Adhesion of Porphyromonas gingivalis strains to cultured epithelial cells from patients with a history of chronic adult periodontitis or from patients less susceptible to periodontitis

BACKGROUND: The present study aimed to explain the interindividual variation in periodontitis susceptibility by differences in the initial adhesion rate of Porphyromonas gingivalis to the pocket epithelium of these individuals, and/or by inter-P. gingivalis strain differences in association capacity (adhesion and internalization). METHODS: Adhesion assays were performed on epithelial monolayers (cultured in vitro from pocket epithelium belonging to patients who were less or more susceptible to chronic adult periodontitis) using 11 genetically different clinical strains of P. gingivalis. RESULTS: Both the disease category (less susceptible versus susceptible) and the interstrain variation were found to have a significant effect (both P <0.05) on the initial bacterial association. The chronic adult periodontitis group showed significantly more association of P. gingivalis when compared to less susceptible patients (4.2 x 10(6) versus 3.5 x 10(6)). Also, the interstrain variation was significant, with strains Pg 4 and 5 representing the least and best associating bacteria (1.8 x 10(6) colony forming units for Pg 4, 9 x 10(6) for Pg 5). CONCLUSIONS: These results indicate that periodontitis susceptibility is influenced by both the interindividual differences in pocket epithelium (allowing more adhesion of P. gingivalis) or by the strain type by which the patient is infected (intra-species differences in adhesion capacity).     

Adhesion of Porphyromonas gingivalis serotypes to pocket epithelium

BACKGROUND: Porphyromonas gingivalis, a key pathogen in periodontitis, is able to adhere to and invade the pocket epithelium. Different capsular antigens of P. gingivalis have been identified (K-serotyping). These P. gingivalis capsular types show differences in adhesion capacity to human cell lines or to cells cultured on a feeder layer or stromal equivalent. METHODS: The adhesion capacity of different P. gingivalis serotypes (6 capsular types and non-encapsulated strains) was compared on in vitro cultured epithelial monolayers from periodontal pockets of patients with periodontitis. The degree of adherence of P. gingivalis was evaluated by both culture and fluorescence microscopy. RESULTS: Non-encapsulated strains adhered significantly more than their capsulated variants. Capsule type 4 (K-4) adhered slightly better than the remaining K-types. CONCLUSION: This study indicates that the presence and type of capsule have a significant influence on the initial adhesion of P. gingivalis to human periodontal pocket epithelial cells.     

不同fimA基因型牙龈卟啉单胞菌黏附和侵入口腔上皮细胞能力的比较

目的 通过比较临床分离的不同fimA型牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)菌株黏附和侵入KB细胞的能力,探讨不同fimA基因型Pg菌株之间致病力差异的原因.方法 应用培养法和抗生素保护法测定不同fimA型Pg菌株黏附和侵入KB细胞的能力,扫描电镜观察不同fimA型Pg对KB细胞的黏附情况.结果 所有菌株均可黏附和侵入KB细胞,黏附率为0.523%～37.125%,侵入率为0.017%～3.750%;各fimA型Pg菌株之间黏附和侵入KB细胞的能力差异无统计学意义(P>0.05);各株Pg之间、4株Ⅱ型菌株之间、5株Ⅳ型菌株之间,黏附和侵入KB细胞的能力差异有统计学意义(P<0.01).结论 Pg的fimA型与其对KB细胞的黏附和侵入能力无相关性,提示可能存在其他影响不同fimA型Pg致病力差异的因子.     

不同fimA基因型牙龈卟啉单胞菌黏附和侵入口腔上皮细胞能力的比较

目的 通过比较临床分离的不同fimA型牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)菌株黏附和侵入KB细胞的能力,探讨不同fimA基因型Pg菌株之间致病力差异的原因.方法 应用培养法和抗生素保护法测定不同fimA型Pg菌株黏附和侵入KB细胞的能力,扫描电镜观察不同fimA型Pg对KB细胞的黏附情况.结果 所有菌株均可黏附和侵入KB细胞,黏附率为0.523%～37.125%,侵入率为0.017%～3.750%;各fimA型Pg菌株之间黏附和侵入KB细胞的能力差异无统计学意义(P>0.05);各株Pg之间、4株Ⅱ型菌株之间、5株Ⅳ型菌株之间,黏附和侵入KB细胞的能力差异有统计学意义(P<0.01).结论 Pg的fimA型与其对KB细胞的黏附和侵入能力无相关性,提示可能存在其他影响不同fimA型Pg致病力差异的因子.     

