康的影响。随机选择36例治疗时间超过6个月的固定矫治患者组成实验组,29例未经正畸治疗者组成对照组。分别检验特定部位牙周临床指数并收集龈下菌斑样本。采用16S rDNA检测9种牙周可疑致病菌。实验组与对照组相比牙龈指数、牙周袋深度、探诊出血差异有明显统计学意义(P<0.05);牙周可疑病原菌中牙龈卟啉菌、齿垢密螺旋体在实验组的检出率明显高于对照组(P<0.05)。固定矫治器戴入可引起患者牙周可疑病原菌的明显变化。     

牙龈卟啉单胞菌菌毛FimA基因型在牙周病患者中的分布情况

目的：探讨牙龈卟啉单胞菌FimA基因型在牙周患者的分布情况。方法：采用PCR技术对40例牙周患者龈下菌斑中牙龈卟啉单胞菌及其基因型进行检测。结果：牙龈卟啉单胞菌在牙周患者的牙周袋内检出率为87．5％,牙龈卟啉单胞菌FimA各基因型在牙龈卟啉单胞菌携带者的检出率分别为：I型22．9％,Ⅱ型60．0％,Ⅲ型17．1％,Ⅳ37．1％．V型未检出;基因I型在牙周袋内未单独检出,与基因Ⅱ型混合感染。结论：牙龈卟啉单胞菌FimA基因Ⅱ型和Ⅵ型是牙龈卟啉单胞菌在牙周病损部位的主要定植菌,两基因型可能与牙周病的发生发展有关。     

血链球菌在不同牙周状态下的分布及相关研究

目的：探讨口腔主要过氧化氢产生菌血链球菌和口腔链球菌在不同牙周健康状态下龈下菌群中的分布,及与牙周健康状态和牙龈卟啉单胞菌群中分布的相互关系。方法：纳入符合标准的受试者30人,受试位点86个,其中健康组11人,位点30个,龈炎组9人,位点29个,慢性牙周炎组10人,位点27个,检查记录牙周健康状态[包括牙龈指数（GI）和牙周袋深度（PD）],采集龈下菌斑标本,经厌氧菌培养基和AP-PCR及PCR鉴定后,将各受试组进行比较分析。结果：共获得草绿色链球菌523株,产黑色素菌241株。经AP-PCR及PCR鉴定后,得到血链球菌112株,口腔链球菌56株,牙龈卟啉单胞菌84株,健康组龈下菌斑中血链球菌,口腔链球菌和牙龈卟啉单胞菌构成比与牙周炎组相比有差异显著性;血链球菌和口腔链球菌与GI、PD呈负相关,牙龈卟啉菌与GI、PD呈正相关;血链球菌的构成与牙龈卟啉单胞菌的构成比呈负相关。结论：血链球菌等过氧化氢产生菌在龈下菌斑中比例的下降。可能是微生态失衡,致病菌过度增殖的重要机制。     

牙周微生态研究进展

１　牙周细菌克隆的多型性与感染模式７０年代以后,随着对厌氧微生物分离、培养和鉴定技术的发展,从口腔内发现了三百多种不同种类的细菌,目前认为最可疑的牙周致病菌有：放线共生放线杆菌、牙龈卟啉菌、中间普氏菌、具核梭杆菌、福氏类杆菌、直形弯曲菌、优杆菌、溶齿艾肯氏菌、微小消化链球菌、月形单胞菌和密螺旋体,其中放线共生放线杆菌作为局限性青少年牙周炎的主要致病菌,牙龈卟啉菌作为成人牙周炎的主要致病菌是被研究得最广泛,证据也是最充足的。在感染微生物学领域,一个非常普遍的现象是致病菌往往存在多种克隆型,有些是毒…     

