Leukotoxic effects of Actinobacillus actinomycetemcomitans

A sonic extract of Actinobarillus actinomycetemcomitans Y4, a microorganism originally isolated from a patient with juvenile periodontitis, contains a leukotoxin (Aa-leukotoxin) which specifically kills human polymorphonuclear leukocytes (PMNs) and monocytes. In the presence of normal human sera, the toxicity of the leukotoxin is enhanced, whereas sera from patients with juvenile periodontitis neutralize leukotoxic activity. In juvenile penodontitis serum immunoglobulin G was identified as the specific inhibitor of the Aaleukotoxin. The leukotoxin enhancing factor(s) in normal human serum appeared to be a large molecular weight protein which was not immunoglobulin. Pooled sera from rabbits immunized with A. actinomycetemcomitans Y4 sonic extract also neutralized Aaleukotoxic activity while normal rabbit serum enhanced toxicity. The presence of antileukotoxin antibody in sera from individuals with juvenile periodontilis suggests that these people have been immunized with A. actittotnyCetemeomitans microorganisms. Monitoring of this antibody may be a convenient tool in the study of the etiology of juvenile periodontitis.     

Actinobacillus actinomycetemcomitans fimbriae

Freshly isolated Actinobacillus actinomycetemcomitans strains were found to possess fimbriae. These appendages appeared to be irreversibly lost after 30 to 40 subcultures in the laboratory. The fimbriated strains were associated with a specific type of colonial morphology designated SP (Star Positive); the non-fimbriated variant colony was designated SN (Star Negative). The fimbriae were approximately 5 nm wide and could be several μm in length. The fimbriated cells tended to aggregate readily whereas the non-fimbriated cells formed uniform suspensions. Three stains of fimbriated A. actinomycetemcomitans isolated from different patients were compared to their non-fimbriated variants for attachment to hydroxyapatite (HA) and saliva coated hydroxyapatite (SHA). Two of the fimbriated variants exhibited a 3- to 4-fold enhancement of attachment to HA and SHA over their non-fimbriated variant. However, there did not appear to be any specificity for SHA over HA. One strain (Pag) showed no major differences in attachment of the fimbriated or non-fimbriated variant, albeit the fimbriae were indistinguishable from those of the other A. actinomycetemcomitans strains. Thus, there may be molecular differences among fimbriae of various A. actinomycetemcomitans strains which determine both their tissue specificity and their affinity for particular substrates.     

Fimbriation of Actinobacillus actinomycetemcomitans

Presence of fimbriae on a bacterium may promote its initial colonization, survival and ability of tissue penetration. Thus, electron microscopy was performed on negatively stained periodontal Actinobacullus actinomycetemcomitans from a patient with Papillon-Lefevre syndrome. The isolates were, in contrast to reference strains found to carry a considerable amount of fimbriae (5 nm in diameter). The presence of fimbriae may support the hypothesis of the invasive ability of this bacterium.     

Intrafamilial transmission of Actinobacillus actinomycetemcomitans

Cross-sectional studies have shown that Actinobacillus actinomycetemcomitans is a frequent member of the oral flora in children with primary teeth. The purpose of the present study was to obtain information of the transmission of A. actinomycetemcomitans. Three types of families were studied for the prevalence and serotype distribution of A. actinomycetemcomitans. First, families whose periodontally healthy child member harbored A. actinomycetemcomitans; second, families of periodontally healthy children who did not harbor A. actinomycetemcomitans; third, families whose adult member harbored A. actinomycetemcomitans and had been referred to treatment for severe periodontitis. As a whole the study included 23 families. All the family members were invited for the examination. The final study population consisted of 38 children and 32 adults. The results showed that when a child was positive for A. actinomycetemcomitans, either the mother or the father was also positive with one exception, whereas when the adult member harbored A. actinomycetemcomitans, the children were infected in only 2 of the 9 families. Using immunodiffusion technique it was found that the child always harbored the same serotype of A. actinomycetemcomitans as the parent. In conclusion, the results suggest the intrafamilial transmittance of A. actinomycetemcomitans.     

