牙髓紫卟啉杆菌血清学特性分析

牙髓类杆菌曾是重要的产黑色素类杆菌群菌株,最近重新命名为牙髓紫卟啉杆菌,与牙髓尖周感染和牙源性脓肿有特殊关系。本文用牙髓紫卟啉杆菌ATCC 35406国际标准菌株免疫Balb/c小鼠制得免疫血清,ELISA法检测该抗血清的特异性。发现与中间型类杆菌、产黑色素类杆菌、赖氏类杆菌、躯体类杆菌、牙类杆菌、牙龈紫卟啉杆菌、脆弱类杆菌、黄褐二氧化碳嗜纤维菌、生痰二氧化碳嗜纤维菌、衣氏放线菌反应均阴性,而同不解糖紫卟啉杆菌呈现弱阳性反应,说明矛髓紫卟啉杆菌与不解糖紫卟啉杆菌具有某种相同抗原结构,存在血清交叉反应,血清学关系较近。牙髓紫卟啉杆茵多克隆抗体直接用于临床该菌的检出与鉴定,尚存一定缺陷。     

产黑色素拟杆菌群菌株的培养,鉴定及其免疫血清的特异性检测

本文报告了6种产黑色素拟杆菌国际标准菌株、2株参考菌株的培养、鉴定特点。厌氧菌药敏试验发现中间型拟杆菌、牙龈拟杆菌、产黑色素拟杆菌对头孢霉素、利福平、灭滴灵、青霉素、洁霉素、氨苄青霉素、羧苄青霉索敏感,可供临床治疗中参考。对中间型拟杆菌、牙龈拟杆菌、产黑色素拟杆菌、不解糖拟杆菌,牙髓拟杆菌制备的免疫血清进行了特异性鉴定,发现牙龈拟杆菌免疫血清特异性强,可用于临床辅助鉴定该菌,其它免疫血清均具有不同程度的交叉反应。     

血液链球菌对牙周可疑致病菌的拮抗作用

本实验观察了血液链球菌对与牙周组织破坏关系密切的５种细菌的拮抗作用,包括：放线共生放线杆菌、牙龈类杆菌１中间类杆菌,产黑色素类杆菌、具核梭杆菌、二氧化碳噬纤维菌和放线菌。结果表明,在体外,血液链球菌除对粘性放线菌无拮抗作用外,对所有参试的这几种牙周可疑致病菌均有拮抗作用。拮抗物质存在于血液链球菌生活的细胞内。     

儿童乳牙根管感染的细菌学分析

对18例3～8岁儿童乳牙的根管感染以无菌技术进行定量取样,按种于12种选择性培养基和2种非选择性培养基上,进行需氧、微需氧和厌氧培养,并对细菌菌落计数。对牙髓拟杆菌和牙龈拟杆菌作半定量免疫荧光染色计数;并对其中9例病牙进行了菌相分析。检出的所有细菌中,厌氧菌占绝对优势;其中检出率较高的菌为:产黑色素拟杆菌属,厌氧革兰氏阳性球菌,微需氧革兰氏阳性球菌等.本试验证明,儿童乳牙根管感染是以厌氧菌为主的混合感染,其中以产黑色素拟杆菌属等最常见.     

钢丝结扎固定外伤牙对牙周组织及龈下微生物丛的影响

牙外伤是口腔科的常见病之一,钢丝结扎固定外牙是临床上最常用的方法本研究观察了钢丝结扎固定前、后的同一部位牙周组织及龈沟内产黑色素类杆菌群的变化情况。结果显示：牙龈指数及龈袋深度均有显著性差异;与牙周组织病关系最为密切的致病菌－产黑色素类杆菌群的检出率明显提高,提示临床应对结扎患者进行了正确的口腔卫生指导,从而进行特异性的预防。     

牙髓拟杆菌的分离,培养,鉴定

牙髓拟杆菌是产黑色素拟杆菌群中的一个新菌种。本试验通过对牙髓腔感染标本中分离、培养、鉴定各种细菌,发现牙髓拟杆菌主要分布在牙髓腔内,其存在与根尖周炎有无症状密切相关,此菌对氧敏感,用常规鉴定及气相色谱法与其它菌无法区别,而间接免疫荧光法是确定牙髓拟杆菌菌种和直接检测牙髓腔标本中有无此菌的一个快速、可靠的方法。     