不同fimA基因型牙龈卟啉单胞菌黏附和侵入口腔上皮细胞能力的比较

目的 通过比较临床分离的不同fimA型牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)菌株黏附和侵入KB细胞的能力,探讨不同fimA基因型Pg菌株之间致病力差异的原因.方法 应用培养法和抗生素保护法测定不同fimA型Pg菌株黏附和侵入KB细胞的能力,扫描电镜观察不同fimA型Pg对KB细胞的黏附情况.结果 所有菌株均可黏附和侵入KB细胞,黏附率为0.523%～37.125%,侵入率为0.017%～3.750%;各fimA型Pg菌株之间黏附和侵入KB细胞的能力差异无统计学意义(P>0.05);各株Pg之间、4株Ⅱ型菌株之间、5株Ⅳ型菌株之间,黏附和侵入KB细胞的能力差异有统计学意义(P<0.01).结论 Pg的fimA型与其对KB细胞的黏附和侵入能力无相关性,提示可能存在其他影响不同fimA型Pg致病力差异的因子.     

Porphyromonas gingivalis invades human pocket epithelium in vitro

The present study examined the adhesive and invasive potential of Porphyromonas gingivalis interacting with human pocket epithelium in vitro . Pocket epithelial tissue, obtained during periodontal surgery of patients with advanced periodontal disease, generated a stratified epithelium in culture. P. gingivalis strains W50 and FDC 381 (laboratory strains), OMGS 712, 1439, 1738, 1739 and 1743 (clinical isolates) as well as Escherichia coli strain HB101 (non-adhering control) were tested with respect to epithelial adhesion and invasion. Adhesion was quantitated by scintillation spectrometry after incubation of radiolabeled bacteria with epithelial cells. The invasive ability of P. gingivalis was measured by means of an antibiotic protection assay. The epithelial multilayers were infected with the test and control strains and subsequently incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). The number of internalized bacteria surviving the antibiotic treatment was assessed after plating lyzed epithelial cells on culture media. All tested P. gingivalis strains adhered to and entered pocket epithelial cells. However, considerable variation in their adhesive and invasive potential was observed. E. coli strain HB101 did not adhere or invade. Transmission electron microscopy revealed that internalization of P. gingivalis was preceded by formation of microvilli and coated pits on the epithelial cell surfaces. Intracellular bacteria were most frequently surrounded by endosomal membranes; however, bacteria devoid of such membranes were also seen. Release of outer membrane vesicles (blebs) by internalized P. gingivalis was observed. These results support and extend previous work from this laboratory which demonstrated invasion of a human oral epithelial cell-line (KB) by P. gingivalis .     

Adhesion and invasion to epithelial cells by fimA genotypes of Porphyromonas gingivalis

Adhesion to and invasion of epithelial cells by the periodontopathogen Porphyromonas gingivalis is promoted by the major fimbriae, encoded by fimA. The microorganism can be classified in six genotypes, based on fimA sequence, and genotype II strains are more prevalent than others in periodontitis patients. This study aimed to determine the adhesive and invasive abilities on KB cells of different fimA allelic variants of P. gingivalis isolates. Twenty-two isolates and six reference strains representing the six fimA genotypes and non-typeable strains were screened for their adhesion and invasion abilities on KB cells, using standard methods. All strains were able to adhere and, except for one, to invade KB cells. However, these properties were not homogeneous among strains belonging to the same genotype. There was no correlation between adhesion and invasion efficiencies. Isolate KdII 865 (fimA genotype II) was the most invasive and the second most adhesive strain, whereas reference strain ATCC 33277 (fimA I) showed a low adhesion ability but was highly invasive. These data indicated that fimA genotypes of P. gingivalis are not related to the adhesion and invasion abilities on KB cells, suggesting that the increased prevalence and proportion of certain genotypes may be attributed to other characteristics besides FimA variation.     