伴放线放线杆菌粘附特性研究

目的分析伴放线放线杆菌的粘附特性及菌毛结构基因tip-1的遗传多样性对菌株粘附活动的影响。方法检测不同孵育条件下5种tip-1基因型临床分离菌株和光滑型菌株的粘附活动。结果临床分离菌株的粘附量随菌液浓度,孵育时间的增加而增加。tip-1基因型Ⅱ型菌株的粘附量高于其它4型菌株,光滑型菌株的粘附量低于临床分离菌株。生理温度下菌株粘附数高,低温下明显降低。厌氧条件和有氧条件下的粘附量无显著性差异。结论伴放线放线杆菌临床分离菌株的粘附存在时间和菌量依赖性,并要求一定新陈代谢活性,粘附效率在氧浓度改变时没有明显变化。伴放线放线杆菌表型影响菌株的粘附作用。不同tip-1基因型菌株粘附能力存在差异,Ⅱ型菌株粘附能力最强。     

Inhibitory effects of rabbit antisera on mitogenic activity of Actinobacillus actinomycetemcomitans lipopolysaccharide

Lipopolysaccharides (LPSs) were isolated from Actinobacillus actinomycetemcomitans strains ATCC 29523 (serotype a), Y4 (b), and NCTC 9710 (c) by the hot phenol-water procedure. Y4 lipid A was obtained by the hydrolysis of Y4 LPS in 1% acetic acid. All the LPS preparations and Y4 lipid A were mitogenic for C3H/HeN mouse spleen cells, but not for C3H/HeJ mouse spleen cells. Immunoglobulin preparations partially purified by ammonium sulfate precipitation at 33% saturation from rabbit antisera against Y4 whole cells inhibited the mitogenic response of C3H/HeN mouse spleen cells to LPSs from all the strains of A. actinomycetemcomitans and Y4 lipid A. Anti-Y4 LPS immunoglobulin preparation inhibited the mitogenic activity of Y4 LPS and Y4 lipid A. Furthermore, anti-Y4 whole cell Fab fragments inhibited the mitogenic activity of both Y4 LPS and Y4 lipid A. These results suggest that antibodies against A. actinomycetemcomitans LPS may modify immune responses of lymphocytes to this organism at periodontal sites.     

放线共生放线杆菌的血清型分布

为了解放线共生放线杆菌（Ａｃｔｉｎｏｂａｃｉｌｕｓａｃｔｉｎｏｍｙｃｅｔｅｍｃｏｍｉｔａｎｓ，Ａａ）不同血清型的分布，作者应用Ａａ菌种及ｂ、ｃ血清型抗体对来自２８人３２个龈下菌斑标本的１３１株Ａａ进行了血清分型。结果：每人只检出一种血清型，未发现复合血清型；２８人中的１９人为血清型ｃ，占６８％；９名青少年牙周炎患者７名为血清型ｃ，２名牙周健康者均为血清型ｂ。研究表明Ａａ不同血清型分布以ｃ型为优势血清型；血清型的分布可能存在地域或种族差异。     

The bactericidal effects of dental ultrasound on Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis

Abstract This study investigated the possible bactericidal acoustic effects of the dental ultrasonic sealer. Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis suspensions, were subjected to the vibrations of a Cavitron PI insert for 2.5 and 5.0 rain in an acoustically-simulated pocket model and the survivors enumerated. The extent of any cavitation occurring within the pocket model to which the statistically significant bactericidal activity observed might be attributed, was determined by ‘sonoluminescence’, which was then investigated by photomultiplication techniques. However, these failed to detect any sonoluminescence within the pocket space and moreover, the necessary deflection of the water coolant away from the insert tip. to avoid flooding of the experimental pocket, proved to result in temperatures of 47.6®C and 52.3®C at the respective time intervals, and thereby constituted an alternative possible bactericidal mechanism. Examination of the effects of such temperature changes on the target bacteria then revealed statistically significant differences in the viable counts of both microorganisms after 5.0-min periods, and as such were comparable to those previously detected in relation to the pocket model. Whilst it must be presumed that the bacteriolytic effect observed in the main investigation was due to the incidental temperature changes, in the absence of acoustic cavitation the influence of any associated acoustic microstreaming cannot be discounted. Further investigations to assess the bactericidal potential of acoustic phenomena using a modified experimental to exclude any hyperthermic effects are therefore necessary.     