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。     

厌氧产黑色素杆菌群超微结构观察

厌氧产黑色素杆菌群在人口腔内的各处感染中起着重要作用，本文利用电子显微镜对八种产黑色素杆菌做了动态的超微结构观察，以进一步揭示菌体结构与其毒性的关系，为口腔感染疾患的细菌病因学机制提供超微形态学的理论依据。     

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     

牙髓类杆菌的分离、培养、鉴定

试验目的是为寻找口腔内产黑色素类杆菌群中新的菌种一牙髓类杆菌(Bacteroides·endodontalis),试验选择牙髓腔感染的成年患者80例,儿童患者(14岁以下)18例,在厌氧环境下用光滑髓针加棉捻方法收集标本,经厌氧培养,对在CDC琼脂培养基(含0.01％Vitk_3及0.05％氯化血红素)上生长的产黑色素的菌落进行生化,直接血凝试验、气相色谱分析、间接荧光免疫(国际参考株     

Characterization of Bacteroides gingivalis by direct fluorescent antibody staining and cellular fatty acid profiles

Bacteroides gingivalis is a newly proposed species which includes strains isolated from the mouth. Thirteen strains of B. gingivalis isolated from three geographic locations in the United States and France were examined with direct fluorescent antibody staining and analysis of total cellular fatty acids and compared with 16 strains of B. asaccharolyticus of nonoral origin by the same methods. Bacteroides gingivalis strains reacted with the B. gingivalis conjugate (fluorescein isothiocyanate labeled antibody reagent) only, while the B, asaccharolyticus strains reacted with the B. asaccharolyticus conjugate only. The B. gingivalis strains showed negative fluorescence with fluorescein isothiocyanate conjugates for other black-pigmented Bacteroides species. The specificity of the B. gingivalis conjugate was demonstrated by its failure to stain 88 strains of aerobic and anaerobic bacteria other than B. gingivalis. The fatty acid profiles of B. gingivalis and B. asaccharolyticus were readily distinguishable. The B. gingivalis profile was also distinguishable from those of other pigmenting Bacteroides species on the basis of concentration ratios among the characteristic components. These results support the species separation of B. gingivalis and B. asaccharolyticus. Further, they indicate the usefulness of cellular fatty acid profiles as an adjunct to the use of specific fluorescent antibody conjugates for identification of Bacteroides species.     

Molecular size variation of the hemagglutinating adhesin HA-Ag2, a common antigen of Bacteroides gingivalis

The array of Bacteroides gingivalis W83 antigens revealed by crossed immunoelectrophoresis includes one antigen that is associated with an erythrocyte-binding capacity, termed the hemagglutinating adhesin HA-Ag2. This antigen was excised from crossed-immunoelectrophoresis plates to produce two polyclonal antisera, VL 011 and WL 303, whose restricted specificity for HA-Ag2 was assessed using crossed immunoelectrophoresis, crossed immunoelectrophoresis with an intermediate gel, and crossed immunoaffinoelectrophoresis. Both antisera, when used to probe blots of an EDTA cell surface extract of B. gingivalis W83, reacted with two bands, at 33 and 38 kDa, which were also detected by a monoclonal antibody (Naito et al. 1985. Infect. Immun. 50: 231-235), specific for a hemagglutinin of B. gingivalis. Antiserum WL 303 was used to examined by immunoblotting the distribution of HA-Ag2 among a variety of human and animal strains of B. gingivalis. All human strains tested showed two major bands at 33 and 38 kDa in the EDTA cell surface extract, and at 43 and 49 kDa in outer membrane preparations. Only one band, at 29 kDa, was detected in EDTA cell surface extracts from the animal strains, while the outer membrane preparation of a single strain showed a positive reaction. We concluded that HA-Ag2 is an antigen common to human and animal strains of B. gingivalis and that its subunits may show heterogeneity in apparent molecular mass.     

