Actinobacillus actinomycetemcomitans and Periodontal Disease (Laboratory and Clinical Studies)

Actinobacillus actinomycetemcomitans has been regarded as one of the major microbiological aetiological agents in certain types of human periodontal disease especially Localized Juvenile Periodontitis. However, relatively few reports are available concerning its prevalence in chronic periodontitis, and there is some doubt concerning the laboratory methods used to isolate and identify A. actinomycetemcomitans from subgingival plaque. In the present investigation the ability of two semi-selective media (TSBV and MGB) to isolate A. actinomycetemcomitans from dental plaque samples was examined. TSBV medium was found to be less inhibitory with pure cultures and more selective in mixed cultures than MGB. In the laboratory, the presence of star-shaped colonies on primary culture plates is commonly used to differentiate A. actinomycetemcomitans from other bacteria, even although non-star-shaped variants are known to occur. Since there is no information concerning the presence of star-shaped and non-star-shaped colonies on primary isolation by A. actinomycetemcomitans in the literature, the prevalence of both colonial forms of A. actinomycetemcomitans on primary culture on TSBV medium was investigated in a subgroup of samples collected from patients with chronic periodontitis (see below). Surprisingly more than half of the isolates (52%) produced non-star-shaped colonies, the remainder having the star-shaped format. As a result if only star-shaped colonies had been selected for identification in this thesis, the prevalence data for the chronic periodontitis patients would have been reduced from 38 to 19 patients. Since no standard method for identifying A. actinomycetemcomitans was available, the API 20 A system was used in this thesis. Four different biochemical profiles were obtained, and the system was found to be simple to use and gave reproducible results. In a clinical study of the prevalence of A. actinomycetemcomitans in 98 patients with chronic periodontitis (302 subgingival samples), the organism was isolated from 39% of patients and from 27% of the total samples collected. However, in a healthy control group of 55 volunteers only one (2%) of the subjects possessed A. actinomycetemcomitans. In the chronic periodontitis group there were 55 females and 43 males, and although A. actinomycetemcomitans was isolated overall from more sites in male patients (60%) compared to female patients (40%), the distribution of A. actinomycetemcomitans was found to be similar in both groups. In general there were no clear differences between the prevalence of A. actinomycetemcomitans in the six different segments sampled in the 98 patients. Although adherence and hydrophobicity are believed to play an important role in the pathogenicity of a number of bacteria, little is known about these properties with regard to A. actinomycetemcomitans. In addition, previously published studies have used small numbers of strains. In the present investigation the ability of 33 strains of A. actinomycetemcomitans to adhere to human buccal epithelial cells in the presence of Saliva Ions Buffer (SIB), clarified mixed saliva and serum, using an epifluorescence technique was assessed in vitro. The hydrophobicity of the same 33 strains was assessed using adsorption to xylene. The adherence results clearly showed that overall A. actinomycetemcomitans can adhere well to buccal epithelial cells in vitro and in comparable numbers when experiments were performed in mixed saliva and in SIB. The adherence values were 12.7 and 12.0 adherent bacteria per buccal epithelial cell respectively. Similarly there was no significant difference between the adherence of fresh isolates (mean 13.3 bacteria) compared to the Type strains (10.7). The addition of human serum to the adherence assay system significantly inhibited the adherence of A. actinomycetemcomitans strains to buccal cells (p <0.0005). All isolates of A. actinomycetemcomitans were hydrophobic and produced a reduction in absorbance which ranged from 39% to 82% (mean 67%). No significant difference between the hydrophobicity of fresh and Type strains of A. actinomycetemcomitans was found. When the adherence to buccal epithelial cells and hydrophobicity results for each strain were compared a significant overall correlation (p