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1.  Cellular fatty acid and soluble protein composition of Actinobacillus actinomycetemcomitans and related organisms. 
Journal of Clinical Microbiology  1981;14(4):376-382.
The cellular fatty acid and protein content of twenty-five representative strains of Actinobacillus actinomycetecomitans isolated from juvenile and adult periodontitis patients was compared to that of 15 reference strains of oral and nonoral Actinobacillus species and Haemophilus aphrophilus. Trimethylsilyl derivatives of the fatty acid methyl esters were analyzed by gas-liquid chromatography. The predominant fatty acids of all 40 strains examined were 14:0, 3-OH 14:0, 16 delta, and 16:0. Actinobacillus seminis (ATCC 15768) was unlike the other strains examined because of a greater amount of 14:0 detected. The soluble protein analysis using polyacrylamide gel electrophoresis revealed that A. actinomycetemcomitans, H. aphrophilus, and nonoral Actinobacillus species possessed distinct protein profiles attesting to the validity of separating these organisms into different species. Established biotypes of A. actinomycetemcomitans could not be differentiated on the basis of fatty acid or protein profiles.
PMCID: PMC271987  PMID: 7287893
2.  Differentiation among closely related organisms of the Actinobacillus-Haemophilus-Pasteurella group by means of lysozyme and EDTA. 
Journal of Clinical Microbiology  1985;22(4):629-636.
Bacteriolysis in Tris-maleate buffer (0.005 M, pH 7.2) supplemented with EDTA (0.01 M) and hen egg white lysozyme (HEWL, 1.0 microgram/ml) was set up to assist differentiation between the taxonomically closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. A. actinomycetemcomitans was more sensitive to lysis in this system than H. aphrophilus. The standard method for bacteriolysis separated the 10 tested strains of A. actinomycetemcomitans into two groups (I and II) based on their lysis patterns, whereas the 7 strains of H. aphrophilus examined were homogeneous. In group I of A. actinomycetemcomitans, EDTA displayed a considerable lytic effect, which was not increased by supplementation with HEWL. In group II, the lytic effect of EDTA was much less, but HEWL had a considerable supplementary lytic effect. When the turbidity of A. actinomycetemcomitans (ATCC 29522) or H. aphrophilus (ATCC 33389) suspended in Tris buffer was monitored at close pH intervals (0.2) from pH 5.2 to 9.2, maximal lysis of ATCC 29522 occurred with EDTA at pH 8.0 and with EDTA-HEWL at pH 7.6, while ATCC 33389 lysed with EDTA at pH 9.0 and with EDTA-HEWL at pH 9.2. When other members of the family Pasteurellaceae (Haemophilus influenzae type b, Haemophilus paraphrophilus, Pasteurella multocida, Pasteurella haemolytica, and Pasteurella ureae) were included for comparison, the group I strains of A. actinomycetemcomitans were the most rapidly lysed by EDTA. H. paraphrophilus was the least sensitive of the gram-negative strains tested, but not as resistant as Micrococcus luteus (control). M. luteus was the organism most sensitive to lysozyme, followed by P. ureae and the group II strains of A. actinomycetemcomitans, while the group I strains of A. actinomycetemcomitans, H. paraphrophilus, and P. haemolytica were the least sensitive organisms.
PMCID: PMC268481  PMID: 3935663
3.  Evaluation of Two Commercial Kits and Arbitrarily Primed PCR for Identification and Differentiation of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus 
Journal of Clinical Microbiology  1999;37(3):742-747.
The closely related species Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus are common findings in oral microbiota. The aims of this study were to evaluate the applicability of the Rapid NH and API ZYM kits and arbitrarily primed PCR (AP-PCR) in the identification and differentiation of the three species from each other. The material included 62 clinical isolates and three reference strains of A. actinomycetemcomitans representing the 5 serotypes and 18 AP-PCR genotypes. Haemophilus species included 12 clinical isolates and 11 reference strains of H. aphrophilus, H. paraphrophilus, and 5 other species. For the PCR amplification, the oligonucleotide 5′-CAGCACCCAC-3′ was used as a primer. Contrary to the consistent performance of API ZYM, the Rapid NH system was able to identify only 10 of 65 (15%) A. actinomycetemcomitans isolates, whereas all Haemophilus species were correctly identified. The API ZYM test differentiated A. actinomycetemcomitans from H. aphrophilus and H. paraphrophilus by negative β-galactosidase and α-glucosidase reactions and a positive esterase lipase reaction. However, the API ZYM test was unable to differentiate H. aphrophilus from H. paraphrophilus, it also could not differentiate A. actinomycetemcomitans serotypes from each other. Among the H. aphrophilus isolates three AP-PCR genotypes and among H. paraphrophilus isolates only one AP-PCR genotype, distinct from those of A. actinomycetemcomitans, were found. The Rapid NH test showed poor ability to identify clinical isolates of all A. actinomycetemcomitans serotypes. Moreover, AP-PCR genotyping proved to be a rapid method for the species differentiation of A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus.