Reduction of vitamin K concentration by salivary Bifidobacterium strains and their possible nutritional competition with Porphyromonas gingivalis

AIMS: To assess the possibility that bifidobacteria compete with Porphyromonas gingivalis for their mutual growth factor vitamin K. This study also examined whether salivary Bifidobacterium species decrease vitamin K concentration in the growth medium. METHODS AND RESULTS: Sixty-five strains of Bifidobacterium were obtained from 20 of 24 periodontally healthy subjects. Bifidobacterium dentium was most frequently detected in the saliva of subjects, followed by Bifidobacterium adolescentis, Bifidobacterium longum, and Bifidobacterium urinalis. The growth of most Bifidobacterium isolates, except that of B. urinalis, was stimulated by vitamin K. Moreover, the isolates were capable of decreasing vitamin K after incubation, which suggests that bifidobacteria compete with P. gingivalis for vitamin K. In a co-culture, a representative strain -B. adolescentis S2-1 - inhibited the growth of P. gingivalis if it was inoculated in the medium before P. gingivalis. CONCLUSIONS: B. adolescentis S2-1 decreased vitamin K concentration and inhibited the growth of P. gingivalis by possibly competing for the growth factor. SIGNIFICANCE AND IMPACT OF THE STUDY: Salivary bifidobacteria may possess the potential to suppress the growth of P. gingivalis by reducing the growth factor(s) in the environment.     

Fatty acid composition and Shwartzman activity of lipopolysaccharides from oral bacteria

The composition and the nature of the linkage of fatty acids and the Shwartzman activity of lipopolysaccharide (LPS) preparations derived from oral gram-negative bacteria including Bacteroides gingivalis, Bacteroides loesheii, Eikenella corrodens, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans were examined. 3-Hydroxylated and nonhydroxy fatty acids of various chain lengths were found in all of the LPS preparations. All nonhydroxy fatty acids were found to be ester-bound, and part of the 3-hydroxy fatty acids in the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans were shown to be involved in ester linkage. It was also suggested that the hydroxy group of the ester-bound 3-hydroxy fatty acid of the LPS of F. nucleatum and A. actinomycetemcomitans is at least partly substituted by another fatty acid, but in the LPS of B. gingivalis and E. corrodens it is not. The main amide-linked fatty acid of the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans was 3-hydroxyheptadecanoic, 3-hydroxydodecanoic, 3-hydroxyhexadecanoic, and 3-hydroxytetradecanoic acid, respectively. The results of the Shwartzman assay showed that the E. corrodens LPS was the most active among the preparations tested, and that the Shwartzman toxicity of Bacteroides LPS is extremely low.     

Diagnostic value of a coagglutination procedure using monoclonal antibodies to Bacteroides gingivalis

A specific monoclonal antibody against Bacteroides gingivalis was bound to particles coated with protein A and evaluated for use in a coagglutination test. B. gingivalis was the only organism tested which gave a specific positive reaction with the CoA reagent. Subgingival plaque samples were collected from 217 patients diagnosed as having periodontitis. Organisms that gave biochemical reactions which indicated they were B. gingivalis were isolated from eleven of the 217 gingival pockets. These eleven strains were the only organisms which gave a positive reaction using the CoA test.     

Detection of fimbriae and fimbrial antigens on the oral anaerobe Bacteroides gingivalis by negative staining and serological methods

We screened 63 clinical isolates of Bacteroides gingivalis from eight different laboratories for the presence of fimbriae by negative staining and by immunological methods. Techniques used were bacterial agglutination, Ouchterlony immunodiffusion and Western immunoblotting analysis using rabbit anti-fimbriae and anti-fimbrilin sera raised against fimbriae and fimbrilin (a constituent protein of B. gingivalis fimbriae) from B. gingivalis strain 381. In 49 of the 51 strains tested, fimbriae were clearly detected by negative staining, and 30 (60%) of the fimbriate strains were positive in all three of the immunological assays. A total of 37 strains (75%) were positive by immunoblotting analysis, which was the most reliable of the serological methods used in this study. The study shows that the majority of B. gingivalis strains are fimbriate, and that these fimbriae are immunologically related to the fimbriae of B. gingivalis strain 381. Molecular heterogeneity of fimbrilin was discovered by the immunoblotting analysis, when different strains were compared. With most of the strains, including strain 381, the antifimbrilin serum reacted with a protein of apparent molecular mass 43 kDa, but with 15 strains the immuno-reactive protein had an apparent molecular mass of 46 kDa.     