Lactoferrin Interaction with Actinobacillus actinomycetemcomitans

The interaction of lactoferrin with Actinobacillus actinomycetemcomitans was examined in a ¹²⁵I-labeled protein binding assay. The binding of human and bovine lactoferrins reached maximum within 1 h. Lactoferrin binding to the bacterium was pH-dependent and reversible. Scatchard analysis indicated the existence of two different types of binding sites on the bacterium, one with a high affinity constant k_α=8.8×10⁻⁷ M) and the other with a low one (k_α=1.8×10⁻⁶ M). Bacteria in the exponential phase of growth showed higher binding than cells in the stationary phase. Bacteria grown in medium containing serum and/or lysed erythrocytes bound lactoferrin to a lesser extent. Heat-inactivated serum, lysed erythrocytes and other proteins such as mucin and laminin inhibited lactoferrin binding to A. actinomycetemcomitans in a competitive binding assay. Sodium dodecyl sulfate polyacrylamidegel electrophoresis and Western blot analysis of the cell envelope as well as the outer membrane of A. actinomycetemcomitans revealed lactoferrin-reactive protein bands at 29 kDa and 16.5 kDa. The 29-kDa band displayed a heat-modifiable lactoferrin-reactive form with a molecular weight of 34 kDa. Neither proteinase K-treated cell envelope nor lipopolysaccharide of this bacterium showed reactivity with lactoferrin. These data suggests a specific interaction of lactoferrin with outer membrane proteins of A. actinomycetemcomitans .     

Environmental influences on Actinobacillus actinomycetemcomitans biofilm formation

Fresh clinical isolates of the periodontal pathogen Actinobacillus actinomycetemcomitans have an adherent, rough colony morphology that transforms into a minimally adherent, smooth colony phenotype during successive in vitro passage. The objectives of this study were: (1) to compare biofilm formation of the rough (RVs) and smooth variants (SVs) of several strains of A. actinomycetemcomitans grown under various environmental conditions and (2) to examine the dynamics of biofilm formation. A microtitre plate biofilm assay was used to evaluate biofilm formation of strains grown in broth with modified salt concentration and pH, and to evaluate the effect of pre-conditioning films. Scanning electron microscopy (SEM) was used to monitor microscopic changes in morphology. Dynamics of biofilm formation were measured in a flowcell monitored by confocal microscopy. The RVs generally produced greater biofilm than the SVs. However, medium-dependent differences in biofilm formation were evident for some rough/smooth pairs. The RVs were more tolerant to changes in salt and pH, and more resistant to chlorhexidine than the SVs. Horse serum virtually eliminated, and saliva significantly reduced, biofilm formation by the SVs in contrast to the RVs. SEM revealed no alteration in morphology with change of environment. In a flowcell, the RVs produced towers of microcolonies anchored by a small contact area, whereas the SVs produced an open architecture of reduced height. After 7 days in a flowcell, the rough to smooth phenotype transition could be demonstrated. In conclusion, strain, growth medium and conditioning film all affect biofilm formation. The RVs produce biofilms of unique architecture that may serve to protect the bacterium from environmental perturbations.     

Killing of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus by human polymorphonuclear leukocytes in serum and saliva

The ability of polymorphonuclear leukocytes from human peripheral blood to kill Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus was examined with fresh isolates and laboratory strains from each species (5 strains within each group) under different conditions. Bacterial cells were mixed with a polymorphonuclear leukocyte suspension in the presence of either active serum or heat-inactivated serum or active serum together with sterile-filtered saliva. Surviving bacteria were determined by counting the number of bacterial colony-forming units in the mixtures after a 60-min incubation at 37°C. Mixtures without polymorphonuclear leukocytes served as controls for the evaluation of the degree of killing of the bacteria. In general, A. actinomycetemcomitans resisted phagocytic killing to a greater extent than H. aphrophilus, and the killing of the former species mainly depended on the presence of heat-labile serum components, probably complement factors. Laboratory strains of A. actinomycetemcomitans were more easily killed than fresh isolates. The presence of saliva in the reaction mixtures decreased the degree of killing. However, strain-dependent variations in the killing were found under either condition. The leukotoxic activity of A. actinomycetemcomitans strains, determined by a [⁵¹Cr]-release assay, was not correlated with the resistance of these strains to the phagocytic killing. The results point out a strain-dependent difference in the ability of A. actinomycetemcomitans to evade the inflammatory response associated with polymorphonuclear leukocytes. This difference may constitute a potential virulence factor for this periodontopathogen. Furthermore, the leukotoxicity of the strains is not the main determinant that modifies the interaction of A. actinomycetemcomitans with human neutrophils.     