PMCID: PMC84540  PMID: 9986843
4.  Identification of Haemophilus aphrophilus and Actinobacillus actinomycetemcomitans by DNA-DNA hybridization and genetic transformation. 
Journal of Clinical Microbiology  1990;28(9):1994-1998.
DNA-DNA hybridization was used to identify clinical isolates as Haemophilus aphrophilus or Actinobacillus actinomycetemcomitans. Some of the isolates were naturally competent for genetic transformation and were also used as DNA recipients for identification of other isolates. The results obtained by hybridization were supported by interstrain-to-intrastrain transformation ratios. Distinction between the closely related species H. aphrophilus and A. actinomycetemcomitans was generally clear-cut by both methods. Distinction of H. aphrophilus and A. actinomycetemcomitans from type and reference strains of a diversity of species in the family Neisseriaceae and other gram-negative species was also demonstrated by both methods. This is the first description of the identification of clinical isolates of H. aphrophilus or A. actinomycetemcomitans by using them as recipients in genetic transformation. The results suggest that this is a reliable system for identification of new clinical isolates belonging to these taxonomic entities.
PMCID: PMC268092  PMID: 2229383
5.  Phylogeny of 54 representative strains of species in the family Pasteurellaceae as determined by comparison of 16S rRNA sequences. 
Journal of Bacteriology  1992;174(6):2002-2013.
Virtually complete 16S rRNA sequences were determined for 54 representative strains of species in the family Pasteurellaceae. Of these strains, 15 were Pasteurella, 16 were Actinobacillus, and 23 were Haemophilus. A phylogenetic tree was constructed based on sequence similarity, using the Neighbor-Joining method. Fifty-three of the strains fell within four large clusters. The first cluster included the type strains of Haemophilus influenzae, H. aegyptius, H. aphrophilus, H. haemolyticus, H. paraphrophilus, H. segnis, and Actinobacillus actinomycetemcomitans. This cluster also contained A. actinomycetemcomitans FDC Y4, ATCC 29522, ATCC 29523, and ATCC 29524 and H. aphrophilus NCTC 7901. The second cluster included the type strains of A. seminis and Pasteurella aerogenes and H. somnus OVCG 43826. The third cluster was composed of the type strains of Pasteurella multocida, P. anatis, P. avium, P. canis, P. dagmatis, P. gallinarum, P. langaa, P. stomatis, P. volantium, H. haemoglobinophilus, H. parasuis, H. paracuniculus, H. paragallinarum, and A. capsulatus. This cluster also contained Pasteurella species A CCUG 18782, Pasteurella species B CCUG 19974, Haemophilus taxon C CAPM 5111, H. parasuis type 5 Nagasaki, P. volantium (H. parainfluenzae) NCTC 4101, and P. trehalosi NCTC 10624. The fourth cluster included the type strains of Actinobacillus lignieresii, A. equuli, A. pleuropneumoniae, A. suis, A. ureae, H. parahaemolyticus, H. parainfluenzae, H. paraphrohaemolyticus, H. ducreyi, and P. haemolytica. This cluster also contained Actinobacillus species strain CCUG 19799 (Bisgaard taxon 11), A. suis ATCC 15557, H. ducreyi ATCC 27722 and HD 35000, Haemophilus minor group strain 202, and H. parainfluenzae ATCC 29242. The type strain of P. pneumotropica branched alone to form a fifth group. The branching of the Pasteurellaceae family tree was quite complex. The four major clusters contained multiple subclusters. The clusters contained both rapidly and slowly evolving strains (indicated by differing numbers of base changes incorporated into the 16S rRNA sequence relative to outgroup organisms). While the results presented a clear picture of the phylogenetic relationships, the complexity of the branching will make division of the family into genera a difficult and somewhat subjective task. We do not suggest any taxonomic changes at this time.
PMCID: PMC205807  PMID: 1548238
6.  Improved selective culture media for Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. 
Journal of Clinical Microbiology  1987;25(10):1985-1988.
