应用16S rDNA结合膜芯片检测3种产黑色素牙周可疑致病菌

目的:探讨采用16SrDNA结合膜芯片检测牙龈卟啉菌Pg、中间普氏菌Pi和变黑普氏菌Pn的价值。方法:利用Genebank中细菌16SrDNA保守区序列,设计一对通用引物,通过已知Pg、Pi和Pn的16SrDNA序列,设计合成对应的特异性寡核苷酸探针。先用通用引物PCR扩增所有标准菌株的DNA,PCR产物和已点样有Pg、Pi和Pn的3种探针的膜芯片杂交,进行分析。结果:膜芯片上的Pg、Pi和Pn3种探针只能与相对应的Pg、Pi和Pn的PCR产物反应,而与其他标准菌株的PCR产物无反应。结论:用16SrDNA结合膜芯片,可准确检测Pg、Pi和Pn,具有很高的特异性,有望成为一种有效的临床检测方法。     

感染根管内产黑色素类杆菌的定植

目的:了解感染根管内产黑色素类杆菌(BPB)的定植情况.方法:采用16S rDNA PCR技术检测5种BPB在牙髓炎、慢性根尖周炎患牙根管内的定植情况.统计学分析5种BPB在感染根管内检出率的差异及菌种间相互关系.结果:BPB在感染根管内总检出率为60%,其中牙髓卟啉单胞菌(Pe)、变黑普氏菌(Pn)、牙龈卟啉单胞菌(Pg)、产黑普氏菌(Pm)、中间普氏菌(Pi)检出率分别为38%、32%、30%、18%、14%.Pe、Pn检出率明显高于Pm、Pi(P<0.05).Pe在慢性根尖周炎组检出率(50%)明显高于牙髓炎组检出率(20%)(P<0.05).Pn、Pg、Pi和Pm在慢性根尖周炎组检出率(43.33%、36.67%、16.67%、16.67%)与牙髓炎组检出率(20%、20%、10%、15%)无明显差异(P>0.05).Pg/Pe、Pg/Pn、Pe/Pn之间存在正相关.结论:BPB是感染根管内的优势菌群,是牙髓炎和慢性根尖周炎共有的致病菌.Pg/Pe、Pg/Pn、Pe/Pn常定植于同一根管.     

基因芯片技术分析慢性根尖周炎患牙产黑色素菌

目的：利用基因芯片技术分析慢性根尖周炎患牙根管中牙髓卟啉单胞菌（Porphyromonas endodontalis,Pg）、牙龈卟啉单胞菌（Porphyromonas gingivalis,聊和中间普氏菌（Irevotella intermedia,Pi）三种产黑色素菌（blackpigmented bacteria,BPB）。方法：收集76例慢性根尖周炎患牙根管细菌学样本,经DNA抽提,PCR荧光标记后,与点样有忍、段和只三种菌特异寡核苷酸探针的基因芯片进行杂交,用激光共聚焦扫描仪分析杂交结果,最后进行统计分析。结果：慢性根尖周炎患牙中Pe、Pg和Pi三种菌的检出率分别为48．68％,32．89％,42．11％。Pe和Pg两菌同时被检出具有统计学意义（P〈0．05）。Pe和Pg与瘘管显著相关（p〈0．01）,Pe和Pg同时感染与瘘管、脓肿显著相关如〈0．01）。结论：产黑色素菌与慢性根尖周炎关系密切,Pe、Pg和瘘管、脓肿显著相关。用基因芯片技术分析慢性根尖周炎患牙中细菌具有快速、灵敏、高效的特点,而且可以通过增加基因芯片上探针数目扩展被检出菌的种类。     

