Outer membrane proteins of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus studied by SDS-PAGE and immunoblotting

This investigation characterized and compared outer membrane proteins (OMP) of the closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus by means of SDS-PAGE patterns and reactions on immunoblots with rabbit antiserum against A. actinomycetemcomitans FDC Y4. Reactions with serum from a patient with Papillon Lefévre syndrome (PLS), from whom periodontal wild strains of A. actinomycetemcomitans had been isolated, were also studied. OMP were purified with selective solubilization from lyophilized cells of 10 wild and 4 reference strains of A. actinomycetemcomitans and 4 reference strains of H. aphrophilus. OMP profiles from wild and reference strains of A. actinomycetemcomitans were similar while those from A. actinomycetemcomitans and H. aphrophilus differed. The most prominent difference was absence of a heat modifiable protein in H. aphrophilus strains. Immunoblotting revealed strong common antigens in most strains, including a heat modifiable protein with mol wt 34 kDa, as well as a 29 kDa and a 16.5 kDa protein. Treatment with pronase and sodium periodate confirmed the protein nature of the major OMP antigens.     

In vivo chemotaxis evoked by Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus

The chemotaxis-evoking capacity of 5 Actinobacillus actinomycetemcomitans and 5 Haemophilus aphrophilus strains were studied in a tissue cage model in rabbits. A significant increase of the total number of polymorphonuclear leukocytes was induced in the tissue cage fluid by both viable and killed bacteria, reaching a maximum after 12-24 h. In parallel, the proportion of viable/non viable leukocytes increased. The leukocyte counts declined during the following 24-48 h in all chambers except in those inoculated with viable cells of H. aphrophilus. The H. aphrophilus strains survived the 72 h experiment while A. actinomycetemcomitans decreased to undetectable levels within 24-72 h. Lactate dehydrogenase and lysozyme activities in cage fluid increased in all but the uninoculated chambers. Viable bacteria induced higher activities of the enzymes than killed ones. It is concluded that both species of bacteria exhibit similar chemotaxis evoking properties. A strain dependent ability to induce release of leukocyte-associated enzymes exists.     

Polymorphonuclear leukocyte chemiluminescence induced by Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus in serum and saliva

The ability of different strains of Actinobacillus actinomycetemcomitans (A.a .) and Haemophilus aphrophilus (H.a .) to trigger activation of an oxidative burst in human polymorphonuclear leukocytes (PMNL) was examined by measuring the luminol-amplified light emission - chemiluminescence (CL) - from these cells. Bacterial cells were incubated with PMNL from one healthy subject, in the presence of either active serum, heat-inactivated serum, saliva, or saliva and active serum. In the presence of active serum, all five H.a . strains and two out of five A.a . strains triggered a CL response. The CL induced in the presence of heat-inactivated serum was considerably less than that achieved with fresh serum. In the presence of only saliva, all strains induced considerably weaker CL responses than those induced in the presence of saliva with active serum. In the presence of serum, intracellular reactions appeared to be the main source of CL, while addition of saliva and active serum increased the extracellular CL. The results indicate that strain-dependent differences exist among A.a . strains in their ability to trigger the oxygen-dependent bactericidal mechanisms of human PNML. In contrast, the CL patterns of H.a . strains were equivalent. Various factors in the environment, such as activated complement and salivary compounds, affect the interaction of these species with neutrophils.     

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.     

Sensitivity of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus to oxidative killing