By modifying the previously described media tryptic soy-serum-bacitracin-vancomycin (TSBV) agar and tryptic soy-serum-bacitracin-vancomycin-fluoride (TSBVF) agar, two improved selective culture media were developed for isolation and enumeration of Actinobacillus actinomycetemcomitans (A medium) and Haemophilus aphrophilus (H medium) in oral specimens. Both media were supplemented with fusidic acid and spiramycin, and carbenicillin was also added to A medium. The growth yields of pure cultures of A. actinomycetemcomitans on A medium and of H. aphrophilus on H medium were comparable with those on the reference media. Compared with blood agar, the selective media inhibited these species about 10-fold or less. In addition, A and H media suppressed the growth of pure cultures of Capnocytophaga spp. and Neisseria spp., commonly found as contaminants on TSBV and TSBVF, 10(5) times or more compared with that on blood agar. In samples from diseased periodontal pockets, the recoveries of A. actinomycetemcomitans on A medium and H. aphrophilus on H medium equaled those on TSBV and TSBVF, respectively. In about 50% of the cultures on the reference media, contaminating bacteria were detected at levels higher than 10(4) CFU/ml of sample. The corresponding value for both A and H media was about 2%.
PMCID: PMC269382  PMID: 3667919
7.  Serology of oral Actinobacillus actinomycetemcomitans and serotype distribution in human periodontal disease. 
Infection and Immunity  1983;41(1):19-27.
Actinobacillus actinomycetemcomitans from the human oral cavity was serologically characterized with rabbit antisera to the type strain NCTC 9710; a number of reference strains, including Y4, ATCC 29522, ATCC 29523, ATCC 29524, NCTC 9709; and our own isolates representative of each of 10 biotypes. Using immunoabsorbed antisera, we identified three distinct serotypes by immunodiffusion and indirect immunofluorescence. Serotype a was represented by ATCC 29523 and SUNYaB 75; serotype b was represented by ATCC 29522 and Y4; and serotype c was represented by NCTC 9710 and SUNYaB 67. Indirect immunofluorescence revealed no reaction between the three A. actinomycetemcomitans serotype-specific antisera and 62 strains representing 23 major oral bacterial species. Distinct from the serotype antigens were at least one A. actinomycetemcomitans species common antigen and an antigen shared with other Actinobacillus species, Haemophilus aphrophilus, and Haemophilus paraphrophilus. All serotype a A. actinomycetemcomitans strains failed to ferment xylose, whereas all serotype b organisms fermented xylose. Serotype c included xylose-positive as well as xylose-negative strains. A total of 301 isolates of A. actinomycetemcomitans from the oral cavity of 74 subjects were serologically categorized by indirect immunofluorescence with serotype-specific rabbit antisera. Each patient harbored only one serotype of A. actinomycetemcomitans. Fourteen healthy subjects, five diabetics, and seventeen adult periodontitis patients exhibited serotypes a and b in approximately equal frequency, whereas serotype c was found less frequently. In contrast, in 29 localized juvenile periodontitis patients, the incidence of serotype b was approximately two times higher than that of serotypes a or c, suggesting a particularly high periodontopathic potential of A. actinomycetemcomitans serotype b strains. In subjects infected with A. actinomycetemcomitans, serum antibodies were detected to the serotype antigens, indicating that these antigens may play a role in the pathogenesis of periodontal disease.
PMCID: PMC264736  PMID: 6407997
8.  Identification of Actinobacillus actinomycetemcomitans by leukotoxin gene-specific hybridization and polymerase chain reaction assays. 
Journal of Clinical Microbiology  1993;31(7):1856-1859.
Eleven strains of Actinobacillus actinomycetemcomitans isolated from cases of systemic infections, local abscesses, and periodontitis were identified by genetic assays using the leukotoxin gene as the target. We have developed a polymerase chain reaction (PCR) assay, based on the leukotoxin structural gene of this pathogen, which clearly identified all tested strains of A. actinomycetemcomitans and separated them from the closely related Haemophilus aphrophilus as well as other bacterial species. Furthermore, DNA-DNA hybridization was performed with the cloned partial leukotoxin structural gene (lktA) as a probe, which again clearly distinguished A. actinomycetemcomitans from H. aphrophilus, parts of the normal oral flora, and species harboring RTX (repeats in toxin) family-related cytotoxins. The PCR fragment amplified from the leukotoxin structural gene gave results similar to those given by the cloned leukotoxin gene when used as a probe in hybridization experiments. The hybridization and PCR assays described here are fundamental improvements for the identification of A. actinomycetemcomitans.
PMCID: PMC265645  PMID: 8349764
9.  Assessment of serum antibody patterns and analysis of subgingival microflora of members of a family with a high prevalence of early-onset periodontitis. 
Infection and Immunity  1985;49(3):742-750.
