牙周菌斑生物膜的体外模型建立

目的 观察血链球菌、牙龈卟啉单胞菌和具核梭杆菌在人工牙根面上随培养时间变化形成单菌种、双菌种和三菌种生物膜的能力,期望在体外建立龈下菌斑生物膜模型.方法 培养血链球菌、牙龈卟啉单胞菌和具核梭杆菌试验菌株,制备胶原包被羟磷灰石的人工牙根面,在人工牙根面上培养形成试验菌株的单菌种、双菌种和多菌种生物膜,并用扫描电镜观察三种试验菌株分别在培养24、48和72 h后形成单菌种、双菌种和三菌种生物膜的情况.结果 单独培养24h的血链球菌在人工牙根面上形成的完整生物膜,具备三维立体结构;培养48和72 h后,细菌密度逐渐增大,生物膜更加成熟.单独培养48 h的牙龈卟啉单胞菌形成的完整生物膜,具备三维立体结构;培养72 h后,细菌密度有所降低.牙龈卟啉单胞菌和具核梭杆菌培养24h形成的完整的双菌种生物膜,具备三维立体结构;培养48和72h后已看不出完整的生物膜结构,两菌的数量大幅度降低.血链球菌、牙龈卟啉单胞菌和具核梭杆菌在培养24h时形成的较完整的三菌种生物膜,初步具备三维立体结构;三菌中,血链球菌、具核梭杆菌所占比列远大于牙龈卟啉单胞菌;培养48 h后,三菌种生物膜更加成熟,牙龈卟啉单胞菌所占比例有所增加,此时的生物膜已具备三维立体结构;培养72 h后,三菌种仍保持较完整的生物膜结构,但数量均有所下降.结论 血链球菌、牙龈卟啉单胞菌和具核梭杆菌在培养24h时间点已基本形成了完整的单菌种、双菌种和三菌种生物膜结构,故在今后建立龈下菌斑生物膜模型时可选取24h这一时间点.     

牙周菌斑生物膜的体外模型建立

目的观察血链球菌、牙龈卟啉单胞菌和具核梭杆菌在人工牙根面上随培养时间变化形成单菌种、双菌种和三菌种生物膜的能力,期望在体外建立龈下菌斑生物膜模型。方法培养血链球菌、牙龈卟啉单胞茵和具核梭杆菌试验菌株,制备胶原包被羟磷灰石的人工牙根面,在人工牙根面上培养形成试验菌株的单菌种、双菌种和多菌种生物膜,并用扫描电镜观察三种试验菌株分别在培养24、48和72h后形成单菌种、双菌种和三菌种生物膜的情况。结果单独培养24h的血链球菌在人工牙根面上形成的完整生物膜,具备三维立体结构;培养48和72h后,细菌密度逐渐增大,生物膜更加成熟。单独培养48h的牙龈卟啉单胞菌形成的完整生物膜,具备三维立体结构;培养72h后,细菌密度有所降低。牙龈卟啉单胞菌和具核梭杆菌培养24h形成的完整的双菌种生物膜,具备三维立体结构;培养48和72h后已看不出完整的生物膜结构,两菌的数量大幅度降低。血链球菌、牙龈卟啉单胞菌和具核梭杆菌在培养24h时形成的较完整的三菌种生物膜,初步具备三维立体结构;三菌中,血链球菌、具核梭杆菌所占比列远大于牙龈卟啉单胞菌;培养48h后,三菌种生物膜更加成熟,牙龈卟啉单胞菌所占比例有所增加,此时的生物膜已具备三维立体结构;培养72h后,三菌种仍保持较完整的生物膜结构,但数量均有所下降。结论血链球菌、牙龈卟啉单胞菌和具核梭杆菌在培养24h时间点已基本形成了完整的单菌种、双菌种和三菌种生物膜结构,故在今后建立龈下菌斑生物膜模型时可选取24h这一时间点。     

血红素浓度对双菌生物膜中牙龈卟啉单胞菌致病潜力的影响

目的:研究血红素浓度对白色念珠菌(C.albicans)-牙龈卟啉单胞菌(P.gingivalis)双菌生物膜中P.gingivalis致病潜力的影响.方法:在0.1μg/mL(低)和5μg/mL(高)血红素培养条件下构建P.gingivalis-C.albicans双菌生物膜以及P.gingi-valis单菌生物膜,...     