甘草提取物对四种牙周常见致病菌的抑菌作用

目的体外评价甘草提取物对牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌和伴放线放线杆菌四种牙周常见致病菌的抑制效果。方法以牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌和伴放线放线杆菌四种牙周常见致病菌作为供试菌,采用液体稀释法,考察甘草提取物对这四种细菌的最小抑菌浓度(MIC)和最小杀菌浓度(MBC);并采用不同浓度的甘草提取物溶液,绘制甘草提取物对四种牙周致病菌的时间-杀菌曲线。结果甘草提取物对牙龈卟啉单胞菌、中间普氏菌、具核梭杆菌和伴放线放线杆菌的MIC值分别为1.50、1.50、0.75和1.50mg/mL,MBC值分别为6、3、3和3mg/mL。当甘草提取物达到对四种细菌的MBC值时,对于牙龈卟啉单胞菌、中间普氏菌、伴放线放线杆菌可在2h后可达到杀菌效果,对于具核梭杆菌可在4h后达到杀菌效果。结论甘草提取物对以上四种牙周常见致病菌具有良好的抑菌及杀菌作用。     

正常口腔中的可疑牙周致病菌

本实验对49例正常口腔的龈上和龈下菌斑内的可疑牙周致病菌(SPB)的分布情况进行了观察分析。 49例研究对象男21例,女28例;6～25岁,无龋,无牙周病的健康人。三月内未服用抗生素,未接受任何牙周治疗。定对在右上颌第一磨牙(No.3)的龈上及龈下菌斑。用BHI培养基厌氧培养37℃,4天以后进行细菌菌落计数,转种分离和鉴定。龈上菌斑内SPB的检出率按其高低顺序依次为:二氧化碳噬纤维菌、梭杆菌、消化链球菌、唾液弯曲杆菌、产黑色素类杆菌、优杆菌,溶蚀艾肯氏菌、生疾月形单胞菌。二氧化碳噬纤维菌中依次为牙龈二氧化碳噬纤维菌、黄褐二氧化碳噬纤维菌及生疾二氧化碳噬纤     

固定矫治器戴入前后牙周可疑病原菌和牙周临床指数的变化

目的:固定矫治器戴入前后的不同时间进行龈缘菌斑的微生物学检查和牙周状况的临床检查,以探讨固定矫治器戴入前后牙周可疑病原菌和牙周状况的动态变化过程.方法:选择18名固定正畸患者,于矫治器戴入前和戴入后1、3、6月分别检查16、41牙位的菌斑指数、牙龈指数、探诊深度、探诊出血,并在近中颊侧颊轴角处采集龈缘菌斑样本,采用细菌培养鉴定方法测定牙周可疑病原菌的检出量(CFU/g)和检出率.结果:与基线相比,观察期内龈缘菌斑的G￣产黑色素厌氧杆菌检出量和检出率在两个牙位均升高(P＜0.05),41的优杆菌、弯曲杆菌、拟杆菌、普氏菌亦有升高(P＜0.05).临床指标中16的牙龈指数(颊侧)和探诊深度(颊、舌侧)升高;41的菌斑指数(舌侧)降低(P＜0.05).结论:固定矫治器戴入后虽然可通过严格的口腔卫生指导有效控制牙面菌斑,但仍可引起牙周可疑病原菌增加.     

用间接荧光免疫法快速检测牙周袋内牙龈类杆菌

本试验选择成年牙周炎致病菌—产黑色素类杆菌群中牙龈类杆菌为指示菌,用牙龈类杆菌47A—1参考株制备标准抗血清,并和羊抗兔IgG荧光抗体,直接与牙周龈下菌斑标本涂片进行间接荧光反应,计数一个视野荧光反应阳性的牙龈类杆菌的菌数(三个视野平均值)。全过程只需1.5～2小时。用此法检查了90例健康人,75例牙龈炎及70例牙周炎患者,结果发现正常牙周人的牙龈类杆菌菌数为     

牙龈卟啉杆菌W83单克隆抗体的研制与鉴定

牙龈类杆菌曾是重要的产黑色素类杆菌群菌株,最近重新命名为牙龈卟啉杆菌,该菌为牙周病龈下菌斑中关键性厌氧菌,与成人慢性牙周炎的发生、发展有密切关系。作者用牙龈卟啉杆菌侵袭型菌株W83,作为免疫原,通过免疫小鼠、细胞融合、筛选、克隆化,最后得到一株能够稳定分泌抗牙龈卟啉杆菌W83的单克隆抗体杂交瘤细胞系,经鉴定该单抗特异性良好,可用于临床该菌的检出和生态学研究。     