牙龈卟啉单胞菌红细胞凝集素A黏附和入侵人牙龈上皮细胞的研究

目的:检测牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)膜表面红细胞凝集素A(hemagglutinin A,HagA)黏附和入侵人牙龈上皮细胞的功能。方法:构建P.g381hagA基因的变异菌株,连接hagA基因到pYA292质粒上,并克隆到无毒性的沙门杆菌x4072菌中,通过比较变异菌株、野生菌株和表达hagA基因的沙门杆菌对人牙龈上皮细胞的黏附和入侵功能,检测HagA在此过程中的作用。结果:P.g381hagA基因变异菌株和野生菌株在对细胞的黏附和入侵过程中没有显著差别,但沙门杆菌x4072HagA表达菌株对细胞的黏附性和入侵性比对照组分别提高了3倍和4倍。结论:HagA参与了P.g381黏附和入侵牙龈上皮细胞的过程。     

纤维蛋白原对牙龈卟啉单胞菌黏附口腔上皮细胞的影响

目的探讨血浆蛋白成分纤维蛋白原（Fg）在牙周病发病机制中的作用。方法体外培养口腔上皮细胞系KB细胞至单层融合，分别与不同浓度Fg溶液共同孵育，并加入以^3H-胸腺嘧啶核苷标记的牙龈卟啉单胞菌（Pg）行细菌感染攻击细胞实验。采用同位素闪烁光谱测定法检测黏附和内化于KB细胞的Pg数量。结果加入Fg的各实验组黏附和内化细菌量及细菌黏附和内化率均显著高于未加入Fg的对照组；不同Fg浓度的各组间黏附和内化细菌量及黏附和内化率差异亦有统计学意义，随着Fg浓度的升高，黏附和内化的细菌量显著增加。结论纤维蛋白原可促进Pg黏附于口腔上皮细胞，在牙周病的发生、发展中可能具有一定的病理学作用。     

Porphyromonas gingivalis lipopolysaccharide delays human polymorphonuclear leukocyte apoptosis in vitro

Apoptosis (programmed cell death) is a mechanism by which superfluous or damaged cells undergo changes that lead to selective removal from organ systems by phagocytic cells. Certain bacterial products delay apoptosis in neutrophils (PMNs). In this study, PMNs were incubated for up to 8 h with varying concentrations of lipopolysaccharide (LPS), lipid A or capsular polysaccharide isolated from 3 strains of Porphyromonas gingivalis (Pg) (strains HG-184, A7A1-28 and 381). Assay runs included controls containing cells and medium but no bacterial products. Fluorescence microscopy was used to evaluate apoptotic changes. PMNs exhibited a time-dependent increase in the number of apoptotic cells. When cells were cultured in the presence of LPS from any of the 3 Pg strains, apoptosis was delayed in a dose-dependent fashion (p < 0.05). The effects of these LPS preparations were similar to each other and to Escherichia coli 0111:B4 LPS. Lipid A from the 3 Pg strains also delayed apoptosis (p < 0.05), but was less potent than LPS or synthetic lipid A. Capsular polysaccharide had no significant effect on apoptosis (p > 0.05). Thus, LPS and lipid A from P. gingivalis appear to modulate the functional lifespan of PMNs. This could potentiate the inflammatory and destructive components of periodontal diseases.     

牙龈卟啉单胞菌影响血管内皮细胞增殖和凋亡的实验研究

目的 观察牙龈卟啉单胞菌(Porphyromonas gingivatis,Pg)对血管内皮细胞增殖和凋亡的影响,探讨Pg在动脉粥样硬化发病机制中的作用.方法 建立体外Pg侵入血管内皮细胞模型,甲基噻唑基四唑(MTT)法观察细胞增殖,碘化丙啶染色、流式细胞仪分析细胞周期,Annexin-V-FITC凋亡试剂盒检测细胞凋亡.结果 PgATCC33277侵入后72 h细胞增殖活性降低12.46%,Pg W83侵入后72 h细胞增殖活性降低10.47%(F=786.68,P<0.01);Pg W83侵入后24 h使G1期细胞增加(F=43.23,P<0.01),ATCC 33277侵入后48 h使G1期细胞增加(F=66.72,P<0.01);Pg侵入后24 h即诱导细胞凋亡(F=1074.56,P<0.01).结论 Pg可能通过细胞毒性及诱导凋亡作用,加重血管内皮细胞的局部炎性反应,在动脉粥样硬化炎性病理反应中有重要意义.     