In a study of members of a large family with a high prevalence of early-onset periodontitis, we sampled the subgingival microflora and characterized 40 isolates from each sample. We surveyed serum samples by enzyme-linked immunosorbent assay for antibodies reacting with any of a panel of 21 periodontal bacteria. The mother and 7 of her 13 children had early-onset periodontitis. Bacteroides gingivalis was not detected in the subgingival flora of any affected or unaffected family member, and Actinobacillus actinomycetemcomitans was isolated from only one affected child. Capnocytophaga ochracea was isolated from five of seven affected children and from none of their normal siblings. We found no significant differences among the floras from family members who had rapidly progressive, juvenile, and prepubertal forms of periodontitis. Elevated levels of serum antibody reacting with one or more of the bacteria tested were found in all family members with disease, but in only one periodontally normal family member. Both children with prepubertal periodontitis had antibodies reacting with C. sputigena, a species not found in their subgingival floras, but with none of the other bacteria tested. All remaining affected family members had antibodies to one or more serotypes of A. actinomycetemcomitans, and four had antibodies reacting with additional bacteria, including C. sputigena, Eikenella corrodens, Fusobacterium nucleatum, and Haemophilus aphrophilus. Sera from patients contained antibodies specific for putative periodontal pathogens not found in their pocket flora, and conversely, putative periodontal pathogens for which no serum antibodies were found frequently comprised a large proportion (10% or more) of the pocket flora. In no case were both the bacterium and its antibody found. These observations are suggestive of sequential infection in the early-onset forms of periodontitis and of induction of protective immunity against reinfection by the same microorganism.
PMCID: PMC261261  PMID: 4030102
10.  Resistance of Actinobacillus actinomycetemcomitans and differential susceptibility of oral Haemophilus species to the bactericidal effects of hydrogen peroxide. 
Infection and Immunity  1984;46(3):644-648.
We compared the sensitivities of oral and nonoral isolates of Actinobacillus actinomycetemcomitans, Haemophilus segnis, H. aphrophilus, and H. paraphrophilus to the bactericidal action of reagent hydrogen peroxide (H2O2). Susceptibility to a range of H2O2 concentrations (10(-6) to 10(-3) M) was assessed by incubating bacterial suspensions for 1 h at 37 degrees C in the presence of H2O2 and plating on chocolate agar to determine the concentration of H2O2 that would produce a 50% reduction in CFU (50% lethal dose). As a group, A. actinomycetemcomitans was more resistant to H2O2 than the oral haemophili, and H. aphrophilus was much more sensitive than all other organisms tested. The range of 50% lethal dose values for A. actinomycetemcomitans was between 8.5 X 10(-5) and 10(-3) M H2O2 or above. In contrast, H. aphrophilus exhibited 50% lethal dose values from below 1 X 10(-6) to 3.4 X 10(-4) M H2O2. The resistance of A. actinomycetemcomitans to H2O2 may be sufficient to protect these organisms from direct H2O2-mediated killing by host phagocytes.
PMCID: PMC261590  PMID: 6500706
11.  Cellular fatty acid composition of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. 
Journal of Clinical Microbiology  1980;11(6):625-630.
Strains of Actinobacillus actinomycetemcomitans isolated from deep pockets of patients with juvenile periodontitis were analyzed for their content of cellular fatty acids. Oral Haemophilus strains, morphologically and biochemically similar to Haemophilus aphrophilus, were also examined for their content of cellular fatty acids. The extractable lipids of the actinobacilli represented approximately 10% of the cell dry weight, with the bound lipids representing 2 to 5%. The major fatty acids consisted of myristic (C14:0) and palmitic (C16:0) acids and a C16:1 acid, possibly palmitoleic acid, accounting for 21, 35, and 31% of the total extractable fatty acids, respectively. Haemophilus strains had a similar cellular fatty acid content.
PMCID: PMC273474  PMID: 7430333
12.  Phosphorylcholine-Dependent Cross-Reactivity between Dental Plaque Bacteria and Oxidized Low-Density Lipoproteins 
Infection and Immunity  2001;69(11):6612-6617.