慢性牙周炎患者洁刮治术后牙周微生物变化及意义

目的:研究洁刮治术后慢性牙周炎可疑致病微生物检出量与临床疗效的关系。方法:随机选取我院2012年1月至2014年5月慢性牙周炎患者36例,均采用洁刮治术治疗。分别于治疗前后检测患者牙周膜附着丧失量、牙周探诊深度、出血率以及微生物数量。结果:治疗前后比较,患者牙周探诊深度与探诊出血率差异具有统计学意义(P0.05),临床附着丧失量无统计学差异(P0.05);A组治疗后中间普氏菌数量显著高于治疗前(P0.05);牙龈卟啉单胞菌、齿垢密螺旋体和福赛氏类杆菌显著低于治疗前(P0.05);B组治疗前后细菌数量无统计学差异(P0.05);牙龈卟啉单胞菌C组牙周探诊深度变化显著高于D组(P0.05);齿垢密螺旋体C组探诊出血率变化显著低于D组(P0.05);福赛氏类杆菌C组牙周探诊深度和出血率与D组存在显著性差异(P0.05)。结论:牙龈卟啉单胞菌、齿垢密螺旋体和福赛氏类杆菌数量与洁刮治术后疗效密切相关,可作为微生物指标。     

Porphyromonas gingivalis prevalence related to other micro-organisms in adult refractory periodontitis.

Forty-six adult periodontal patients, selected on the basis of clinical examination, and 46 adult healthy subjects were examined. The subgingival plaque samples from one inflammatory and one non-inflammatory site of each periodontal patient were studied to determine Porphyromonas gingivalis prevalence related to other periodontal micro-organisms and to periodontal tissue destruction. The results showed Porphyromonas gingivalis as the main pathogenic micro-organism isolated in the inflammatory sites together with Bacteroides forsythus. Peptostreptococcus sp., Actinomyces sp. and Prevotella sp. were found as a normal oral flora in the healthy subjects. Fusobacterium nucleatum, Prevotella intermedia, Campylobacter rectus and Eikenella corrodens were detected both in inflammatory and in non-inflammatory sites of periodontal patients as well as in the healthy subjects.     

Microbiological and clinical effects of a 1% chlorhexidine-gel in untreated periodontal pockets from adult periodontitis patients.

The aim of this study was to report the microbiological and clinical effects of repeated subgingival administration of a 1% Chlorhexidine-gel in periodontal pockets from 10 patients with adult periodontitis. Results showed that the experimental treatment significantly improved clinical parameters (Plaque Index, Gingival Bleeding Index, and Pocket Probing Depth). Direct subgingival administration of Chlorhexidine-gel also produced a remarkable modification in the proportions of putative periodontopathic microorganisms, such as Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Veillonella parvula, Fusobacterium nucleatum, and Peptostreptococcus micros, in subgingival bacterial plaque from periodontitis patients.     

Comparison of culture methods and multiplex PCR for the detection of periodontopathogenic bacteria in biofilm associated with severe forms of periodontitis

Conventional culture methods and Multiplex PCR, both of which we have been used for a long time in our clinical microbiology laboratory, were compared for their ability to detect a selected panel of periodontopathic bacteria: Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia. Tests were performed in a single subgingival sample taken from a periodontal diseased site with a probing depth equal to or greater than 6mm. The results were compared site-by-site, taking into account the quality and the presence or absence of pathogens. 529 samples of subgingival plaque were analysed and the prevalence of the six species monitored varied in relation to the species itself and the method of detection. The most represented species is F. nucleatum, with a percentage of positive variability between 44.9% PCR and 46.5% culture test. Generally, the lowest prevalence was determined by culture test, with the exception of E. corrodens and F. nucleatum, which, unlike other bacteria, have been seen in higher percentages in culture with respect to PCR. For both methods, there was a good degree of accuracy in the determination of A. actinomycetemcomitans, C. rectus, E. corrodens, and P. gingivalis. It becomes weak for F. nucleatum and P. intermedia. Both culture and PCR techniques introduced many methodological problems when applied in oral microbiology, but the ideal technique for accurate detection of pathogens in subgingival plaque samples has yet to be developed.     