Porphyromonas gingivalis invades oral epithelial cells in vitro

The aim of the present study was to analyze the adhesive and invasive potential of a number of P. gingivalis strains, in an in vitro system utilizing cultures of human oral epithelial cells (KB cell line, ATCC CCL 17). P. gingivalis strains W50 and FDC 381 (laboratory strains) and OMGS 1738, 1743 and 1439 (clinical isolates) as well as E. coli strain HB 101 (non-adhering, non-invasive control) were used. Adherence was assessed by means of scintillation counting and light microscopy, after incubation of radiolabelled bacteria with epithelial cells. In the invasion assay, monolayers were infected with the P. gingivalis and E. coli strains and further incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). Invasion was evaluated by (i) assessing presence of bacteria surviving the antibiotic treatment, and (ii) electron microscopy. All P. gingivalis strains adhered to and entered into the oral epithelial cells. After 3 hours of incubation, bacteria were frequently identified intracellularly by means of electron microscopy. The cellular membranes, encapsulating the microorganisms in early stages of the invasive process, appeared later to disintegrate. The presence of coated pits on the epithelial cell surfaces suggested that internalization of P. gingivalis was associated with receptor-mediated endocytosis (RME). Formation of outer membrane vesicles (blebs) by intracellular bacteria indicated that internalized P. gingivalis was able to retain its viability. E. coli strain HB 101 neither adhered to nor invaded epithelial cells.     

根周细菌对胶原包被羟磷灰石粘附的体外实验研究

目的：细菌对牙面的粘附能力与其致龋性密切相关，本研究旨在比较对牙面具有粘附能力的变形链球菌ATCC25175、粘性放线菌ATCC15987、乳杆菌ATCC4546、牙龋卟啉菌ATCC33277及中间普氏菌ATCC25611对胶原包被羟磷灰石实验膜（C－HA）的粘附能力，探讨口腔细菌在根周疾病中的作用。方法：采用同位素闪烁计数法测定上述五种细菌对胶原处理的羟基磷灰石（C－HA）的粘附能力，以[^3H]胸腺嘧啶核苷为标记对细菌的粘附进行定量观察，用每分钟的同位素放射量CPM表示（counts per mintue）。结果：所有细菌对C－HA表面的粘附率均有统计学意义（P＜0．01），粘性放线菌对C－HA表面的粘附率显著高于其它细菌组，牙龋卟啉菌及乳杆菌对C－HA表面的粘附率次之，变形链球菌及中间普氏菌对C－HA表面的粘附能力最弱。结论：不同的根周细菌对胶原包被的羟磷灰石的选择性粘附作用不同，粘性放线菌、乳杆菌及牙龋卟啉菌对胶原具较强的亲和作用，在细菌的局部定植过程及疾病的进展中发挥重要作用。     

牙龈卟啉单胞菌对牙龈上皮细胞焦点黏附成分paxillin和FAK的作用

目的:研究牙龈卟啉单胞菌(P.gingivalis)对牙周组织的破坏机制,探讨菌毛的致病作用.方法:应用Western blot法检测P.gingivalis ATCC 33277及其fimA缺陷突变株对Hela细胞及永生化人牙龈上皮细胞 (immortalized human gingival epithelial cells, IHGE细胞) 的焦点黏附成分——焦点黏附蛋白paxillin和焦点黏附激酶 (focal adhesion kinase,FAK) 蛋白表达的影响.结果:P.gingivalis ATCC 33277野生株和fimA缺陷突变株能够使HeLa细胞和IHGE细胞的paxillin和FAK发生降解,fimA缺陷突变株对paxillin和FAK的降解能力较野生株显著减弱.在IHGE细胞中,paxillin和FAK的降解呈时间依赖性和MOI依赖性.结论:菌毛介导的P.gingivalis的黏附和侵入可能对焦点黏附成分的降解起促进作用,IHGE细胞较Hela细胞更适用于牙周致病菌的研究.     