Studies of mixed anaerobic infections involving Bacteroides gingivalis

The infectiousness of several combinations of bacteria containing Bacteroides gingivalis and other bacteria associated with periodontal diseases was evaluated by subcutaneous injection to guinea pigs. Among the seven mixtures studied, only one permitted the development of an infection easily transmissible to a second guinea pig; this bacterial mixture was composed of B. gingivalis, Fusobacterium nucleatum, Eubacterium saburreum, and Capnocytophaga ochracea (formerly Bacteroides ochraceus). Owing to its ability to synthesize many lytic enzymes and potentially cytotoxic products, B. gingivalis represented the most virulent species of the mixture. Results from in vitro studies suggest that the development of B. gingivalis in the infected animal depends on the growth of C. ochracea. Succinic acid produced in large amount by C. ochracea seems to be one of the growth factors used by B. gingivalis.     

Purification and immunochemical characterization of a recombinant outer membrane protein from Bacteroides gingivalis.

1. Bacteroides gingivalis is thought to be one of the most virulent microorganisms in relation to adult periodontitis. A gene clone, MD125, is an Escherichia coli host which produces an outer membrane protein of B. gingivalis. 2. The recombinant outer membrane protein (rOMP) was purified to homogeneity from cell sonicate of MD125 by four chromatographic steps. The molecular weight of the purified rOMP was estimated to be approximately 40 kDa. 3. Immunodiffusion analysis showed that antiserum against whole cells of B. gingivalis reacted not only with B. gingivalis cells but also with other Bacteroides cells. Antiserum against the purified recombinant protein reacted with cells of B. gingivalis, whereas this antiserum did not react with all of the other Bacteroides species tested. 4. These data suggest that the rOMP may be a B. gingivalis-specific antigen and that the purified rOMP will be useful material for serodiagnosis and for the development of a vaccine against B. gingivalis infection.     

Chemical, immunobiological and antigenic characterizations of lipopolysaccharides from Bacteroides gingivalis strains.

Lipopolysaccharides (LPS) were extracted from whole cells of seven strains of Bacteroides gingivalis--381, ATCC 33277, BH18/10, OMZ314, OMZ406, 6/26 and HW24D-1--by the phenol/water procedure, and purified by treatment with nuclease and by repeated ultracentrifugation. These LPS were composed of hexoses, hexosamines, fatty acids, phosphorus and phosphorylated 2-keto-3-deoxyoctonate (KDO). The major components of the lipid portion of these LPS were hexadecanoic, 3-hydroxyhexadecanoic, branched 3-hydroxypentadecanoic and branched 3-hydroxyheptadecanoic acids. All the LPS preparations induced marked mitogenic and in vitro polyclonal B cell activation responses in spleen cells from both C3H/HeN and C3H/HeJ mice, exhibited no definitive preparatory activity in the local Shwartzman reaction in rabbits, but were active in the chromogenic Limulus amoebocyte lysate test. A monoclonal antibody (mAb) raised against the LPS from B. gingivalis strain 6/26 reacted with LPS from all other B. gingivalis strains tested. Other mAbs raised against LPS from B. gingivalis strains 381 and 6/26 reacted with the LPS from strains 381, ATCC 33277, BH18/10 and 6/26 (these strains were termed LPS serogroup I), as revealed by ELISA and immunodiffusion. The LPS from these strains except for 6/26 showed almost identical patterns in SDS-PAGE stained with ammoniacal silver. A mAb raised against the LPS from B. gingivalis HW24D-1 reacted with the LPS from strains OMZ314, HW24D-1 and OMZ409 (LPS serogroup II). These LPS, except OMZ409, exhibited very similar profiles in SDS-PAGE. These results indicate that there are at least two different antigenic groups present among LPS from B. gingivalis strains, as well as a common, species-specific antigen.     

Proteolytic activity of black-pigmented Bacteroides strains

Abstract The proteolytic activity of several black-pigmented Bacteroides species was measured. Bacteroides gingivalis was the only species having collagenolytic activity. General proteolytic activity on gelatin and Azocoll was shown in cultures of B. gingivalis, B. asaccharolyticus, B. endodontalis, B. intermedius, B. corporis and to a lesser extent B. melaninogenicus; B. loescheii did not show proteolytic activity. When culture filtrates were tested, B. gingivalis showed high cell free proteolytic activity, whereas the other species had only very weak cell free activity. Growth curves of B. gingivalis revealed two distinct proteolytic activities; general proteolytic activity was found during the logarithmic growth phase, whereas a second peak containing high collagenolytic activity was found after prolonged incubation of cells showing autolysis.