Bacterial diversity in human subgingival plaque

The purpose of this study was to determine the bacterial diversity in the human subgingival plaque by using culture-independent molecular methods as part of an ongoing effort to obtain full 16S rRNA sequences for all cultivable and not-yet-cultivated species of human oral bacteria. Subgingival plaque was analyzed from healthy subjects and subjects with refractory periodontitis, adult periodontitis, human immunodeficiency virus periodontitis, and acute necrotizing ulcerative gingivitis. 16S ribosomal DNA (rDNA) bacterial genes from DNA isolated from subgingival plaque samples were PCR amplified with all-bacterial or selective primers and cloned into Escherichia coli. The sequences of cloned 16S rDNA inserts were used to determine species identity or closest relatives by comparison with sequences of known species. A total of 2,522 clones were analyzed. Nearly complete sequences of approximately 1,500 bases were obtained for putative new species. About 60% of the clones fell into 132 known species, 70 of which were identified from multiple subjects. About 40% of the clones were novel phylotypes. Of the 215 novel phylotypes, 75 were identified from multiple subjects. Known putative periodontal pathogens such as Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola were identified from multiple subjects, but typically as a minor component of the plaque as seen in cultivable studies. Several phylotypes fell into two recently described phyla previously associated with extreme natural environments, for which there are no cultivable species. A number of species or phylotypes were found only in subjects with disease, and a few were found only in healthy subjects. The organisms identified only from diseased sites deserve further study as potential pathogens. Based on the sequence data in this study, the predominant subgingival microbial community consisted of 347 species or phylotypes that fall into 9 bacterial phyla. Based on the 347 species seen in our sample of 2,522 clones, we estimate that there are 68 additional unseen species, for a total estimate of 415 species in the subgingival plaque. When organisms found on other oral surfaces such as the cheek, tongue, and teeth are added to this number, the best estimate of the total species diversity in the oral cavity is approximately 500 species, as previously proposed.     

Rapid detection and quantification of five periodontopathic bacteria by real-time PCR

The quantity of periodontopathic bacteria in plaque samples is an important determinant for understanding the etiologic role of bacteria. The real-time PCR method was used to detect and quantify periodontopathic bacteria, such as Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii, in saliva and subgingival plaque samples. There was good agreement between the results of conventional PCR and real-time PCR methods. Using the LightCycler system we were able to determine the amount of periodontopathic bacteria within an hour. The real-time PCR method was linear for samples containing from 10(3) to more than 10(8) cells (r2 = 0.999). The application of the real-time PCR method should be useful in the rapid detection and quantification of periodontopathic bacteria in clinical samples.     

Use of fluorescence microscopy for monitoring periodontal disease state

Samples of subgingival plaque from patients with periodontal disease and control subjects were stained with the Fluoretec Fluorescent test kits (Pfizer Inc., New York) developed for the rapid detection of members of the Bacteroides fragilis and B. melaninogenicus groups of anaerobes. The same fluorescent fields were also examined by dark-field microscopy for the total count of bacteria. Bacteroides fragilis and B. melaninogenicus were found in plaque samples of healthy subjects and periodontally diseased patients with no significant difference in percent of total flora. Oral spirochetes also fluoresced with the antisera used. Samples from healthy sites showed virtually no spirochetes; spirochetes were present in diseased sites. Tests with other antisera also showed that fluorescein-labelled antibodies can be adsorbed nonspecifically to the surface of spirochetes. Such a phenomenon can be used to monitor an individual's periodontal disease state.     

