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1.  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
2.  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
3.  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.
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PMCID: PMC268481  PMID: 3935663
4.  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.
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PMCID: PMC265645  PMID: 8349764
5.  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
6.  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
7.  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
8.  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.
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PMCID: PMC269598  PMID: 2312676
9.  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.
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PMCID: PMC551351  PMID: 7439996
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.  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
12.  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
13.  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
14.  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.
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PMCID: PMC272154  PMID: 7068837
15.  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
16.  Cardiobacterium hominis endocarditis: two cases and a review of the literature 
Cardiobacterium hominis, a member of the HACEK group (Haemophilus parainfluenzae, Haemophilus aphrophilus, and Haemophilus paraphrophilus, Actinobacillus actinomycetemcomitans, C. hominis, Eikenella corrodens, and Kingella species), is a rare cause of endocarditis. There are 61 reported cases of C. hominis infective endocarditis in the English-language literature, 15 of which involved prosthetic valve endocarditis. There is one reported case of C. hominis after upper endoscopy and none reported after colonoscopy. Presented here are two cases of C. hominis prosthetic valve endocarditis following colonoscopy and a review of the microbiological and clinical features of C. hominis endocarditis. Patients with C. hominis infection have a long duration of symptoms preceding diagnosis (138±128 days). The most common symptoms were fever (74%), fatigue/malaise (53%), weight loss/anorexia (40%), night sweats (24%), and arthralgia/myalgia (21%). The most common risk factors were pre-existing cardiac disease (61%), the presence of a prosthetic valve (28%), and history of rheumatic fever (20%). Of the 61 cases reviewed here, the aortic valve was infected in 24 (39%) and the mitral valve in 19 (31%) patients. The average duration of blood culture incubation before growth was detected was 6.3 days (range, 2–21 days). Complications were congestive heart failure (40%), central nervous system (CNS) emboli (21%), arrhythmia (16%), and mycotic aneurysm (9%). C. hominis is almost always susceptible to β-lactam antibiotics. Ceftriaxone is recommended by the recently published American Heart Association guidelines. The prognosis of C. hominis native valve and prosthetic valve endocarditis is favorable. The cure rate among 60 patients reviewed was 93% (56/60). For prosthetic valve endocarditis, the cure rate was 16/17 (94%). Valve replacement was required in 27 (45%) cases.
doi:10.1007/s10096-006-0189-9
PMCID: PMC2276845  PMID: 16955250
17.  Natural Transformation and DNA Uptake Signal Sequences in Actinobacillus actinomycetemcomitans 
Journal of Bacteriology  2002;184(13):3442-3449.
Actinobacillus actinomycetemcomitans is a member of the family Pasteurellaceae and a major causative agent of periodontitis. While several genera from this family are known to be competent for transformation, A. actinomycetemcomitans has yet to be fully characterized. Here we show that the competence of A. actinomycetemcomitans is remarkably similar to that of Haemophilus influenzae. In addition to having a similar frequency of transformation as H. influenzae, A. actinomycetemcomitans competence could also be induced at least 100-fold by cyclic AMP, suggesting that, as in H. influenzae, at least some competence genes are regulated by catabolite repression. Even more intriguing was the discovery of a putative A. actinomycetemcomitans DNA uptake signal sequence (USS) virtually identical to the USS of H. influenzae. Moreover, we provide evidence that this sequence functions in the same capacity as that from H. influenzae; the sequence appears to be required and sufficient for DNA uptake in a variety of assays. Finally, we have taken advantage of this system to develop a simple, highly efficient competence-based method for generating site-directed mutations in the wild-type fimbriated A. actinomycetemcomitans.
doi:10.1128/JB.184.13.3442-3449.2002
PMCID: PMC135135  PMID: 12057937
18.  Heterogeneity of antibodies reactive with the dominant antigen of Actinobacillus actinomycetemcomitans. 
Infection and Immunity  1997;65(9):3794-3798.