Antibodies reactive with phosphorylcholine (PC) are ubiquitous in human sera, but the antigens stimulating their production and their function are not clear. Previous studies have shown that a significant proportion of dental plaque bacteria contain PC as determined by reactivity with PC-specific mouse myeloma proteins and monoclonal antibodies. Additionally, serum antibody concentrations of immunoglobulin (IgG) G anti-PC are higher in sera of individuals who have experienced periodontal attachment loss than those who are periodontally healthy. These data implicate the oral microflora as a source of antigen-stimulating anti-PC responses. Recent data also indicate that antibodies with specificity for PC are elevated in ApoE-deficient mice, a model for studies of athersclerosis, and that such antibodies bound oxidized low-density lipoproteins (LDL) (oxLDL) in atherosclerotic plaques. These data prompted the hypothesis that human anti-PC could bind to both oral bacteria and human oxLDL, and that these antigens are cross-reactive. We therefore examined the ability of human anti-PC to bind to PC-bearing strains of oral bacteria using enzyme-linked immunosorbent inhibition assays and by assessment of direct binding of affinity-purified human anti-PC to PC-bearing Actinobacillus actinomycetemcomitans. Our results indicated that PC-bearing strains of Streptococcus oralis, Streptococcus sanguis, Haemophilus aphrophilus, Actinomyces naeslundii, Fusobacterium nucleatum, and A. actinomycetemcomitans, as well as a strain of Streptococcus pneumoniae, absorbed up to 80% of anti-PC IgG antibody from human sera. Furthermore, purified anti-PC bound to a PC-bearing strain of A. actinomycetemcomitans but only poorly to a PC-negative strain. OxLDL also absorbed anti-PC from human sera, and oxLDL but not LDL reacted with up to 80% of the anti-PC in human sera. Furthermore, purified anti-PC bound directly to oxLDL but not to LDL. The data indicate that PC-containing antigens on a variety of common oral bacteria are cross-reactive with neoantigens expressed in oxLDL. We propose that PC-bearing dental plaque microorganisms may induce an antibody response to PC that could influence the inflammatory response associated with atherosclerosis.
PMCID: PMC100034  PMID: 11598029
13.  Species-specific oligodeoxynucleotide probes for the identification of periodontal bacteria. 
Journal of Clinical Microbiology  1990;28(2):319-323.
Oligodeoxynucleotide probes were developed for identification of the periodontal bacteria Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, B. intermedius types I and II, B. forsythus, Eikenella corrodens, Fusobacterium nucleatum, Haemophilus aphrophilus, Streptococcus intermedius, and Wolinella recta. Probes were designed by sequencing the 16S rRNA for each bacterium, identifying hypervariable regions, and chemically synthesizing species-specific probes. These probes were specific when tested against a panel of nucleic acids from closely related bacteria.
PMCID: PMC269598  PMID: 2312676
14.  Epithelial cell invasion by Actinobacillus actinomycetemcomitans strains from restriction fragment-length polymorphism groups associated with juvenile periodontitis or carrier status 
Oral microbiology and immunology  1998;13(6):341-347.
The epithelial cell invasiveness of Actinobacillus actinomycetemcomitans strains of different restriction fragment-length polymorphism (RFLP) groups associated with disease conversion and asymptomatic carrier status in localized juvenile periodontitis was examined. Twenty clinical isolates were studied for their ability to invade KB monolayers, using the quantitative gentamicin killing assay. Five isolates were found to be invasive; five were not invasive; and the other 10 did not invade better than an invasion negative control Haemophilus aphrophilus strain ATCC 19415. Using probe-specific DNA fingerprinting, 11 strains were assigned to RFLP group II (disease–associated); 4 to RFLP type XIII (carrier status-associated); and the others to groups III, IV, V and VII. Eight isolates, all RFLP group II, were leukotoxin producers as determined by PCR amplification of the lkt promoter region. No correlation was found between invasiveness and RFLP group. Leukotoxin production was more associated with noninvasive than invasive strains.
PMCID: PMC3528405  PMID: 9872109
Actinobacillus actinomycetemcomitans; invasion; KB epithelial cell; restriction fragment-length polymorphism; leukotoxin
15.  Microtubules Are Associated with Intracellular Movement and Spread of the Periodontopathogen Actinobacillus actinomycetemcomitans 
Infection and Immunity  1999;67(12):6518-6525.
Actinobacillus actinomycetemcomitans SUNY 465, the invasion prototype strain, enters epithelial cells by an actin-dependent mechanism, escapes from the host cell vacuole, and spreads intracellularly and to adjacent epithelial cells via intercellular protrusions. Internalized organisms also egress from host cells into the assay medium via protrusions that are associated with just a single epithelial cell. Here we demonstrate that agents which inhibit microtubule polymerization (e.g., colchicine) and those which stabilize polymerized microtubules (e.g., taxol) both increase markedly the number of intracellular A. actinomycetemcomitans organisms. Furthermore, both colchicine and taxol prevented the egression of A. actinomycetemcomitans from host cells into the assay medium. Immunofluorescence microscopy revealed that protrusions that mediate the bacterial spread contain microtubules. A. actinomycetemcomitans SUNY 465 and 652, strains that are both invasive and egressive, interacted specifically with the plus ends (growing ends) of the filaments of microtubule asters in a KB cell extract. By contrast, neither A. actinomycetemcomitans 523, a strain that is invasive but not egressive, nor Haemophilus aphrophilus, a noninvasive oral bacterium with characteristics similar to those of A. actinomycetemcomitans, bound to microtubules. Together these data suggest that microtubules function in the spread and movement of A. actinomycetemcomitans and provide the first evidence that host cell dispersion of an invasive bacterium may involve the usurption of host cell microtubules.
