Oral Haemophili Research Articles

Immunoproteomics of Actinobacillus actinomycetemcomitans outer-membrane proteins reveal a highly immunoreactive peptidoglycan-associated lipoprotein

In a search for novel bioactive cell surface structures of periodontal pathogens, it was found that sera from two patients with Actinobacillus actinomycetemcomitans-associated infections reacted strongly at 17 kDa on immunoblots of A. actinomycetemcomitans outer-membrane protein (OMP) preparations. The 17 kDa antigen was also recognized by anti-CsgA (Escherichia coli curli major subunit) antibody. The 17 kDa A. actinomycetemcomitans protein was identified as peptidoglycan-associated lipoprotein (PAL; AaPAL) by two-dimensional immunoblotting and subsequent sequence analysis by mass spectrometry and bioinformatics tools. AaPAL was an OMP and a lipoprotein, and it had an OmpA-like domain. In a group of middle-aged subjects (n = 26), serum reactivity to AaPAL was associated with the presence of periodontitis but not with the oral detection of A. actinomycetemcomitans. Both human sera and rabbit antisera against three different types of antigens, the gel-purified AaPAL, A. actinomycetemcomitans whole-cell antigens, and CsgA, recognized putative PALs of oral haemophili in addition to AaPAL. The results demonstrated that the novel AaPAL is a conserved bacterial lipoprotein. It is expressed in vivo and is strongly immunoreactive. The antigenic cross-reactivity found between AaPAL and oral haemophili may enhance local and systemic immuno-inflammatory reactions in periodontitis.

Relationships among isolates of oral haemophili as determined by DNA-DNA hybridization.

In order to assess the relationships among strains of the genera Actinobacillus and Haemophilus, DNAs from 50 strains of these genera were isolated and purified. The guanine plus cytosine (G + C) content of DNAs from strains of Haemophilus segnis and Haemophilus parainfluenzae were determined by thermal denaturation. DNA-DNA homologies were measured using labelled probes from one strain representing Haemophilus segnis (strain ATCC 10977), and two strains representing Haemophilus parainfluenzae (strains ATCC 9796 and ATCC 7901). Strains isolated as H. segnis had a G + C content of 39.0 to 42.9% and were 49-92% homologous with the ATCC 10977 DNA probe. All of the strains freshly isolated as H. parainfluenzae were 70-81% homologous with the ATCC 9796 DNA probe and had a G + C content of 34.9 to 38.3%. Strain ATCC 7901 was 11% homologous with the ATCC 9796 DNA probe, had a G + C content of 42.4%, and was 65-78% homologous to DNA from strains identified as Haemophilus aphrophilus and Haemophilus paraphrophilus. From these results we conclude that strain ATCC 7901 is a mislabelled strain of H. paraphrophilus. The results of multiple DNA-DNA hybridizations indicated that separate species designations were appropriate for H. segnis, H. parainfluenzae, Actinobacillus actinomycetemcomitans ("Haemophilus actinomycetemcomitans"), and H. aphrophilus. H. aphrophilus and H. paraphrophilus were closely related organisms and did not fulfill the generally accepted criteria for designation as separate species.

Serum and Gingival Fluid Antibodies as Adjuncts in the Diagnosis of Actinobacillus actinomycetemcomitans Associated Periodontal Disease.