We examined the killing of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus by oxygen metabolites generated by the xanthine-xanthine oxidase (X-XO) system. This system generates a mixture of oxidants, including superoxide radical, hydrogen peroxide, hydroxyl radical, and possibly singlet oxygen. Differential sensitivity to the X-XO system was observed among strains of A. actinomycetemcomitans; notably, 2 catalase-deficient strains and 2 strains representative of serotypes b and c were the most susceptible. H. aphrophilus was not sensitive. The amount of oxidants produced by the X-XO system more closely correlated with killing than the ratio of oxidant production. Cytochrome c, superoxide dismutase, catalase, dimethyl sulfoxide, and desferrioxamine were used to determine the role of superoxide radical, hydrogen peroxide and hydroxyl radical in the bactericidal process. Hydrogen peroxide was the major bactericidal agent against A. actinomycetemcomitans. Superoxide anion participated in killing of A. actinomycetemcomitans to varying but lesser degrees. The intracellular generation of hydroxyl radical was implicated in the killing of several strains. We conclude that (i) strains of A. actinomycetemcomitans are differentially sensitive to the bactericidal effects of the X-XO system and (ii) of the oxidants produced by the X-XO system, hydrogen peroxide is the most bactericidal against A. actinomycetemcomitans.     

Rapid identification of Actinobacillus actinomycetemcomitans based on analysis of 23S ribosomal RNA

Actinobacillus actinomycetemcomitans is a key microorganism in the pathogenesis of several different forms of periodontal diseases. Identification of this bacterium from clinical specimens may often be complicated by the fact that the colony morphology on TSBV selective medium closely resembles that of Haemophilus aphrophilus and a key differentiating characteristic, catalase reaction, may be variable. Recent genetic studies have shown that the 23S ribosomal RNA molecule is split into two smaller forms in A. actinomycetemcomitans, but is intact in H. aphrophilus. Based on this finding, we describe a new, rapid method for identifying A. actinomycetemcomitans in which single colonies isolated from culture on TSBV agar in 5% CO2 in air are lysed, electrophoresed on 1.5% submarine agarose gels and visualized by staining with ethidium bromide. Using this assay, A. actinomycetemcomitans can be easily distinguished from morphologically similar colonies such as H. aphrophilus strains by differences in 23S rRNA within 2 h.     

Cell surface hydrophobicity of Actinobacillus actinomycetemcomitans Y4

Abstract Oral bacteria colonize the dento-gingival tissues in a selective manner. Hydrophobic reactions have been suggested as one of the major mechanisms of adhesion. Hydrophobicity of Actinobacillus actinomycetemcomitans Y4 (Aa) cells was studied in vitro using adherence to the liquid hydrocarbon, octane. Adherence of Aa cells to octane varied from 60–90%, depending on the medium in which they were grown, age of the culture and the buffer in which the assay was carried out. These data suggest that Aa is a hydrophobic bacterium, the hydrophobicity of which is expressed to a varying degree, and may have a role in its adherence to oral tissues.     

The binding of delmopinol and chlorhexidine to Streptococcus mutans and Actinobacillus actinomycetemcomitans strains with varying degrees of surface hydrophobicity

This study evaluated the binding of chlorhexidine and the new surface-active anti-plaque agent delmopinol hydrochloride to Streptococcus mutans and Actinobacillus actinomycetemcomitans cells with various cell surface hydrophobicities. The influence of saliva concentration on the binding of these compounds was also investigated. The radiolabeled compounds were incubated with bacteria and the cells were recovered using a centrifugal filtering technique. Delmopinol had higher binding to the hydrophilic variant strains than to the hydrophobic parent strains; chlorhexidine had higher binding to hydrophobic than to hydrophilic A. actinomycetemcomitans strains and higher binding to hydrophilic than to hydrophobic S. mutans strains. The presence of salivary films decreased the binding of both compounds. Both delmopinol and chlorhexidine had stronger affinity to A. actinomycetemcomitans cells than to S. mutans cells. At equimolar concentrations, delmopinol had a lower binding to all strains tested than chlorhexidine. The high reversibility of the delmopinol binding might be related to a higher diffusion rate and solubility compared with that of chlorhexidine. The amphiphilicity of both molecules is an important feature in their retention to S. mutans and A. actinomycetemcomitans strains of varying hydrophobicities and could play an important role in the substantivity of delmopinol or chlorhexidine in the oral cavity.     