The serotype b-specific carbohydrate antigen (SbAg) of Actinobacillus actinomycetemcomitans Y4 is reported to be the O antigen of lipopolysaccharide, and the highest titers of serum antibody reactive with A. actinomycetemcomitans in early-onset periodontitis (EOP) patients bind SbAg. These high titers of serum antibody reactive with SbAg are associated with a lesser extent and severity of periodontal disease. The aim of this study was to determine if a limited number of genes code for anti-SbAg antibodies as has been shown for immunoglobulin G (IgG) reactive with the type b polysaccharide from Haemophilus influenzae. Serum IgG reactive with the SbAg was prepared from 20 high-titer EOP patients by affinity chromatography. The IgG subclass concentrations were determined, and heterogeneity was analyzed by isoelectric focusing (IEF). IgG2 was the dominant subclass (83% of total IgG) in the anti-SbAg IgG fraction and represented an average of 1.33% of total serum IgG2. The IgG2 reactive with SbAg was isolated from the affinity-purified IgG fraction by affinity chromatography with protein A and subclass-specific monoclonal antibodies. On IEF gels, only 4 to 20 bands were observed in the anti-SbAg IgG fractions, indicating limited heterogeneity. N-terminal amino acid sequence analysis of eight representative anti-SbAg IgG2 preparations indicated that variable heavy and light chains consisted largely of V(H)III and V(kappa)II, respectively. However, a significant fraction of anti-SbAg may use V(H) and V(lambda) genes with blocked N termini. In short, these findings indicate that IgG reactive with SbAg is very much like the antibody reactive with H. influenzae type b polysaccharide. Similarities include IgG2 dominance, limited bands on IEF gels, supporting an oligoclonal response, and use of genes from V(H)III and V(kappa)II regions.
PMCID: PMC175541  PMID: 9284154
19.  Rapid identification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus by restriction enzyme analysis of PCR-amplified 16S rRNA genes. 
Journal of Clinical Microbiology  1997;35(6):1630-1632.
Restriction enzyme analysis of PCR-amplified 16S rRNA genes was used to distinguish among clinical isolates of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus which were originally identified by conventional phenotypic methods. This PCR-based method is a reliable and rapid alternative to conventional methods for identification of these bacterial species.
PMCID: PMC229808  PMID: 9163503
20.  Haemophilus Aphrophilus Associated Spleen Abscess: An Unusual Presentation of Subacute Endocarditis 
The HACEK group of bacteria (Haemophilus spp., Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella spp.), is uncommon pathogens of infective endocarditis, but can cause life-threatening events such as heart failure or formation of lethal emboli. Here we report a 58-year-old Asian man with a past history of congenital valvular heart disease who presented with sudden onset of left flank pain followed by fever with chills for 2 weeks. Abdominal computed tomography (CT) indicated a 1.6 cm abscess in the spleen. Culturing indicated the presence of Haemophilus aphrophilus. We diagnosed the patient with subacute endocarditis complicated with spleen abscess. The patient recovered fully after two weeks antibiotic (Ceftriaxone) treatment. Clinicians should give further attention to infective endocarditis caused by bacteria in the HACEK group in patients with metastatic infection such as spleen abscess with suspected valvular heart disease.
doi:10.4021/jocmr803w
PMCID: PMC3376880  PMID: 22719808
Endocarditis; HACEK; Haemophilus aphrophilus; Spleen abscess
21.  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.
doi:10.1128/IAI.71.9.5005-5011.2003
PMCID: PMC187330  PMID: 12933843
22.  Acute Haemophilus parainfluenzae endocarditis: a case report 
Introduction
Numerous pathogens can cause infective endocarditis, including Haemophilus parainfluenzae. H. parainfluenzae is part of the H. aphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae group that may cause about 3% of the total endocarditis cases, and is characterized by a subacute course and large vegetations.
Case presentation
Acute H. parainfluenzae endocarditis developed in a 54-year-old woman, with no underlying predisposing factors. The patient presented with fever of 3 days duration and a severe headache. Magnetic resonance imaging of the brain revealed multiple cerebral emboli with hemorrhagic foci. Upon suspicion of endocarditis, cardiac transesophageal ultrasonography was performed and revealed massive vegetations. The patient underwent emergency mitral valve replacement, and was further treated with ceftriaxone. Blood cultures grew H. parainfluenzae only after valve replacement, and a 6-week course of ceftriaxone was prescribed.
Conclusion
We underline the typical presentation of large vegetations in H. parainfluenzae endocarditis, which are associated with embolic phenomena and resulting severity. Although the majority of the few cases reported in the literature are subacute in progress, our case further underlines the possibility that H. parainfluenzae endocarditis may develop rapidly. Thus, awareness of the imaging characteristics of the pathogen may enhance early appropriate diagnosis and therapeutic response.
doi:10.4076/1752-1947-3-7494
PMCID: PMC2737790  PMID: 19830211
23.  Nucleotide Sequence and Analysis of Conjugative Plasmid pVT745 
Journal of Bacteriology  2001;183(5):1585-1594.