成人牙面色素沉积与口腔产黑菌关系的研究

目的:对口腔常见4类产黑菌进行检测,研究口腔产黑菌与口腔色素沉积的关系。方法:分别采集18～40岁牙面有色素沉积(非食用含色素食物和吸烟)和无色素沉积各30名受试者的牙菌斑样本,通过DNA抽提和二步PCR扩增方法,对口腔常见4类产黑菌:牙龈卟啉单胞菌(porphyromonas gingivalis,Pg)、牙髓卟啉单胞菌(porphyromonas endodontalis,Pe)、中间普氏菌(porphyromonas intermedius,Pi)、变黑普氏菌(Pre-votella nigrescens,Pn)进行检测。结果:所有样本均检测到目标细菌,30例无色素沉积者的标本中Pg、Pe、Pi、Pn的检出率分别为27%、40%、30%、87%;30例有色素沉积标本中的检出率分别为60%、80%、73.3%、80%。有色素沉积者的Pg、Pe、Pi的检出率明显高于无色素沉积者,差异有统计学意义(P<0.05);Pn二组无统计学差异(P﹥0.05)。结论:牙面有色素沉积者口腔产黑色素杆菌检出率高。     

PCR检测慢性根尖周炎临床标本中牙髓卟啉单胞菌

目的:检测慢性根尖周炎临床标本中牙髓卟啉单胞菌(Porphyromonas endodontalis, Pe),以反映Pe在慢性根尖周炎中的存在情况. 方法:采用聚合酶链反应(PCR)检测100例慢性根尖周炎临床标本中Pe 16S rDNA,电泳,照相后计算其检出率. 结果:PCR检测慢性根尖周炎临床标本中Pe的检出率为50%.结论:Pe较高的检出率提示Pe可能与根管感染密切相关.     

牙龈卟啉单胞菌与牙周病相关性的聚合酶链反应研究

目的利用PCR检测慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌(Porphyromonas gingivalis,P. g)的16S rDNA水平,通过检测该基因水平来探讨牙龈卟啉单胞菌的水平与牙周病的相关性.方法采集慢性牙周炎患者12例共36个龈下菌斑标本,记录临床指标(探诊深度、临床附着水平、牙龈指数、菌斑指数、龈沟出血指数),PCR检测龈下菌斑标本中的P. g 16S rDNA基因,扩增产物经琼脂糖电泳、拍照后,应用计算机软件GeneTools扫描并计量其中的相对核酸含量.结果P. g16S rDNA水平与探诊深度(P＜0.001)及牙龈指数(P＜0.01)之间存在正相关关系.结论P.g16S rDNA水平与牙周状态密切相关,对于P.g16S rDNA水平的监测有望成为牙周病诊断和治疗方案制定的辅助检查手段.     

AP-PCR法鉴别中间普里沃菌和变黑普里沃菌

目的　用引物OPA - 13的随机引物聚合酶链法 (arbitrarilyprimedpolymerasechainreaction ,AP -PCR)区分中间普里沃菌 (Prevotellaintermedia ,Pi)和变黑普里沃菌 (Prevotellanigrescens ,Pn)。 方法　用引物OPA - 13,采用AP -PCR法分析 97株来自 2 5例成年人牙周炎 (adultperiodontitis,AP)的Pi和 4 9株来自 2 1例AP的Pn的基因型。结果　 97株Pi中仅有 1株没有 90 0bp的特异性片段 ,4 9株Pn中也只有 1株没有产生 1.3kb的特异性片段。牙龈卟啉单胞菌 (Pg)ATCC 332 77,PgW 83,产黑色素普里沃氏菌ATCC 2 5 84 5 ,不解糖卟啉单胞菌ATCC 2 5 2 6 0 ,躯体普里沃氏菌ATCC 335 4 7,小齿普里沃氏菌ATCC 33185均不产生相关片段。结论　用引物OPA - 13的AP -PCR法可以区分Pi和Pn。     

牙周致病微生物与药物性牙龈肥大的关系

目的:探讨菌斑微生物在硝苯地平诱导的药物性牙龈肥大中的作用。方法:采用Real-time PCR技术对服用硝苯地平后出现药物性牙龈肥大的高血压患者(A组,23例)、服用硝苯地平后未出现药物性牙龈肥大的高血压患者(B组,23例)、慢性牙周炎者(C组,23例)龈下菌斑内牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)、中间普氏菌(Prevotella intermedia,Pi)、齿垢密螺旋体(Treponema denticola,Td)和福赛坦氏菌(Tannerella forsythia,Tf)进行相对定量检测,比较3组患者龈下菌斑中4种牙周致病微生物相对含量的差异。结果:Pg、Td、Tf、Pi在A组明显高于B组,两者之间有统计学差异(P<0.05), A组与C组比较无统计学差异。结论:菌斑微生物在硝苯地平诱导的药物性牙龈肥大中起着重要作用。     