维吾尔族慢性牙周炎中牙龈卟啉单胞菌菌毛fimA毒力基因型的分布

目的:分析维吾尔族慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌菌毛fimA毒力基因型的分布情况.方法:收集52例维吾尔族慢性牙周炎患者的龈下菌斑,采用16S rRNA PCR法检测牙龈卟啉单胞菌,并根据菌毛fimA毒力基因型的特异引物,用聚合酶链反应(PCR)检测Ⅱ型fimA和Ⅳ型fimA菌株的分布.结果:16S rRNA PCR法检测牙龈卟啉单胞菌在龈下菌斑中阳性检出率是76.9%.牙周袋PPD＞6 mm位点龈下菌斑标本的P.gingivalis检出率高于4＜PPD≤6 mm采样的位点,2组差异有统计学意义(P＜0.05).牙龈卟啉单胞菌菌毛fimA毒力基因型在牙龈卟啉单胞菌感染者的检出率分别是:ⅡfimA型为37.5%,ⅣfimA型为22.5%.结论:维吾尔族慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌有较高的检出率.牙龈卟啉单胞菌存在fimA毒力基因多态性.     

慢性根尖周炎感染根管内牙龈卟啉单胞菌和福赛斯拟杆菌的定植

目的：研究慢性根尖周炎患牙感染根管内牙龈卟啉单胞菌和福赛斯拟杆菌的定植情况,探讨两者间的定植关系.方法：采集31例慢性根尖周炎患者的38颗患牙根管内标本,加热裂解法获得细菌DNA,菌种特异引物16SrDNA PCR法检测标本的牙龈卟啉单胞菌和福赛斯拟杆菌,四格表确切概率检验福赛斯拟杆菌在有、无牙龈卟啉单胞菌定植根管内的检出率,比数比（odds ratio,OR）单因素分析两者间的定植关系.结果：牙龈卟啉单胞菌和福赛斯拟杆菌的检出率分别为39.5%、26.3%,其中前者单独检出7颗患牙,后者2颗患牙,两者同时检出8颗患牙.福赛斯拟杆菌在有、无牙龈卟啉单胞菌定植根管内的检出率分别为53.33%、8.70%（P=0.0036）,相关分析两菌间在慢性根尖周炎根管中的OR＞2（OR=12）,呈正相关关系（P＜0.05）.结论：牙龈卟啉单胞菌和福赛斯拟杆菌为慢性根尖周炎感染根管的定植菌,两者呈正相关定植.     

黏性放线菌和牙龈卟啉单胞菌间共聚对黏性放线菌黏附于S-HA的影响

目的:研究黏性放线菌(A.viscosus)和牙龈卟啉单胞菌(P.gingivalis)间共聚对A.viscosus黏附于唾液包被的羟基磷灰石(S-HA)的影响.方法:将0.5mL 5×108CFU/mL A.viscosus分别与0.5mL 1×109、3×109、5×109CFU/mL的P.gingivalis菌液混合,室温旋转,SEM观察P.gingivalis与A.viscosus间的共聚;将不同浓度的P.gingivalis和用放射性核素标记的A.viscosus混合,室温旋转1.5h,荧光闪烁记数法检测A.viscosus对S-HA的黏附量,以单菌种的A.viscosus作为对照,设定其黏附量为100%.采用SPSS10.07软件包对数据进行单因素方差分析,比较不同浓度P.gingivalis对A.viscosus黏附的影响.结果:1个A.viscosus的细胞表面可黏附多个P.gingivalis.P.gingivalis浓度较小时,A.viscosus对S-HA黏附量相对于对照组略增加;随P:gingivalis浓度增加,A.viscosus黏附量显著下降(P＜0.05).结论:A.viscosus和P.gingivalis可发生细菌之间的共集.这种共集可影响A.viscosus对S-HA的黏附.     