伴放线放线杆菌菌落生长形态变化的观察

目的：观察伴放线放线杆菌（Actinobacillus actinomycetemcomitans,Aa)从粗糙型到光滑型的转变过程，认别Aa在实验室传代过程中出现的不同生长形态。方法：从牙周炎患者龈下菌班中分离出的原代菌株8株，应用固体及液体培养基连续传代，液体培养每次传代的同时接种固体培养基观察相应的菌落形态。结果：液体培养获得3株光滑型转变株。菌落的变化从粘附的小菌落到沉淀的大菌落到完全的均匀生长，转化过程大约需要7－8代。在这一过程中相应传至固体培养基上生长的Aa从粘附的半透明的小菌落变大、不透明并失去粘附的特性，又随着边缘的扩散变为扁平，透明度也增加；与此同进内部的星形结构逐渐变简单、变小，最后消失。固体培养未获得典型的转变株。结论：Aa从粗糙型到光滑型的转变是一个菌落湿度逐渐增加，体积逐渐增大，并逐渐失去内部结构的过程。这一过程至少可以看到半透明突起的粗糙型，不透明突起的光滑型和近乎透明的扁平光滑型3种菌落形态。     

臭氧水对伴放线放线杆菌的灭活效果观察

目的探讨臭氧水对牙周可疑致病菌伴放线放线杆菌（Aa）的灭活效果。方法采用悬液定量杀菌方法和和化学方法在实验室进行观察。用4、8、15mg／L的臭氧水分别对悬液中A。作用1、2、3min。结果当臭氧水浓度为4mg／L对悬液中An没有杀灭作用。当臭氧水浓度为8mg儿时,对悬液中An作用1min,杀灭率为57％,当浓度上升至15mg／L时,对悬液中Aa作用1min．杀灭率上升至98％．而延长杀菌时间至3min．杀菌率维持在97％～99％。结论在25℃的室温条件下．臭氧水浓度达到15mg／L．臭氧水温控制在15℃～18℃时对悬液中Aa有快速、有效的杀灭作用。     

抗伴放线放线杆菌IgY的制备及抑菌试验研究

目的:观察伴放线放线杆菌诱导母鸡产生特异性IgY抗体情况,以及其抑制伴放线放线杆菌(A.a)和牙龈二氧化碳噬纤维菌(C.g)生长效果。方法:应用免疫接种法、水稀释法、盐析法、液体培养抑菌法、以及ELISA法,诱导、提取和纯化IgY抗体,取一定量抗体与细菌共同培养,测定抑制伴放线放线杆菌和牙龈二氧化碳噬纤维菌生长效果。结果:两步硫酸铵盐析沉淀的IgY抗体纯度达85.6%～90.3%;抗原结合效价为1∶32000;抗伴放线放线杆菌IgY抗体与牙龈二氧化碳噬纤维菌交叉免疫反应的抗原结合效价为1∶8000;当抗伴放线放线杆菌IgY抗体浓度在5.0、1.0、0.1g/L时,细菌浓度在5×108CFU/L培养24h其抑菌率分别为31.60%(P=0.004)、10.24%(P=0.024)、-3.30%,培养72h其抑菌率分别为64.20%(P=0.004)、53.21%(P=0.002)、11.20%。细菌浓度在1×108CFU/L培养24h其抑菌率分别为35.71%(P=0.004)、30.95%(P=0.012)、11.11%,培养72h其抑菌率分别为65.11%(P=0.005)、54.04%(P=0.002)、16.17%;5.0g/L的抗伴放线放线杆菌IgY与1×108CFU/L牙龈二氧化碳噬纤维菌培养24h其抑菌率为41.61%(P=0.005),培养72h抑菌率为86.99%(P=0.014)。结论:伴放线放线杆菌能够诱导母鸡产生高效价的特异性IgY抗体,该抗体在一定的浓度内有抑制伴放线放线杆菌和牙龈二氧化碳噬纤维菌生长的作用;伴放线放线杆菌与牙龈二氧化碳噬纤维菌存在着共同抗原。     