牙周源性牙周牙髓联合病变常见病原菌的分布及其意义

目的研究牙周源性牙周牙髓联合病变常见病原菌分布及其相关性,为临床治疗提供依据。方法选择2018年1月至2020年6月于新疆维吾尔自治区人民医院口腔科就诊的牙周源性牙周牙髓联合病变患者43例43颗牙作为实验组,重度牙周炎患者41例41颗牙作为对照组。分别采集根管内组织和龈下菌斑,构建含有8种待测细菌基因片段的重组质粒,建立定量标准,应用实时荧光定量PCR技术检测福赛斯坦纳菌(Tannerella forsythia,Tf)、牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)、具核梭杆菌(Fusobacterium nu?cleatum,Fn)、中间普氏菌(Prevotella intermedia,Pi)、齿垢密螺旋体(Treponema denticola,Td)、消化性链球菌(Digestive streptococcus,Ds)、粪肠球菌(Enterococcus faecalis,Ef)、牙髓卟啉单胞菌(Porphyromanus endodontics,Pe)数量。结果实验组根管内组织与其龈下菌斑中Ds、Pe数量差异无统计学意义(Ρ>0.05),其余6种病原菌数量差异均有统计学意义(P<0.05);实验组与对照组龈下菌斑中Ds数量差异无统计学意义(P=0.241),其余7种病原菌数量差异均有统计学意义(P<0.05);实验组根管内组织与其龈下菌斑中Ef、Pe、Pg、Td、Tf细菌数量密切相关,Ef(r=0.347,Ρ<0.05)、Pe(r=0.363,Ρ<0.05)、Pg(r=0.437,Ρ<0.01)、Td(r=0.471,Ρ<0.01)、Tf(r=0.679,Ρ<0.01)。结论牙周源性牙周牙髓联合病变常见病原菌在根管内组织中数量低于龈下菌斑但根管内病原菌数量与龈下菌斑密切相关,临床治疗期间在控制牙周组织感染的同时,还应重视牙髓组织感染的控制。     

实时荧光定量PCR法检测治疗后龈下菌斑中优势菌的变化

目的：运用实时荧光定量PCR(qRT-PCR)技术检测慢性牙周炎患者治疗前、后龈下菌斑中牙龈卟啉单胞菌(Pg)和中间普氏菌(Pi)的拷贝数,以了解qRT-PCR对2种牙周致病菌检测的敏感性和牙周治疗的疗效。方法：选取62例中、重度牙周炎患者,分别采用龈下刮治、牙周袋内盐酸米诺环素软膏(派丽奥)给药和两者综合治疗,采集治疗前、后(7d)龈下菌斑,提取细菌基因组DNA,合成针对Pg和Pi的16S rRNA基因的特异引物,运用qRT-PCR法检测Pg和Pi的拷贝数。采用SAS 9.1.3软件包对数据进行Kruskal-Wallis和 Wilcoxon 秩和检验。结果：Pg、Pi在不同样品组中检测到的绝对拷贝数数量分别为103～106和102～106,在慢性牙周炎患者龈下菌斑中的检出率和绝对拷贝数量显著高于健康对照组(P<0.05);3组治疗后的Pg数量比治疗前下降,其中综合治疗组和派丽奥治疗组下降率显著高于龈下刮治组。Pi的数量在派丽奥治疗组和龈下刮治组治疗后无显著减少,但在综合治疗组显著下降(P<0.05)。结论：qRT-PCR法能快速鉴定和精确定量Pg和Pi;综合治疗法比单一疗法能更有效抑制Pg和Pi。     

Detection of Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, and Prevotella nigrescens in chronic endodontic infection