变异链球菌绿色荧光蛋白报告株在双菌种生物膜研究的应用

目的探讨变异链球菌（S.mutans）绿色荧光蛋白（GFP）报告株在单、双菌种生物膜的形成、代谢和抗药性研究的应用。 方法采用基因重组技术构建S.mutans GFP报告株C67-1 pDM15和UA159 pDM15;通过荧光显微镜和荧光光度计,评估报告株转化效率、生长能力和荧光表达规律;构建S.mutans GFP报告株与戈登链球菌（S.gordonii）的单、双菌种生物膜,比较单、双菌种生物膜中S.mutans的生物膜形态、糖代谢活力及氯已定（CHX）作用下的抗药性差异。数据运用单因素方差分析、Pearson相关性分析与独立样本t检验进行统计学分析。 结果S.mutans GFP报告株可稳定地表达gfp基因,生长能力和生物膜形成能力与野生株相似;生物膜加入0.2%的葡萄糖后荧光量迅速增加,可检测细菌代谢改变,4 h的相对荧光增长值（ΔRLU）可反映生物膜量,二者显著相关（r=0.9818~0.9985,P<0.001）;S.gordonii改变S.mutans C67-1和UA159的生物膜结构,抑制UA159菌株生物膜的形成;剩余荧光增长率[ΔRLU（%）]反映耐药能力,单菌种生物膜C67-1和UA159的ΔRLU（%）相似,分别为（70.2 ± 8.0）%和（72.3 ± 7.9）%（t=-0.521,P=0.630）,在双菌种生物膜中S.mutans的ΔRLU（%）发生改变,与各自单菌种生物膜相比差异有统计学意义：C67-1升高至（85.6 ± 4.3）%（t=-2.872,P=0.045）,UA159降低至（41.2 ± 10.1）%（t=3.551,P=0.024）。 结论S.gordonii对S.mutans生物膜形成能力和抗药性的改变具有亚型差异。GFP报告株可作为多菌种生物膜定量与空间结构研究的模式菌。     

黏性放线菌对牙龈卟啉单胞菌生长的影响

目的:研究黏性放线菌(Actinomyces viscosus,A.viscosus)对牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)生长的影响。方法:将细菌分为3组,即A.viscosus组、P.gingivalis组和2种细菌等量、等浓度混合组。3组细菌专性厌氧培养48h,绘制生长曲线;混合细菌组每隔2h取菌悬液梯度稀释,接种于BHI血琼脂平板,培养72h,计数P.gingivalis占菌落总数的百分比;制备P.gingivalis BHI血琼脂平板,将不同浓度的A.viscosus菌悬液及除菌后的上清液滴注于已经接种P.gingivalis的固体血琼脂平板,厌氧条件下培养72h,测量抑菌圈直径。实验数据采用SPSS10.0软件包进行单因素方差分析。结果:3组中,A.viscosus2h已处于对数生长期,8h达平台期;P.gingivalis在12h进入对数生长期,32h达平台期;混合细菌组在2h开始进入对数生长期,12h达平台期;混合细菌组前8h P.gingivalis所占总菌落数的百分比随时间延长呈下降趋势﹙P<0.05﹚,8h后平板上已经没有P.gingivalis的存在;1×109、108、107、106A.viscosus对P.gingivalis的抑菌圈平均直径(mm)为19.3、17.4、13.2和9.8,差异具统计学意义﹙P<0.05﹚;A.viscosus的上清液对P.gingivalis无抑制作用。结论:A.viscosus对P.gingivalis生长的抑制作用是由于前者生长速度快,造成营养性竞争所致。     

Nod/Rip2信号通路参与牙龈卟啉单胞菌诱导牙髓细胞白细胞介素-6表达

目的探讨牙龈卟啉单胞菌刺激牙髓细胞产生细胞因子的信号转导通路。方法厌氧培养牙龈卟啉单胞菌（P.gingivalis）,胞内感染原代培养牙髓细胞,RNA抽提,实时荧光定量聚合酶链反应（qPCR）检测Nods、Rip2,ELISA检测白细胞介素-6（IL-6）的表达水平。结果牙髓细胞基础表达Nods、Rip2 mRNA及IL-6。P.gingivalis感染后2 h, Nods和Rip2 mRNA增高,达到高峰,6 h出现下降趋势。而P.gingivalis活菌刺激牙髓细胞能增强IL-6表达水平。结论P.gingivalis能激活牙髓细胞固有免疫反应,通过Nod/Rip2途径进行信号转导上调细胞因子IL-6表达。     

牙龈卟啉单胞菌对铁和亚铁血红素的摄取机制

牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)是慢性牙周病重要病原菌之一.作为绝对需铁菌,牙龈卟啉单胞菌不能产生铁载体.所以在人体中,牙龈卟啉单胞菌主要利用外源的铁/亚铁血红素,通过特异性外膜受体结合含铁蛋白中的铁/亚铁血红素,并将其转运至细胞内.铁/亚铁血红素对于牙龈卟啉单胞菌的生长和毒性都起着重要作用.本文主要介绍牙龈卟啉单胞菌对铁和亚铁血红素的摄取机制.     