PMCID: PMC97062  PMID: 10569770
16.  Identification and characterization of the major cell envelope proteins of oral strains of Actinobacillus actinomycetemcomitans. 
Infection and Immunity  1983;39(1):253-261.
The major cell envelope protein compositions of seven Actinobacillus actinomycetemcomitans strains of human origin were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major envelope polypeptides were homogeneous, in relation to molecular weight, in all of the strains that were examined. The characterization of the five major proteins, designated Env1 through Env5, in the leukotoxic strain Y4 revealed that proteins Env2 to -5 may reside in the outer membrane as suggested by differential detergent extractions and 125I-labeling experiments. The proteins did not demonstrate covalent or ionic interactions with the peptidoglycan; however, one protein, Env2, displayed heat-modifiable properties, having apparent molecular weights of 32,000 and 45,000 when heated in sodium dodecyl sulfate at 50 and 100 degrees C, respectively. The protein composition of the extracellular "bleb" material, normally released by strain Y4, was determined, and proteins Env1 to -4 were the predominant protein species found. A comparison of the cell envelope proteins of strain Y4 with those of other members of the human oral flora, including species within the genera Capnocytophaga, Bacteroides, and Fusobacterium, revealed distinct differences on the basis of molecular size and heat-modifiable properties. However, the membrane proteins of Haemophilus aphrophilus showed a remarkable degree of homology with those of A. actinomycetemcomitans.
PMCID: PMC347934  PMID: 6401694
17.  DNA from Periodontopathogenic Bacteria Is Immunostimulatory for Mouse and Human Immune Cells 
Infection and Immunity  2003;71(2):850-856.
Although bacterial DNA (bDNA) containing unmethylated CpG motifs stimulates innate immune cells through Toll-like receptor 9 (TLR-9), its precise role in the pathophysiology of diseases is still equivocal. Here we examined the immunostimulatory effects of DNA extracted from periodontopathogenic bacteria. A major role in the etiology of periodontal diseases has been attributed to Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Peptostreptococcus micros. We therefore isolated DNA from these bacteria and stimulated murine macrophages and human gingival fibroblasts (HGF) in vitro. Furthermore, HEK 293 cells transfected with human TLR-9 were also stimulated with these DNA preparations. We observed that DNA from these pathogens stimulates macrophages and gingival fibroblasts to produce tumor necrosis factor alpha and interleukin-6 in a dose-dependent manner. Methylation of the CpG motifs abolished the observed effects. Activation of HEK 293 cells expressing TLR-9 which were responsive to bDNA but not to lipopolysaccharide confirmed that immunostimulation was achieved by bDNA. In addition, the examined bDNA differed in the ability to stimulate murine macrophages, HGF, and TLR-9-transfected cells. DNA from A. actinomycetemcomitans elicited a potent cytokine response, while DNA from P. gingivalis and P. micros showed lower immunostimulatory activity. Taken together, the results strongly suggest that DNA from A. actinomycetemcomitans, P. gingivalis, and P. micros possesses immunostimulatory properties in regard to cytokine secretion by macrophages and fibroblasts. These stimulatory effects are due to unmethylated CpG motifs within bDNA and differ between distinct periodontopathogenic bacteria strains. Hence, immunostimulation by DNA from A. actinomycetemcomitans, P. gingivalis, and P. micros could contribute to the pathogenesis of periodontal diseases.
PMCID: PMC145359  PMID: 12540566
18.  Serum antibody in Actinobacillus actinomycetemcomitans-infected patients with periodontal disease. 
Infection and Immunity  1991;59(5):1795-1802.
This study was designed to (i) delineate the characteristics of serum antibody responses to Actinobacillus actinomycetemcomitans in patients with periodontitis who are infected with A. actinomycetemcomitans; irrespective of disease classification; (ii) assess the relationship of the elevated antibody levels to colonization of the oral cavity by A. actinomycetemcomitans; and (iii) describe the serotype distribution of A. actinomycetemcomitans and antibodies to the microorganism in infected patients with various clinical classifications. To compare the levels of various isotype-specific antibodies to the different antigens, studies were performed that allowed quantitation of each isotype-specific antibody in a human reference standard. By using this reference standard, it was shown that the levels of immunoglobulin G (IgG), IgM, and IgA responses to A. actinomycetemcomitans were similar among the infected patients, irrespective of disease classification. Also, we demonstrated that the serum antibody response to serotype b was quantitatively greater in all isotypes. Our findings indicate that b was the most frequent A. actinomycetemcomitans serotype detected in the patients and appears to be capable of initiating a substantial serum IgG antibody response that may contain cross-reactive antibodies to other serotypes of A. actinomycetemcomitans. Generally, in cases in which the response to a single serotype was elevated, only that type of A. actinomycetemcomitans was detected in the plaque. Individuals exhibiting elevated antibodies to multiple serotypes were most consistently colonized by the serotype b microorganism. This study represents the first report detailing the distribution of IgG subclass antibodies to A. actinomycetemcomitans in periodontal disease. The results demonstrated that the primary responses of patients with periodontitis to A. actinomycetemcomitans were of the IgG1 and IgG3 subclasses, which is consistent with elicited responses to protein antigens. In contrast, the primary subclass response in normal subjects was limited to the IgG2 subclass and may represent broader cross-reactivity to polysaccharide antigens-lipopolysaccharide from the bacteria.