Growth and Activities of Sulfate-Reducing and Methanogenic Bacteria in Human Oral Cavity

Viable counts and activities of sulfate-reducing bacteria (SRB) and methanogenic bacteria were determined in the oral cavities of eight volunteers. Of these, seven harbored viable SRB populations, and six harbored viable methanogenic bacterial populations. Two volunteers classified as type III periodontal patients had both SRB and methanogenic bacteria. Six separate sites were sampled: posterior tongue, anterior tongue, mid-buccal mucosa, vestibular mucosa, supragingival plaque, and subgingival plaque. The SRB was found in all areas in one volunteer, and it was mostly present in posterior tongue, anterior tongue, supragingival, and subgingival plaques in many volunteers. The methanogenic bacteria were mostly found in supragingival and subgingival plaques. The activities of sulfate reduction and methane production were determined in randomly selected isolates. Received: 27 July 2002 / Accepted: 27 August 2002     

Quantitative detection of periodontopathogens by real-time PCR

Specific bacteria are believed to play an important role in chronic periodontitis, yet the significance of their relative numbers in initiation and progress of the disease is still unclear. We report here the development of a sensitive, quantitative PCR technique for enumerating Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Dialister pneumosintes (Dp) and Micromonas micros (Mm) as well as total eubacteria in subgingival plaque samples from subjects with periodontitis. Quantification was performed with specific 16S rRNA target sequences with double fluorescence labeled probes and serial dilutions of plasmid standard by real-time PCR. This method showed a broad quantification range from 10(2) to 10(8) and accurate sensitivity and specificity. Fifty subgingival plaque samples from periodontitis patients and 33 from periodontally healthy subjects were subsequently examined. Higher levels of total bacteria numbers, Aa, Pg, Dp and Mm were found in samples from periodontitis subjects in comparison to samples from periodontally healthy subjects. Quantitative real-time PCR thus provides a reliable and valuable method for quantification of periodontopathogens in subgingival plaque samples.     

Periodontal status in an Italian young adult population. Prevalence and relationship with periodontopathic bacteria

To determine the prevalence of periodontitis in an Italian young adult population and the relationship with Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in subgingival plaque. A full-mouth periodontal and oral examination was performed in 70 subjects. Dental and behaviour habits were assessed with a standardised questionnaire. Subgingival plaque samples were collected from the deepest pocket of the first molars in each quadrant with a sterile curette. A. actinomycetemcomitans, P. gingivalis and P. intermedia were detected using a multiplex polymerase chain reaction. At subject level, the prevalence of bleeding on probing, calculus, normal pocket depth (PD), PD > 5mm and bacterial positivity were 44.8%, 43.3%, 22.9%, 11.4% and 95.7%, respectively. At quadrant level bacterial prevalence was 79.4%; P. intermedia was the most common bacteria (79.0%); A. actinomycetemcomitans had a prevalence of 40.8%. A significant linear trend across categories of gingival conditions (healthy, bleeding on probing, calculus presence) was detected for P. intermedia (p = 0.0038) and A. actinomycetemcomitans (p = 0.00005) proportions. No significant association was observed between pathogenic bacteria and PD, nor with behavioural attitudes. Gingival conditions are found to be a good predictors (VPP = 85%) for periodontopathic bacteria. For the Italian population, as no data are present, prospective longitudinal studies are needed to examine the relationship between PD and bacteria presence with periodontal disease onset and/or progression.     

Oral Microbiome of Deep and Shallow Dental Pockets In Chronic Periodontitis

We examined the subgingival bacterial biodiversity in untreated chronic periodontitis patients by sequencing 16S rRNA genes. The primary purpose of the study was to compare the oral microbiome in deep (diseased) and shallow (healthy) sites. A secondary purpose was to evaluate the influences of smoking, race and dental caries on this relationship. A total of 88 subjects from two clinics were recruited. Paired subgingival plaque samples were taken from each subject, one from a probing site depth >5 mm (deep site) and the other from a probing site depth ≤3mm (shallow site). A universal primer set was designed to amplify the V4–V6 region for oral microbial 16S rRNA sequences. Differences in genera and species attributable to deep and shallow sites were determined by statistical analysis using a two-part model and false discovery rate. Fifty-one of 170 genera and 200 of 746 species were found significantly different in abundances between shallow and deep sites. Besides previously identified periodontal disease-associated bacterial species, additional species were found markedly changed in diseased sites. Cluster analysis revealed that the microbiome difference between deep and shallow sites was influenced by patient-level effects such as clinic location, race and smoking. The differences between clinic locations may be influenced by racial distribution, in that all of the African Americans subjects were seen at the same clinic. Our results suggested that there were influences from the microbiome for caries and periodontal disease and these influences are independent.