Actinobacillus actinomycetemcomitans in human periodontal disease

Recent evidence implicates Actinobacillus actinomycetemcomitans in the etiology of localized juvenile periodontitis. This paper reviews the morphological, biochemical and serological charcteristics of A. actinomycetemcomitans, evidence incriminating it as a periodontopathogen, its importance in human nonoral infections, and virulence factors which may be involved in the pathogenesis of A. actinomycetemcomitans infections. A. actinomycetemcomitans is a non-motile, gram-negative, capnophilic, fermentative coccobacillus which closely resembles several Haemophilus species but which does not require X or V growth factors. The organism has been categorized into 10 biotypes based on the variable fermentation of dextrin, maltose, mannitol, and xylose and into 3 serotypes on the basis of heat stable, cell surface antigens. A. actinomycetemcomitans' primary human ecologic niche is the oral cavity. It is found in dental plaque, in periodontal pockets, and buccal mucosa in up to 36% of the normal population. The organism can apparently seed from these sites to cause severe infections throughout the human body such as brain abscesses and endocarditis. There is a large body of evidence which implicates A. actinomycetemcomitans as an important micro-organism in the etiology of localized juvenile periodontitis including: (1) an increased prevalence of the organism in almost all localized juvenile periodontitis patients and their families compared to other patient groups; (2) the observation that localized juvenile periodontitis patients exhibit elevated antibody levels to A. actinomycetemcomitans in serum, saliva and gingival crevicular fluid; (3) the finding that localized juvenile periodontitis can be successfully treated by eliminating A. actinomycetemcomitans from periodontal pockets; (4) histopathologic investigations showing that A. actinomycetemcomitans invades the gingival connective tissue in localized juvenile periodontitis lesions; (5) the demonstration of several pathogenic products from A. actinomycetemcomitans including factors which may: (a) facilitate its adherence to mucosal surfaces such as capsular polysaccharides; (b) inhibit host defense mechanisms including leukotoxin, a polymorphonuclear leukocyte chemotaxis inhibiting factor, and a lymphocyte suppressing factor (c) cause tissue destruction such as lipopolysaccharide endotoxin, a bone resorption-inducing toxin, acid and alkaline phosphatases, collagenase, a fibroblast inhibiting factor and an epitheliotoxin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Actinobacillus actinomycetemcomitans induces lethal effects on the macrophage-like human cell line U937

We examined the cytotoxicity in culture medium of Actinobacillus actinomycetemcomitans against the human monocyte-macrophage-like cell line U937 using the trypan blue exclusion test and WST-1 test. We found that A. actinomycetemcomitans Y4 showed the highest cytotoxic activity among the three different serotype strains and the cytotoxic effects of both bacterial cells and culture supernatants in A. actinomycetemcomitans Y4 were stronger on phorbol-12-myristate 13-acetate (PMA)-induced U937 cells than uninduced U937 cells. Morphological changes in PMA-induced U937 cells treated with culture supernatants differed from those treated with leukotoxin, and a difference in the susceptibility to 56 degrees C heat treatment was found between culture supernatants and leukotoxin. The cytotoxic activity by WST-1 was determined more rapidly and strongly than that by trypan blue assay. These findings suggested that the cytotoxic effect of A. actinomycetemcomitans was influenced by the differentiation of U937 cells and may be more potent on the respiratory chain than the cell membrane.     

伴放线放线杆菌外膜蛋白的研究进展

伴放线放线杆菌与牙周炎,特别是与局限性侵袭性牙周炎有着密切的关系.伴放线放线杆菌外膜蛋白作为其重要毒力因子,在牙周病的发病中起着重要的作用.本文就近年来有关伴放线放线杆菌外膜蛋白的结构特征、外膜蛋白的表现型、外膜蛋白与血清型、外膜蛋白的致病性等研究进展作一综述.