Serum antibody titers to Actinobacillus actinomycetemcomitans were measured in 200 subjects by an enzyme-lined immunosorbent assay (ELISA) using whole microorganisms as antigen. Comparisons were made between titers found in periodontally normal subjects and titers in subjects with localized juvenile periodontitis (LJP), postlocalized juvenile periodontitis, generalized juvenile periodontitis or adult periodontitis. It was found that titers to all three serotypes of A. actinomycetemcomitans were elevated in LJP patients' sera, while serum antibody levels in other diseased groups were not significantly elevated to any of the serotypes. Patient sera were also examined for serum antibody to oral Haemophili previously shown to cross-react with A. actinomycetemcomitans. Similar antibody titers were found in both normal subjects and in patients with various forms of periodontal disease to Haemophilus aphrophilus, H. influenzae and H. parainfluenzae. The A. actinomycetemcomitans antibodies which were elevated in LJP patients could not be correlated with antibody titers to cross-reacting Haemophili, suggesting that these antibodies are A. actinomycetemcomitansspecific. Serum antibody responses in six of the LJP patients were assessed to autologous strains of A. actinomycetemcomitans. Each patient was found to be infected with only a single serotype of A. actinomycetemcomitans, and specific antibodies to the infecting serotype were found in the patients' sera. In families, the LJP patients had significantly elevated IgG, IgA and IgM serum antibody titers to A actinomycetemcomitans, while the IgG and IgA antibody titers in periodontally normal siblings were at levels comparable to those found in normal subjects. However, IgM serum antibodies were elevated in the periodontally normal siblings of LJP patients suggesting that the formation of IgM antibodies to A. actinomycetemcomitans may precede the clinical appearance of localized juvenile periodontitis. Gingival crevicular fluid and serum antibody levels to A. actinomycetemcomitans were compared in LJP patients. Comparable titers of IgG, IgA and IgM antibodies were found in serum and gingival fluid in most subjects; however, gingival fluid samples sometimes showed higher titers than serum, likely resulting from local antibody synthesis. The value of serum antibody determinations to A. actinomycetemcomitans in the diagnosis of Actinobacillus-associated periodontitis was also assessed. The predictive value of a positive test (significantly elevated anti-A actinomycetemcomitans IgG) was 86%, while the specificity was 89%. These results suggest that the measurement of serum or gingival crevicular fluid antibodies to A. actinomycetemcomitans may be valuable in the diagnosis of Actinobacillusassociated periodontal disease.

Influence of endogenous catalase activity on the sensitivity of the oral bacterium Actinobacillus actinomycetemcomitans and the oral haemophili to the bactericidal properties of hydrogen peroxide

Actinobacillus actinomycetemcomitans and the genetically-related oral haemophili ( Hae mophilus segnis, Haemophilus aprhophilus and Haemophilus paraphrophilus) exhibit a range of sensitivities to the lethal effect of hydrogen peroxide (H 2O 2), A. actinomycetemcomitans being the most resistant. To extend this information, susceptibility to a range of H 2O 2 concentrations (10 −6-10 −3 M) was assessed by incubating bacterial suspensions for l h at 37°C in the presence of H 2O 2 and spreading the suspensions on chocolate agar plates to determine the concentration of H 2O 2 producing a 50 per cent reduction in colony-forming units (LD 50). Catalase activity was quantified with a dark-type oxygen electrode, which polarographically monitored the formation of dissolved oxygen in bacterial suspensions or sonicates following addition of reagent H 2O 2. Sensitivity to H 2O 2 did not correlate with catalase activity, either in intact cells or in bacterial sonicates. Specifically, some bacterial strains with undetectable catalase activity were highly resistant to H 2O 2. Micromolar concentrations of sodium azide which completely inhibited cell-associated catalase activity did not affect the resistance of A. actinomycetemcomitans to H 2O 2. Thus, the endogenous catalase activity of A. actinomycetemcomitans and certain oral haemophili is not an important determinant of resistance to the bactericidal effects of H 2O 2.

Resistance of Actinobacillus actinomycetemcomitans and differential susceptibility of oral Haemophilus species to the bactericidal effects of hydrogen peroxide.

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.

Distribution of oral Haemophilus species in dental plaque from a large adult population.