The complete nucleotide sequence and genetic map of pVT745 are presented. The 25-kb plasmid was isolated from Actinobacillus actinomycetemcomitans, a periodontal pathogen. Two-thirds of the plasmid encode functions related to conjugation, replication, and replicon stability. Among potential gene products with a high degree of similarity to known proteins are those associated with plasmid conjugation. It was shown that pVT745 derivatives not only mobilized a coresident nontransmissible plasmid, pMMB67, but also mediated their own conjugative transfer to different A. actinomycetemcomitans strains. However, transfer of pVT745 derivatives from A. actinomycetemcomitans to Escherichia coli JM109 by conjugation was successful only when an E. coli origin of replication was present on the pVT745 construct. Surprisingly, 16 open reading frames encode products of unknown function. The plasmid contains a conserved replication region which belongs to the HAP (Haemophilus-Actinobacillus-Pasteurella) theta replicon family. However, its host range appears to be rather narrow compared to other members of this family. Sequences homologous to pVT745 have previously been detected in the chromosomes of numerous A. actinomycetemcomitans strains. The nature and origin of these homologs are discussed based on information derived from the nucleotide sequence.
doi:10.1128/JB.183.5.1585-1594.2001
PMCID: PMC95043  PMID: 11160089
24.  Aae, an Autotransporter Involved in Adhesion of Actinobacillus actinomycetemcomitans to Epithelial Cells  
Infection and Immunity  2003;71(5):2384-2393.
The periodontal pathogen Actinobacillus actinomycetemcomitans possesses myriad virulence factors, among them the ability to adhere to and invade epithelial cells. Recent advances in the molecular manipulation of this pathogen and the sequencing of strain HK 1651 (http://www.genome.ou.edu/act.html) have facilitated examination of the genetics of its interaction with epithelial cells. The related gram-negative organism, Haemophilus influenzae, possesses autotransporter adhesins. A search of the sequence database of strain HK 1651 revealed a homologue with similarity in the pore-forming domain to that of the H. influenzae autotransporter, Hap. A. actinomycetemcomitans mutants deficient in the homologue, Aae, showed reduced binding to epithelial cells. A method for making A. actinomycetemcomitans SUNY 465 transiently resistant to spectinomycin was used with conjugation to generate an isogenic aae mutant. An allelic replacement mutant was created in the naturally transformable A. actinomycetemcomitans strain ATCC 29523. Lactoferrin, an important part of the innate host defense system, protects against bacterial infection by bactericidal and antiadhesion mechanisms. Lactoferrin in human milk removes or cleaves Hap and another autotransporter, an immunoglobulin A1 protease, from the surface of H. influenzae, thereby reducing their binding to epithelial cells. Human milk whey had similar effects on Aae from A. actinomycetemcomitans ATCC 29523 and its binding to epithelial cells; however, there was little effect on the binding of SUNY 465. A difference in the genetic structure of aae in the two strains, apparently due to the copy number of a 135-base repeated sequence, may be the cause of the differential action of lactoferrin. aae is the first A. actinomycetemcomitans gene involved in adhesion to epithelial cells to be identified.
doi:10.1128/IAI.71.5.2384-2393.2003
PMCID: PMC153273  PMID: 12704108
25.  Influence of anti-Actinobacillus actinomycetemcomitans Y4 (serotype b) lipopolysaccharide on severity of generalized early-onset periodontitis. 
Infection and Immunity  1996;64(9):3908-3910.
The objective of this study was to determine if a relationship exists between antibody reactive with the Actinobacillus actinomycetemcomitans serotype b lipopolysaccharide (LPS) and severity of periodontal disease in generalized early-onset periodontitis (G-EOP). The concentration of antibody reactive with A. actinomycetemcomitans serotype b LPS was determined for 102 G-EOP subjects. Analysis of the relationship between antibody reactive with A. actinomycetemcomitans serotype b LPS and measures of periodontal attachment loss indicated that the patients with the highest concentrations of antibody reactive with A. actinomycetemcomitans serotype b LPS had significantly less attachment loss. These high-responder subjects also had anti-A. actinomycetemcomitans serotype b LPS with significantly higher relative avidity. The results suggest that antibody reactive with A. actinomycetemcomitans serotype b LPS is protective in G-EOP patients.
PMCID: PMC174312  PMID: 8751948

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