PMCID: PMC257918  PMID: 2019443
19.  Morphology and ultrastructure of oral strains of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. 
Infection and Immunity  1980;30(2):588-600.
Selected human oral and nonoral strains of the genera Actinobacillus and Haemophilus were examined by transmission and scanning electron microscopy. The strains examined were morphologically identical to recognized Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus. By transmission electron microscopy, the cells were typically gram negative in morphology, with several strains possessing some extracellular ruthenium red-staining polymeric material. Numerous vesicular structures, morphologically identical to lipopolysaccharide vesicles, were seen to originate from and be continuous with the surface of the outer membrane. Large numbers of these vesicles were also found in the external environment. Scanning electron microscopic observations revealed that both actinobacilli and haemophili possessed surface projections and an amorphous surface material which connected and covered adjacent cells.
PMCID: PMC551351  PMID: 7439996
20.  Structural and Genetic Analyses of O Polysaccharide from Actinobacillus actinomycetemcomitans Serotype f 
Infection and Immunity  2001;69(9):5375-5384.
The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP). A. actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from A. actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed of l-rhamnose and 2-acetamido-2-deoxy-d-galactose (molar ratio, 2:1) with the structure →2)-α-l-Rhap-(1–3)-2-O-(β-d-GalpNAc)-α-l-Rhap-(1→. O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known A. actinomycetemcomitans serotypes. Strain CU1000 was mutagenized with transposon IS903φkan, and three mutants that were deficient in O-PS synthesis were isolated. All three transposon insertions mapped to a single 1-kb region on the chromosome. The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of A. actinomycetemcomitans serotype b, c, and e O-PS antigens. The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five known A. actinomycetemcomitans serotypes. These data indicate that strain CU1000 should be assigned to a new A. actinomycetemcomitans serotype, designated serotype f. A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored A. actinomycetemcomitans strains carrying the serotype f gene cluster. The finding of an A. actinomycetemcomitans serotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted.
PMCID: PMC98647  PMID: 11500407
21.  Selective medium for isolation of Actinobacillus actinomycetemcomitans. 
Journal of Clinical Microbiology  1982;15(4):606-609.
A selective medium, TSBV (tryptic soy-serum-bacitracin-vancomycin) agar, was developed for the isolation of Actinobacillus actinomycetemcomitans, TSBV agar contained (per liter) 40 g of tryptic soy agar, 1 g of yeast extract, 100 ml of horse serum. 75 mg of bacitracin, and 5 mg of vancomycin. The TSBV medium suppressed most oral species and permitted significantly higher recovery of A. actinomycetemcomitans than nonselective blood agar medium. The distinct colonial morphology and positive catalase reaction of A. actinomycetemcomitans easily distinguished this bacterium from Haemophilus aphrophilus, Capnocytophaga species, and a few other contaminating organisms. With the TSBV medium, even modestly equipped laboratories will be able to isolate and identify A. actinomycetemcomitans from clinical specimens.
PMCID: PMC272154  PMID: 7068837
22.  Distribution of protein D, an immunoglobulin D-binding protein, in Haemophilus strains. 
Infection and Immunity  1991;59(4):1231-1238.