Electron microscopy of phages in serotypes of Actinobacillus actinomycetemcomitans

Actinobacillus actinomyetemcomitans, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzas, Haemophilus parainfluenzae, Pasteurella haemolytica and Pasteurella multocida strains were examined by transmission electron microscopy for the presence of bacteriophages. Phages were detected in serotype a (SUNY 75) and e (UOH 1705) and in the fresh clinical isolates UOH Q1243 and UOH Q1247 of A. actinomycetemcomitans. Phages were not found in serotype b, c and d strains of A. actinomycetemcomitans , in the fresh clinical isolate UOH Q1244 of this species or in old strains (including reference strains) of related species from the Actinobacillus-Haemophilus-Pasteurella group.     

伴放线放线杆菌形态变化对菌体表面疏水性影响的研究

目的研究伴放线放线杆菌形态变化对菌体表面疏水性的影响。方法采用碳氢化合物法检测伴放线放线杆菌粗糙型和光滑型的菌体表面疏水性,观察同一菌株不同表型疏水性的变化。结果伴放线放线杆菌粗糙型和光滑型菌体表面具有疏水性。14株粗糙型伴放线放线杆菌菌体表面疏水率高于4株光滑型,差异有统计学意义（P〈0．05）。4株同源的粗糙型与其光滑型转变株比较得出除1株外,其余3株菌两种表型的菌体表面疏水率差异无统计学意义（P〉0．05）。结论伴放线放线杆菌形态变化可引起菌体表面疏水性的改变,粗糙型转变为光滑型后菌体表面疏水性减弱。     

Specificity and levels of oral and systemic antibodies to Actinobacillus actinomycetemcomitans

Abstract Salivary and gingival crevicular fluid antibodies and systemic antibodies were analysed for levels and specificity against Actinobacillus actinomycetemcomitans components. The major reactivity of salivary and serum IgA1 and IgA2 antibodies to the periodontal pathogen A. actinomycetemcomitans was against bands between 14 and 83 kD for IgA1 and bands between 14 and 68 kD for IgA2 in Western blot. In addition to specific binding, there was also a hitherto unrecognized Fc-mediated binding of IgG antibodies to an A. actinomycetemcomitans component around 50 kD. Serum IgG antibodies to A. actinomycetemcomitans leukotoxin displayed the highest median value and only 1 individual showed salivary IgM antibodies in ELISA. Elevated levels of gingival crevicular fluid lgA2 antibodies indicated a local production of IgA from periodontal tissues. Using synthetic peptides, several distinct epitopes on the leukotoxin were recognized by both salivary and serum IgA antibodies.     

Specific antibodies against Actinobacillus actinomycetemcomitans in serum and saliva of patients with advanced periodontitis

The aim of the present study was to discover any possible correlation between specific antibodies against Actinobacillus actinomycetemcomitans (A.a.) in serum and saliva. The test group consisted of 38 patients aged 31–68 yr (mean 49) with advanced periodontitis. Twenty-nine subjects aged 23–67 yr, without periodontal destruction, formed a control group with a reference level of specific salivary antibodies against A.a. A subgingival plaque sample for culturing A.a. , a specimen of stimulated whole saliva, and a sample of venous blood were taken from each subject of the test group. Specific IgG and IgA antibodies against A.a. were determined from serum and stimulated whole saliva by means of the ELISA test. Fifteen of the patients (39%) had cultivable A.a. Six of the 15 A.a. culture-positive patients and one of the 29 reference subjects exhibited very high antibody titers against A.a. in saliva. Specific IgG and IgA antibodies in saliva correlated highly significantly with the corresponding antibody values in serum among the patients in the test group. It was concluded that among patients with severe adult periodontitis, the less invasive saliva sample has a diagnostic value equal to that of the serum sample concerning specific antibodies against A.a.     