The periodontal status of maxillary first molars in 284 young adults demonstrating near-health to early disease was evaluated, and supragingival and subgingival plaque samples were collected. Plaque samples were processed anaerobically, enumerated microscopically for bacterial morphotypes, and cultivated on various media to enumerate the microflora. Although haemophili were ubiquitous (recovered in 98.5 and 96.2% of the supragingival and subgingival plaque samples, respectively), 50% of the respective samples had proportions of less than or equal to 1.5% and less than or equal to 0.33% total Haemophilus spp. based on total cultivable microflora. To study the distribution of Haemophilus spp., 377 colonies were identified from modified chocolate agar (selective for oral haemophili) from 14 supragingival and corresponding subgingival samples from 14 subjects. The most prevalent species, Haemophilus parainfluenzae, was found in significantly higher proportions, based on total haemophili on modified chocolate agar, in supragingival and subgingival samples from teeth with shallower probing depths (less than or equal to 3.0 mm) versus deeper probing depths (greater than or equal to 3.0 mm). Additional statistically significant findings included Haemophilus segnis in higher proportions in supragingival samples from deeper sites, Haemophilus aphrophilus in higher proportions in subgingival samples from deeper sites, and Haemophilus paraphrophilus in higher proportions in subgingival samples from shallower sites. Scatter diagrams illustrating the bivariate distributions of proportions of haemophili with proportions of dark-pigmented Bacteroides spp., spirochetes, and streptococci demonstrated that high proportions of haemophili were never recovered from sites with high proportions of Bacteroides spp. or spirochetes. All levels of haemophili, however, were recovered from sites with all levels of streptococci. Two potential systems for interpreting haemophili data were hypothesized for predicting periodontal probing depths. There was highly significant agreement between the two systems. Small but statistically significant correlations were found between the gingival index, probing depth, and attachment level, and proportions of total Haemophilus species in the respective samples.

The acid end-products of glucose metabolism of oral and other haemophili.

The acids produced in broth culture by various species of oral haemophili and by stock strains of capsulated and other haemophili were identified and measured by gas-liquid chromatography. Succinic acid was the major acid end-product of all strains, with acetic acid also being regularly produced but in smaller amounts. A stock strain, Haemophilus parainfluenzae NCTC 4101, produced less succinic acid than other strains of haemophili. Strain NCTC 4101 possessed all the enzymes of the tricarboxylic acid cycle, as previously reported, but in the other haemophili examined only succinic dehydrogenase, fumarase and malate dehydrogenase could be detected. No other enzymes of the tricarboxylic acid cycle were detected and isocitrate lyase, malate synthase and pyruvate carboxylase were also absent. Phosphoenolpyruvate-carboxylase was present in all strains. A partial tricarboxylic acid cycle and marked malate dehydrogenase activity appear to be characteristic of haemophili. The pathway to succinate in haemophili appears to be via carboxylation of phosphoenolpyruvate to oxalacetate and thence via malate and fumarate. The results of tracer studies on a single oral strain of H. parainfluenzae using various labelled substrates were in keeping with this proposed metabolic pathway.

Aspects of the pathogenicity of some oral and other haemophili.

The pathogenicity of the predominantly non-capsulated, V-factor requiring haemophili that are commonly recovered from oral infections has been explored by studies of their endotoxins and infectivity as compared with those of Haemophilus influenzae. Similar yields of endotoxin (2.40-2.59% w/w) were obtained from all the haemophili examined except H. haemolyticus (1.61% w/w). The lipopolysaccharide (LPS) extracts all contained heptoses but not 2-keto-3-deoxyoctonate (KDO). The fatty acid compositions of the lipid-A fractions were essentially similar but comprised 76% of the LPS in the H. influenzae type d strain tested and only 20% in the H. influenzae type b strain and some strains of H. parainfluenzae. All extracts contained arachidic acid, which may be unique to haemophili. The endotoxins from all strains produced characteristic pyrogenic and polymorph effects in rabbits. The endotoxins from the pharyngeal X- and V-factor-requiring strains had LD50 values for actinomycin-D-sensitised mice of 2.4-2.7 micrograms/kg, and were about eight times more potent than those from the oral V-factor-requiring strains (LD50 values 17.2-22.4 micrograms/ml). Approximately ten thousand times more free endotoxin was detected in broth cultures of H. influenzae type b than in those of oral haemophili, and this greater endotoxin release was not associated with a greater degree of autolysis. Endotoxin release from viable cells may be important in the pathogenicity of this organism. H. influenzae type b was much more potent in producing infection in chambers implanted subcutaneously in guinea pigs than were oral strains of haemophili; only 10 type b organisms were required, compared with 9 X 10(5) of H. parainfluenzae. However, in infections maintained for 90 days, the numbers of haemophili--c. 10(7)/ml of chamber fluid--were similar for all the test strains. Thus, although the oral haemophili lack special attributes of invasiveness and resistance to host defences, they are not devoid of pathogenic potential and, if allowed to proliferate, may become an important element in an infection.