Biofilm formation by Porphyromonas gingivalis and Streptococcus gordonii

Confocal scanning laser microscopy (CSLM) was used to visualize and quantify biofilm formation by the oral bacteria Streptococcus gordonii and Porphyromonas gingivalis , A saliva-coated glass coverslip under continuous bacterial challenge and conditions of low shear force was used to investigate attachment to the salivary pellicle and also the effect of cell-cell interactions on the extent of colonization and biofilm development. S. gordonii bound to the salivary pellicle and outcompeted P. gingivalis for attachment sites. Both P. gingivalis and S. gordonii failed to establish substantial biofilm formation independently. However, biofilm formation did occur subsequent to initial adherence of P. gingivalis to S. gordonii cells deposited on the salivary pellicle. The commensal species S. gordonii may. therefore, provide an attachment substrate for colonization and biofilm accretion by the potential pathogen, P. gingivalis.     

Involvement of Porphyromonas gingivalis fimbriae in adherence to Streptococcus gordonii

Adherence of Porphyromonas gingivalis to early plaque bacteria, such as Streptococcus gordonii , is considered an important colonization mechanism. The molecules that mediate this interspecies binding have not been determined. Fimbriae were prepared from P. gingivalis 33277 by mild agitation, ammonium sulfate precipitation and DEAE-Sepharose chromatography. In a nitrocellulose blot adherence assay, purified fimbriae inhibited S. gordonii G9B- P. gingivalis 33277 binding by up to 54%. In addition, fimbriae bound to S. gordonii cells in a dot-blot assay. Incubation of fimbriae with S. gordonii cells followed by washing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotting and probing with P. gingivalis antibodies also revealed that the fimbriae bind to S. gordonii. In contrast, S. gordonii did not interact with fimbriae that were first subjected to SDS-PAGE and electroblotting or deposited on a nitrocellulose membrane, suggesting that conformational determinants of the fimbriae may be important in binding. The results indicate that binding between P. gingivalis and S. gordonii is mediated, at least in part, by the porphyromonads' fimbriae.     

Efficacy of antibiotics to strains of periodontopathogenic bacteria within a single species biofilm - an in vitro study

OBJECTIVES: This study examined differences in the efficacy of antibiotics against a single strain of three periodontal pathogens grown in an artificial biofilm. METHODS: Single species biofilms were established with artificial saliva and one of the following bacterial strains: Actinobacillus actinomycetemcomitans Y4, Streptococcus constellatus 384b (a clinical isolate) and Porphyromonas gingivalis ATCC 33277. The efficacy of the antibiotics clindamycin, doxycycline, metronidazole, and moxifloxacin to these bacteria was determined using concentrations up to 100-fold minimal inhibitory concentration (MIC) to planctonic bacteria over 48 h. RESULTS: The ability of the bacteria to form a biofilm varied. The biofilms of S. constellatus 384b and A. actinomycetemcomitans Y4 contained more viable bacteria and showed a larger thickness in SEM photographs than those of P. gingivalis ATCC 33277. The antibiotics tested showed different efficacy for the different strains. Moxifloxacin was the most efficient antibiotic: onefold MIC was sufficient to eliminate A. actinomycetemcomitans Y4 and P. gingivalis ATCC 33277 after 48 h. However, only the 50-fold MIC completely eradicated S. constellatus 384b. SEM photographs underlined the damaging effect of moxifloxacin on the biofilm structure. CONCLUSION: The complete removal of bacteria by the use of antibiotics alone seems to be impossible when taking into account MIC values and the level of antibiotics in gingival fluid.     

Identification of 40-kDa outer membrane protein as an aggregation factor of Porphyromonas gingivalis to Streptococcus gordonii

Porphyromonas gingivalis, an important pathogen in periodontitis, aggregates with other oral microorganisms such as Streptococcus gordonii. We previously succeeded in gene cloning the 40-kDa outer membrane protein (OMP) from P. gingivalis. Although recombinant (r) 40-kDa OMP itself did not show aggregation activity, the affinity-purified antibody against 40-kDa rOMP inhibited the aggregation activity of P. gingivalis cells toward S. gordonii which is one of the first oral bacteria to colonize on tooth surfaces and can be expected to support subsequent colonization of other bacteria. In this study, in order to clarify the pathological role of 40-kDa OMP, we used a cross-linking reagent to construct a polymeric form of r40-kDa OMP and examined its aggregation activity. The polymeric r40-kDa OMP significantly expressed aggregation activity with S. gordonii cells. Moreover, the antibody against r40-kDa OMP inhibited the aggregation activity of the polymeric r40-kDa OMP. These findings clearly demonstrate that 40-kDa OMP, as a multivalent form, is one of the important aggregation factors on the cell surface of P. gingivalis.     