Oral and systemic immunoglobulin G-subclass antibodies to Actinobacillus actinomycetemcomitans leukotoxin

Salivary, gingival crevicular fluid and serum-specific immunoglobulin G (IgG)-subclass antibodies to Actinobacillus actinomycetemcomitans leuktoxin were quantified by enzyme-linked immunosorbent assay. Samples were taken from six patients with periodontal pockets > or = 5 mm, harboring A. actinomycetemcomitans in subgingival plaque and from six healthy, sex- and age-matched controls, who did not harbor A. actinomycetemcomitans. In individuals suffering from periodontitis, the median values of specific IgG1- and IgG2-subclass antibodies in saliva, gingival crevicular fluid and serum were, respectively IgG1 147 ng/ml, 5226 ng/ml and 7318 ng/ml and IgG2 4.8 ng/ml, 934 ng/ml and 860 ng/ml. In the patients, specific IgG3 antibodies were detected in one out of six individuals in saliva, in two individuals in gingival crevicular fluid and in five out of six patients in serum with a median value of 561 ng/ml. The median values of specific IgG4 antibodies in saliva, gingival crevicular fluid and serum were below detectable levels. The median values of the total IgG subclasses in saliva and serum were 14622 ng/ml and 10.3 g/l respectively. Individuals with periodontitis had, compared with controls, a higher ratio of specific IgG1 antibodies to total IgG1 in saliva (P < 0.05) and in serum (P < 0.05) and a higher ratio of specific IgG antibodies to total IgG in saliva (P < 0.05) and in serum (P < 0.01). The results show an elevation of both oral and systemic specific antibodies to A. actinomycetemcomitans leukotoxin.     

Occurrence of Actinobacillus actinomycetemcomitans in patients wearing orthodontic appliances

Abstract The aim of the present study was to assess: (1) the occurrence of Actinobacillus actinomycetemcomitans (Aa) in subgingival plaque from young patients undergoing orthodontic treatment with fixed appliances; (2) a possible relationship between the presence of Aa and the clinical conditions; (3) a relation between the duration of orthodontic treatment and the microbiological and clinical parameters; (4) whether differences exist when taking into consideration the different type of appliances, i.e., bands or brackets. 34 subjects aged between 12 and 20 years participated in the study. Of these, 20 subjects had worn orthodontic appliances (test group), while the remaining 14 subjects served as matched control (control group). 4 to 8 sites in each patient were available for clinical and microbiological examination. Clinical parameters consisted of presence/absence of plaque and gingival bleeding index (GBT), Microbiological sampling was performed in the same sites as in the clinical examination. A statistically significant difference was present when comparing %s of GB1 positive scores between teeth from the test group (57.5%) and teeth from the control group (25%). Plaque was present in 53% of test sites and 37% of control sites, but this difference was not statistically significant. Aa was detected, from at least one site in 85% of test subjects and in 15% of the control subjects (p<0.001). Among the subjects, 41% harboured Aa at a concentration between 0.1% and 1.0%, whereas another 40% yielded Aa at a concentration greater than 1.0%. Finally, a positive correlation was noted between the % of sites positive for Aa and the % of sites displaying a positive GB1 score (r=0.41; p<0.005). No relation was found between the duration of orthodontic treatment and the microbiological or clinical parameters; neither were statistically significant differences found when we compared results from sites wearing bauds or brackets. In conclusion, the present study showed that young subjects wearing orthodontic appliances harbour Aa with a remarkable frequency of detection, although plaque levels do not significantly differ from those of a matched control group.     

Distribution of biotypes and antimicrobial susceptibility of Actinobacillus actinomycetemcomitans

Eighty isolates of Actinobacillus actinomycetemcomitans from 30 Brazilian periodontitis patients were examined to determine the distribution of biotypes and in vitro antimicrobial susceptibility. Seventy-seven percent of the isolates belonged to biotype X. All A. actinomycetemcomitans isolates were susceptible to cefoxitin, imipenem and tetracycline.     