Haemophili in developing dental plaque.

Evidence for a possible role played by oral haemophili in the development of dental plaque was sought by studying the occurrence of these bacteria in early dental plaque of smooth surfaces and occlusal fissures in six dental students. The mean number of haemophili per 10(3) anaerobes in early smooth surface plaque (18 h) and fissure plaque (7 d) was 95 and 22 respectively. Examination of 988 Haemophilus isolates revealed that H. parainfluenzae was the only species in samples of fissure plaque, whereas some samples from smooth surfaces, in addition to the predominating and ubiquitous H. parainfluenzae, yielded growth of the two species H. segnis and H. aphrophilus. It is concluded that haemophili are among the primary colonizers of smooth surfaces of teeth.

Haemophili and related bacteria in the human oral cavity

The occurrence of haemophili and related bacteria in saliva and dental plaque has been studied using a selective medium. Haemophili were isolated from all samples studied. The mean salivary count for 57 saliva specimens was 3.9 × 10 7, whereas the relative numbers found in 15 specimens of dental plaques were about three to five times lower. More than 1000 isolates were subjected to a high number of biochemical tests. Several species of Haemophilus were encountered, but H. influenzae was practically absent from all samples studied. Haemophilus parainfluenzae comprised the overwhelming majority of the isolates from saliva, whereas H. segnis constituted a significant proportion of the population of haemophili in dental plaques. H. paraphrophilus was present in a minority of the samples. Haemophilus aphrophilus, Actinobacillus actinomycetemcomitans and Eikenella corrodens were isolated from dental plaques only, and the former species only from 27 per cent of the samples. A practical scheme for the identification of oral haemophili and related bacteria is provided.

Lack of association between haemophili and human periodontal disease

Comparison of viable counts of haemophili in samples of saliva and periodontal pocket fluid, obtained from 20 subjects with periodontal disease, showed that there were significantly fewer haemophili in periodontal pockets. There were also significant differences in the types of haemophili isolated. The numbers of Haemophilus influenzae and beta-haemolytic strains of H. parainfluenzae were proportionately increased in periodontal pockets, whilst capsulated strains of H. parainfluenzae were often entirely absent. Although the numbers of non-haemolytic, non-capsulated, H. parainfluenzae were less in pockets than in saliva, most of the haemophili, in both locations, were of this type. The sera of the 20 subjects with periodontal disease and those from 10 healthy control subjects were examined for the presence of antibodies reactive with oral haemophili. All 30 sera were bactericidal for haemolytic and non-haemolytic, oral strains of H. parainfluenzae. The complement fixation, haemagglutination and indirect fluorescent antibody tests showed that all sera contained low levels of antibody, probably confined to the IgG class, reactive with oral haemophili. There were no significant differences in antibody titres between the group with periodontal disease and the controls. Antibodies reactive with capsule of the oral H. parainfluenzae (NCTC 10665) were not detected in any of the 30 sera.

Pathogenicity of human oral strains of haemophili: Enzymes, anaerobiosis and effects on mice and rabbits

Oral strains of Haemophilus parainfluenzae, including beta-haemolytic and capsulated strains, and H. influenzae were found to produce hyaluronidase when tested by the plate method. These organisms had no effect on azocoll powder or fibrin, and no chondroitin sulphatase, deoxyribonuclease, lecithinase or lipase activity could be demonstrated. In sealed Petri-dishes, haemophili were sufficiently effective in absorbing the available oxygen that the growth of Actinomyces israelii, Clostridium welchii and Veillonella alcalescens, but not Clostridium tetani, was facilitated. When mice were injected with saline suspensions of oral haemophili, the results were haphazard. However, when the organisms were suspended in dextran sulphate, a reproducible doseresponse relationship was established and ld 50's ranging between 2.8 × 10 6 and 29.2 × 10 6 were determined. Oral haemophili elicited the Shwartzman reaction in rabbits.

Continuous recording of blood oxygen tensions by polarography.

Continuous recording of blood oxygen tensions by polarography.