Effects of chlorhexidine digluconate and hydrogen peroxide on Porphyromonas gingivalis hemin binding and coaggregation with oral streptococci

Porphyromonas gingivalis, a gram-negative anaerobe, is one of the major causative agents of periodontal disease. In this study, the effects of chlorhexidine digluconate and hydrogen peroxide on the hemin binding of P. gingivalis and coaggregation of this bacterium with oral streptococci were examined. The pretreatment of P. gingivalis W50 and 381 with chlorhexidine digluconate and hydrogen peroxide increased the hemin binding of these bacteria. The hemin binding of P. gingivalis was increased by the subminimal inhibitory concentration (MIC) of chlorhexidine digluconate. However, concentrations of hydrogen peroxide below the MIC had no effect on the hemin binding of P. gingivalis W50 and 381. Coaggregation of P. gingivalis 381 with Streptococcus oralis ATCC 9811 and Streptococcus gordonii DL1 was diminished by chlorhexidine digluconate. The coaggregation-inhibitory effect was concentration-dependent. Hydrogen peroxide also showed inhibitory effects on the coaggregation of P. gingivalis 381 with S. oralis 9811 and S. gordonii DL1 at concentrations below that used clinically. Concentrations of chlorhexidine digluconate below the MIC inhibited coaggregation. However, concentrations of hydrogen peroxide below the MIC were not effective in reducing the coaggregation of P. gingivalis with oral streptococci. These observations show that chlorhexidine digluconate and hydrogen peroxide could confer variable effects on P. gingivalis hemin binding and coaggregation of this bacterium with oral streptococci.     

Epithelial cell pro-inflammatory cytokine response differs across dental plaque bacterial species

Aim: The dental plaque is comprised of numerous bacterial species, which may or may not be pathogenic. Human gingival epithelial cells (HGECs) respond to perturbation by various bacteria of the dental plaque by production of different levels of inflammatory cytokines, which is a putative reflection of their virulence. The aim of the current study was to determine responses in terms of interleukin (IL)-1 β , IL-6, IL-8 and IL-10 secretion induced by Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Streptococcus gordonii in order to gauge their virulence potential.
Materials and Methods: HGECs were challenged with the four bacterial species, live or heat killed, at various multiplicity of infections and the elicited IL-1 β , IL-6, IL-8 and IL-10 responses were assayed by enzyme-linked immunosorbent assay.
Results: Primary HGECs challenged with live P. gingivalis produced high levels of IL-1 β , while challenge with live A. actinomycetemcomitans gave high levels of IL-8. The opportunistic pathogen F. nucleatum induces the highest levels of pro-inflammatory cytokines, while the commensal S. gordonii is the least stimulatory.
Conclusion: We conclude that various dental plaque biofilm bacteria induce different cytokine response profiles in primary HGECs that may reflect their individual virulence or commensal status.     

牙周可疑致病菌对胶原包被羟磷灰石黏附的体外实验研究

目的比较对牙根面具有黏附能力的具核梭杆菌、牙龈卟啉单胞菌、中间普氏菌和伴放线菌嗜血菌对胶原包被的羟磷灰石实验膜（C- HA）的黏附能力,初步探讨以上牙周可疑致病菌在牙根表面形成菌斑生物膜的能力。方法采用同位素闪烁计数法测定上述4种细菌黏附至C- HA表面的黏附量及黏附率,比较其黏附能力。结果培养相同时间,不论是培养24 h还是培养48 h,4种不同细菌两两比较,具核梭杆菌ATCC 10953和牙龈卟啉单胞菌ATCC 33277对C- HA表面黏附率的差异无统计学意义;中间普氏菌ATCC 25611和伴放线菌嗜血菌ATCC 29523的黏附率之间差异也无统计学意义,但是具核梭杆菌ATCC 10953和牙龈卟啉单胞菌ATCC 33277对C- HA表面的黏附率显著高于中间普氏菌ATCC 25611和伴放线菌嗜血菌ATCC 29523（P<0.001）。同一种细菌,在培养不同时间即培养24 h和48 h,对C- HA表面黏附率的差异均无统计学意义。结论不同的牙周可疑致病菌对胶原包被的羟磷灰石的选择性黏附作用不同,具核梭杆菌和牙龈卟啉单胞菌对胶原有较强的亲和作用,在细菌的局部定植过程和牙周炎的进展和复发中可能发挥重要作用。