Black-pigmented anaerobic rods such as Prevotella spp. and Porphyromonas spp. are involved in the etiology and perpetuation of endodontic infections. The aim of this study was to evaluate the prevalence of these species in chronic endodontic infections by using culture and polymerase chain reaction (PCR) techniques. Samples of 100 patients with root canals displaying chronic endodontic infections were obtained by sterilized paper points. Bacterial identification was performed by using culture and PCR techniques. By culture, in 33% of the samples, P. intermedia-P. nigrescens (75.8%), P. gingivalis (27.3%), and P. endodontalis (9.1%) were identified, and by PCR 60% of the samples harbored P. nigrescens (43.3%), P. gingivalis (43.3%), P. intermedia (31.7%), and P. endodontalis (23.3%). The presence of these black-pigmented anaerobic rods alone or in association in chronic endodontic infections seems to be frequent. PCR is a very sensitive technique for detecting DNA from bacterial cells. Culturing is only able to reveal living bacteria and is less sensitive for the identification of low numbers of bacterial cells.     

Human lactoferrin binding to Porphyromonas gingivalis, Prevotella intermedia and Prevotella melaninogenica

Human isolates of Porphyromonas gingivalis (n = 16), Prevotella intermedia n = 82) and Prevotella melaninogenica (n = 18) from diseased periodontal pockets were examined for interaction with human lactoferrin (HLf) in a standardized 125I-labeled protein binding assay. The highest HLf binding was found in P. intermedia strains, followed by P. gingivalis and P. melaninogenica. Further characterization of the interaction was performed with 1 representative strain from each species. HLf binding to P. gingivalis reached a saturation instantly and was optimal at pH 5.0-6.5. The corresponding values for P. melaninogenica were 90 min and pH 3.0-5.5. The HLf binding to the 2 strains seem to be nonspecific. In contrast, P. intermedia demonstrated specific binding, and a time-saturability within 60 min with an optimal uptake at pH 6.0-7.5. Scatchard analysis implied 45,000 receptors per cell with an affinity constant of 5.5 x 10(-7) M on P. intermedia strain 4H. The binding capacity in all 3 strains was affected by the culture medium. HLf binding components in these strains were susceptible to heat or proteases. Binding was eliminated in P. gingivalis and was enhanced in P. intermedia and P. melaninogenica by periodate treatment. Unlabeled HLf or bovine lactoferrin effectively displaced labeled HLf binding. Various proteins and carbohydrates did not inhibit HLf binding. Our data suggest that HLf binds to these periodontitis-associated species and that this mechanism is distinct from the previously known ligand interactions in oral bacteria.     

Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia and Prevotella nigrescens in endodontic lesions detected by culture and by PCR

he aim of this study was to investigate the presence of four black-pigmented bacteria, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia and Prevotella nigrescens, in endodontic infections by culture and polymerase chain reaction (PCR) analyses. Microbial samples were obtained from 50 teeth with untreated necrotic pulps (primary infection) and from 50 teeth with failing endodontic treatment (secondary infection). Microbiological strict anaerobic techniques were used for serial dilution, plating, incubation, and identification. For PCR detection, the samples were analyzed using species-specific primers of 16S rDNA and the downstream intergenic spacer region. Culture and PCR detected the test species in 13/100 and 50/100 of the study teeth, respectively. The organisms were cultured from 11/50 (22%) of primarily infected root canal samples and from 2/50 (4%) of secondary root canal samples. PCR detection identified the target species in 32/50 (64%) and 18/50 (36%) of primary and secondary infections, respectively. P. gingivalis was rarely isolated by culture methods (1%), but was the most frequently identified test species by PCR (38%). Similarly, P. endodontalis was not recovered by culture from any tooth studied, but was detected by PCR in 25% of the sampled teeth. PCR-based identification also showed higher detection rates of P. intermedia (33%) and P. nigrescens (22%) than culture (13%). In conclusion, P. gingivalis, P. endodontalis, P. intermedia, and P. nigrescens were identified more frequently in teeth with necrotic pulp than in teeth with failing endodontic treatment. Also, a higher frequency of black-pigmented species was detected by PCR than by culture.     