伴放线放线杆菌血清型分析

目的 PCR法检测伴放线放线杆菌（Actinobacillus actinomy＇etemcomitans，Aa）临床分离菌株血清型，分析其与flp-1基因型的关系。方法用血清型特异性引物，通过普通PCR和多重PCR的方法对60株Aa临床分离菌株的血清型进行鉴定，并分析其与flp-1基因型的关系。结果 60株Aa临床分离菌株中血清型c型63，33％，e型23．33％，b型6.67％，a，f型各占3．33％，未检测到d型菌株；在24名被检测者中，15名检测到c型An菌株，3名检测到b型菌株，各有2名分别检测到a、e、f型菌株。fip-1基因型Ⅰ型菌株的血清型均为a型，40株Ⅱ型菌株中38株为c型，Ⅳ型菌株均为b型，11株Ⅴ型菌株中9株为e型，Ⅵ型菌株均为e型。结论 Aa血清型分布以c型为主，fip-1基因型与菌株血清型存在一定对应关系。     

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 and Porphyromonas gingivalis in young Chinese adults

The aim of this study was to determine the presence or absence of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in young Chinese adults and to examine the A. actinomycetemcomitans isolates from positive subjects with regard to the serotype distribution, presence of the leukotoxin gene lktA and the promoter for the leukotoxin operon as well as the incidence of phage Aa phi 23. Sixty subjects, working in a knitting factory in the Province of Guangzhou, People's Republic of China, were investigated. Subgingival microbial samples were taken from both upper first molars. They were cultured both anaerobically and in 5% CO2. P. gingivalis was found in 33 subjects. On average, it constituted 7% of the total anaerobic cultivable counts. A. actinomycetemcomitans was detected in 37 subjects of which seven yielded counts > 10(5). Twenty-one subjects were positive for both organisms. A. actinomycetemcomitans serotype a was found in 9 subjects, serotype c was found in 23 and serotype e in 5. A. actinomycetemcomitans serotypes b and d were not detected in any subjects. Presence of the leukotoxin gene lktA was demonstrated for all A. actinomycetemcomitans isolates; however, none of the A. actinomycetemcomitans strains from the present study had a deletion in the promoter region of the leukotoxin operon. The results of this investigation show a high frequency of the putative periodontal pathogens P. gingivalis and A. actinomycetemcomitans and corroborate the concept that there is variation in virulence and pathogenic potential among isolates from different subjects.     

Actinobacillus actinomycetemcomitans may utilize either actin-dependent or actin-independent mechanisms of invasion

Actinobacillus actinomycetemcomitans is an important pathogen implicated in juvenile and adult periodontal diseases. An important virulence factor of A. actinomycetemcomitans is the ability to invade human oral epithelial cells. A clinical isolate, A. actinomycetemcomitans SUNY 465, has previously been shown to enter epithelial cells by an actin-dependent mechanism. The internalized bacteria are surrounded by an actin halo upon entry. These data are consistent with the mode of entry associated with many enteric pathogens. We tested the effects of cytochalasin D, an inhibitor of the actin microfilament network, on bacterial entry to determine whether this mode of entry was common to other A. actinomycetemcomitans clinical isolates. Cytochalasin D was added prior to infection. A. actinomycetemcomitans SUNY 523 and A. actinomycetemcomitans 4065 exhibited enhanced ability to enter epithelial cells in the presence of cytochalasin D. Immunofluorescent labeling of bacteria and host cell actin confirmed that actin was not being mobilized by the entry of A. actinomycetemcomitans SUNY 523. Inhibitors of receptor-mediated endocytosis inhibited invasion of A. actinomycetemcomitans SUNY 523 and A. actinomycetemcomitans 4065. Microtubule effectors did not inhibit invasion of A. actinomycetemcomitans. A. actinomycetemcomitans SUNY 523, but not A. actinomycetemcomitans 4065, was deficient in exit from epithelial cells as determined by the absence of organisms in the assay medium. These data suggest that A. actinomycetemcomitans strains utilize at least two distinct mechanisms for entry into epithelial cells, and that A. actinomycetemcomitans SUNY 523 may be defective in exit and cell-to-cell spread.     

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。