嗜酸乳杆菌及其灭活菌粘附及拮抗牙周致病菌特性研究

目的研究体外共培养时,嗜酸乳杆菌（lactobacillus acidophilus,La）活菌及其灭活菌对牙龈角化上皮细胞株KB细胞的粘附,以及La对口腔致病菌具核梭杆菌（fusobacteriurn nuclearum,Fn）和牙龈卟啉单胞菌（porphyromonas gingivalis,Pg）的粘附拮抗作用,以期为牙周疾病益生菌防治提供实验依据。方法选取不同生命状态（活菌、灭活菌）的La4356作为研究菌,运用光镜以及革兰染色法,分析不同状态的嗜酸乳杆菌对口腔牙龈上皮细胞株KB细胞的粘附指数,以及La作用于Pg和Fn后,Pg和Fn粘附指数的变化。结果两种生命状态的嗜酸乳杆菌对KB细胞均有粘附作用,活菌的粘附指数略高于灭活菌。La和其灭活菌均可与Pg和Fn竞争对KB细胞的粘附,显著降低Pg和Fn对KB细胞的粘附指数;同时在允许浓度范围内,高浓度的益生菌的粘附拮抗作用较低浓度时明显。当益生菌和致病菌浓度比大于1：1时,La灭活菌抑菌作用略强于La活菌。结论益生菌La及其灭活菌均可粘附于上皮细胞,且均对Pg、Fn具有较强的粘附拮抗作用,可用作牙周生态防治。     

Influence of nicotine and cotinine on epithelial colonization by periodontopathogens

BACKGROUND: Since smoking is an established risk factor for the development of periodontitis, the present study investigated whether nicotine and cotinine can make epithelial cells more prone to colonization by periodontopathogens. METHODS: Primary epithelial cell mono-layers were inoculated with nicotine and cotinine prior to adhesion experiments with Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. The number of bacteria associated with cells inoculated or not with nicotine or cotinine were assessed by an indirect culture viability assay. The same experimental set-up was used for assessing HeLa cells exposed to cigarette smoke extract (CSE). RESULTS: Primary epithelial cells inoculated with concentrations of nicotine and cotinine, found in smokers and non smokers, did not show significant differences (P>0.05) in colonization susceptibility to A. actinomycetemcomitans. When these concentrations were increased to 1 mg/ml, a significant (P<0.05) and species-specific effect of the colonization susceptibility of epithelial cells was observed: It increased for A. actinomycetemcomitans, while it decreased for P. gingivalis. For both species the effects were more pronounced for nicotine, although this was not statistically significant. The change in colonization susceptibility did not result from alterations of the bacterial viability due to nicotine or cotinine. Treatment of HeLa cells with CSE also led to a species-specific variation in colonization tendency; i.e., increased for A. actinomycetemcomitans (P<0.05), but not for P. gingivalis. CONCLUSIONS: The susceptibility of epithelial cells to become colonized by either A. actinomycetemcomitans or P. gingivalis could be altered by nicotine, cotinine, or CSE in a time-dependent, species-specific manner. Whether these findings that support the hypothesis of an increased patient susceptibility for bacterial adhesion to epithelial cells in smokers are clinically relevant remains to be proven.     

F.nucleatum和P.gingivalis对种植体周龈沟液sICAM-1表达的影响

目的 :研究种植体周围龈沟中F .nucleatum (Fn)和P .gingivalis(Pg)对其龈沟液 (PISF)可溶性细胞间黏附分子 -1(sICAM -1)表达的影响。方法 :用PCR法检测种植体周龈沟中的细菌标本 ,根据Fn和Pg检出情况将 3 2名患者的 5 1枚钛种植体分为 3组 ,考察mPLI和PD ,用 periopaper滤纸条收集PISF ,用ELISA方法检测sICAM -1。结果 :检出Pg和 (或 )Fn组与未检出Pg和Fn组之间的PD值有统计学差异 (P <0 .0 5 ) ,单独检出Fn组的sICAM -1与未检出和同时检出Pg及Fn组之间有统计学差异 (P <0 .0 1)。 结论 :细菌是调节PISF中sICAM -1表达水平的重要因素 ,Fn可能是上升调节的作用 ,Pg是一种下降调节的作用     

常用抗菌药物对牙龈卟啉单胞菌体外抗菌活性的影响

目的:探讨牙周炎主要病原菌牙龈卟啉单胞菌(Pg)对常用抗菌药物的敏感性。方法:从成人牙周炎患者龈下菌斑中分离、通过PCR鉴定106株牙龈卟啉单胞菌的临床分离株,通过Kirby-Bauer纸片扩散法测定该菌对克林霉素、复方磺胺甲唑、四环素、头孢唑啉、乙酰螺旋霉素、头孢氨苄和青霉素G的敏感性。结果:Pg对上述药物敏感率分别是9314%、7813%、6014%、5616%、4016%、1014%和0。结论:抗牙龈卟啉单胞菌药物以其作用大小依次为克林霉素、复方磺胺甲唑、四环素、头孢唑啉、乙酰螺旋霉素和头孢氨苄,该菌对青霉素G不敏感。