Possible potentiation of toxins from Prevotella intermedia, Prevotella nigrescens, and Porphyromonas gingivalis by cotinine

BACKGROUND: Smoking is a recognized risk factor for the initiation and progression of periodontitis. However, the mechanism by which smoking induces its negative effects on the periodontium is not clear. This study aimed to test the hypothesis that synergy may occur between cotinine and bacterial products isolated from 3 putative periodontopathogens. METHODS: A chick embryo toxin assay was used to investigate bacterial toxins (cell-free extracellular toxins and cell-free cell lysates) from 5 species with and without cotinine. A total of 9 putative periodontopathogens (3 species) and 2 non-oral controls (2 species) were studied. The periodontal species were: Prevotella intermedia (n = 4), Prevotella nigrescens (n = 4), and Porphyromonas gingivalis (n = 1). The control species tested were: Staphylococcus aureus (n = 1) and Escherichia coli (n = 1). RESULTS: The toxicity kill was significantly greater than expected by simple addition alone (P <0.05, Fisher's exact test) between cotinine (800 ng/ml) and 1) the cell-free extracellular toxins of P. nigrescens MH1 and 2) the cell-free cell lysates of P. intermedia MH2. Synergy occurred with cotinine plus the cell-free extracellular toxins in all but 3 periodontal isolates, and the cell-free cell lysates in all but 2 periodontal isolates. Cotinine significantly (P <0.05, Fisher's exact test) enhanced the effects of cell-free extracellular toxins and cell lysates from one control species (E. coli), but not the other (S. aureus). CONCLUSIONS: These findings indicate that synergy in an in vitro assay can occur between cotinine and toxins from putative periodontopathogens. This may be one important mechanism by which smoking increases the severity of periodontitis.     

Presence and antibiotic resistance of Porphyromonas gingivalis,Prevotella intermedia,and Prevotella nigrescens in children

BACKGROUND/AIMS: Only limited information exists about the prevalence in children of pathogens associated with periodontitis. The aim of the present study was to determine by culture whether 8-11-year-old children carry Porphyromonas gingivalis, Prevotella intermedia, and/or P. nigrescens in samples from the gingiva and/or the buccal mucosa taken before, and after caries treatment and oral hygiene instruction. A second aim was to assess the proportion of subjects who had gram-negative anaerobes carrying the tet(Q) and erm(F) genes, suggesting antibiotic resistance to tetracycline or erythromycin. METHOD: A total of 150 children provided gingival and buccal swab bacterial samples that were cultured for P. gingivalis, P. intermedia, and P. nigrescens. The species was verified using DNA-DNA hybridization with species-specific probes made from the variable region of the 16S rRNA sequences. Antibiotic-resistant genes, tet(Q) and erm(F), were identified using specific DNA-DNA hybridization with specific DNA probes. RESULTS: A total of 116 isolates of black-pigmented bacteria were cultured from 47 (31%) of 150 children. Five isolates were identified as P. gingivalis, 29 as P. intermedia, 33 as P. nigrescens, and 49 as other species. In general, the bacteria were not culturable at more than one time period. We found that 55% of these 47 children harbored black pigmented bacteria that carried either one or both of the two antibiotic-resistant genes studied (tet(Q), and erm(F)). CONCLUSION: The present study demonstrated that children not exposed to regular dental treatment carry bacteria outside the gingival sulcus that have been associated with periodontitis, and that standard treatment procedures may not clear the presence of the putative pathogens. In addition, antibiotic-resistant genes are common in identifiable gram-negative anaerobes, including putative pathogens.     

Dipeptide utilization by the periodontal pathogens Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum

Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum, which can frequently be isolated from periodontal pockets, preferentially utilize proteins and peptides as growth substrates. In this study, we determined the size of peptide that is preferentially utilized as a source of energy and material for cell growth by P. gingivalis, P. intermedia, P. nigrescens and F. nucleatum using various sizes of poly amino acids consisting of two to approximately 100 molecules of aspartate or glutamate. Resting cells of P. gingivalis, P. intermedia and P. nigrescens utilized aspartylaspartate, while cells of P. gingivalis and F. nucleatum utilized glutamylglutamate. The addition of aspartylaspartate to the culture medium increased the growth of P. gingivalis, P. intermedia and P. nigrescens, while the addition of glutamylglutamate promoted the growth of P. gingivalis and F. nucleatum. These results clearly indicate that dipeptides such as aspartylaspartate and glutamylglutamate can be utilized as growth substrates for P. gingivalis, P. intermedia, P. nigrescens and F. nucleatum.     