Protein D, a novel surface protein of the bacterial species Haemophilus influenzae with specific affinity for human immunoglobulin (Ig) D was detected in all 127 H. influenzae strains studied. All strains representing different serotypes of encapsulated strains and different biotypes of nonencapsulated strains bound 125I-labeled IgD to a high degree (38 to 74%). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis showed that protein D from all H. influenzae strains had the same apparent molecular weight (i.e., 42,000) and reacted with all three different anti-protein D monoclonal antibodies. By Scatchard analysis, the number of protein D residues on a nontypeable H. influenzae strain was estimated to be approximately 2,800 per organism. The equilibrium constant for the reaction between a human IgD myeloma protein and IgD was found to be 5.8 x 10(8) M-1. Also, all strains of H. haemolyticus and H. aegypticus strains tested bound IgD, 21 to 28% and 41 to 48%, respectively. In extracts of those bacteria, a 42,000-molecular-weight protein reactive with IgD and all three anti-protein D monoclonal antibodies was found. In H. parainfluenzae, H. aphrophilus, H. paraphrophilus, and Actinobacillus actinomycetemcomitans, a 42,000-molecular-weight protein that was reactive with one to three of three anti-protein D monoclonal antibodies but not reactive with human IgD was detected with Western blot analysis. Other Haemophilus species (H. ducreyi, H. parasuis, H. parahaemolyticus, H. segnis, and H. haemoglobinophilus) did not react with human monoclonal IgD or anti-protein D antibodies. On the basis of the wide distribution of protein D among H. influenzae strains, we suggest that protein D could be a vaccine candidate.
PMCID: PMC257832  PMID: 1900807
23.  Enzymatic characterization of some oral and nonoral gram-negative bacteria with the API ZYM system. 
Journal of Clinical Microbiology  1981;14(3):288-294.
The API ZYM system (Analytab Products, Plainview, N.Y.), containing 19 chromogenic substrates, was utilized semiquantitatively to detect extracellular acid and alkaline phosphatases, aminopeptidases, proteases, esterase-lipase, phosphoamidase, and glycosidases in 128 oral and nonoral isolates of black-pigmented Bacteroides, Actinobacillus, Haemophilus aphrophilus, Capnocytophaga, Fusobacterium nucleatum, Wolinella recta, and Veillonella parvula. In the black-pigmented Bacteroides group of organisms, a strong trypsin reaction was present in Bacteroides gingivalis (oral species) but not in Bacteroides asaccharolyticus (nonoral species). Bacteroides melaninogenicus subsp. melaninogenicus, in contrast to Bacteroides melaninogenicus subsp. intermedius, exhibited strong N-acetyl-beta-glucosaminidase activity. H. aphrophilus produced beta-galactosidase and alpha-glucosidase, but the closely related Actinobacillus actinomycetemcomitans did not. Capnocytophaga was distinct with respect to strong aminopeptidase reactions. This study showed that a wide range of enzymes which have the potential of causing tissue injury and inflammation can be elaborated from major oral gram-negative species. Also, the API ZYM system appears to be a valuable adjunct to traditional biochemical testing in identifying oral gram-negative species.
PMCID: PMC271956  PMID: 7026598
24.  Vertebral osteomyelitis due to coccobacilli of the HB group. 
Three cases of pyogenic vertebral osteomyelitis occurred in which unusual, fastidious, Gram negative coccobacilli belonging to the "HB" group were isolated. The organisms were Haemophilus aphrophilus in case 1, intermediate between H aphrophilus and Actinobacillus actinomycetemcomitans in case 2, and Eikenella corrodens in case 3. All HB bacteria are sensitive to a wide range of antibiotics.
PMCID: PMC1550110  PMID: 6416539
25.  Actinobacillus actinomycetemcomitans Serotype d-Specific Antigen Contains the O Antigen of Lipopolysaccharide  
Infection and Immunity  2003;71(9):5005-5011.
Actinobacillus actinomycetemcomitans is a gram-negative, facultatively anaerobic bacterium which is associated especially with aggressive forms of periodontitis. Contradictory results on the localization of the A. actinomycetemcomitans serotype-specific antigen have been reported. The aim of the present study was to characterize the A. actinomycetemcomitans serotype d-specific antigen. The antigen was isolated by affinity chromatography. The affinity column was prepared from immunoglobulin G isolated from rabbit antiserum raised against A. actinomycetemcomitans serotype d. The isolated antigen was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and silver staining, all of which revealed a ladder-like structure typical for the O antigen of lipopolysaccharide (LPS). In a displacement enzyme-linked immunosorbent assay (ELISA), the isolated antigen displaced in a concentration-dependent manner the binding of the polyclonal rabbit antiserum raised against A. actinomycetemcomitans serotype d to the competing whole-cell serotype d antigen. The isolated antigen contained LPS, and an equal concentration of LPS isolated from A. actinomycetemcomitans serotype d gave a similar displacement curve in the ELISA. In order to test the immunogenic properties of the isolated antigen, it was used to immunize a rabbit. The antiserum raised against the isolated antigen displayed specificity in Western blotting and ELISA similar to that of antibody raised against LPS isolated from A. actinomycetemcomitans serotype d. In conclusion, our results show that the A. actinomycetemcomitans serotype d-specific antigen contains the O-antigenic structure of LPS.
PMCID: PMC187330  PMID: 12933843

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