Killing of Fusobacterium nucleaturn,Porphyromonas gingivalis and Prevotella intermedia by protegrins

Protegrins are broad spectrum antibiotic peptides isolated from porcine leukocytes. In this study, we (i) examine the sensitivity of Gram-negative, anaerobic periodontal pathogens to synthetic protegrins; (ii) determine the relative potencies of protegrin congeners against these bacteria; and (iii) compare the potency of protegrins with other antibiotic peptides, including magainin MSI-78, tachyplesin I, cecropin P1, human defensins HNP-1-3, and clavanin A. Synthetic l - and d -enantiomers of protegrin 1 (PG-1 and D-PG-1, respectively), and L-enantiomers of protegrins 2, 3 and 5 (PG-2, PG-3 and PG-5) were tested against Fusobacteriurn nucleatum, and black-pigmented organisms including Porphyromonas gingivalis and Prevotella intermedia. Strains of both F. nucleatum and the black-pigmented organisms were sensitive to PG-1, and exhibited mean ED₉₉ of 2.2-2.3 μg/ml and 3.4-9.9 μg/ml, respectively. The D-form was statistically more potent than the L-form against these oral anaerobes, and although this difference in potency is unlikely to be of decisive therapeutic significance, the d -form may be of value given ability to resist microbial and host-derived proteases. PG-1 was more potent than magainin, tachyplesin, cecropin, defensins and clavanin under test conditions. Hypertonic saIt concentrations and heat-inactivated serum were found to be inhibitory to the bactericidal activity of PG-1. PG-1 was found to induce morphologic alterations in the ultrastructural appearance of F. nucleatum consistent with damage to the bacterial membranes. We conclude that protegrins may be useful antimicrobial agents in therapy against Gram-negative anaerobic bacteria believed to be involved in chronic, adult forms of periodontal infections.     

Occurrence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia in progressive adult periodontitis

BACKGROUND: Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia are the major periodontal bacteria species in most forms of progressive periodontitis in Scandinavia and the United States. The occurrence of periodontal pathogens appears to be different in subjects of different ethnic origin, and geographical factors may influence the distribution of these species. METHODS: The occurrence of A. actinomycetemcomitans, P. gingivalis, and P. intermedia was determined using a DNA probe in progressive adult periodontitis in Chileans. Sixty patients (mean age 43.6 +/- 8 years) who had not previously received any type of periodontal therapy were selected. Bleeding on probing, probing depth, and clinical attachment level measurements were made with an automated probe. Patients were monitored at 2-month intervals until at least 2 sites exhibited > or =2 mm attachment loss. Two subgingival plaque samples from active sites were taken in 56 subjects and matched with 2 plaque samples from inactive sites in the same individuals. RESULTS: P. gingivalis was found in 75% of active sites and in 59.7% of inactive sites in 96% of the patients (P = 0.022). P. gingivalis at high levels of detection was significantly more frequent in active sites (48.2%) than in inactive sites (31.2%) (P = 0.014). A. actinomycetemcomitans was detected in 6.25% of active sites and in 12.5% of inactive sites in 11.6% of patients. P. intermedia was found in 33% of patients and at a significantly higher proportion in active sites (49.1%) than in inactive sites (30.3%) (P = 0.006). There was a significantly higher proportion of inactive sites (34.8%) than active sites (19.6%) without any of the 3 pathogens (P = 0.016). Bleeding on probing was significantly more associated with active sites with high levels of P. gingivalis and with active sites with P. intermedia than with inactive sites. CONCLUSIONS: A high prevalence of P. gingivalis and P. intermedia was found in adult periodontitis, and the occurrence of these bacteria appears to be higher in Chileans than in other populations. No apparent association exists between A. actinomycetemcomitans and progressive adult periodontitis in Chileans.