Molecular Genetic Analysis of Actinobacillus actinomycetemcomitans Epidemiology

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Abstract
Research over the past decade has identified many of the microorganisms involved in the etiology of human periodontitis such as Actinobacillus actinomycetemcomitans. Efforts are now directed toward defining these species' role in the pathogenic process. Since microbial colonization of host tissues is a key first step in developing a bacterial infection, determining the source of the periodontal pathogens and their route of transmission is likely to be crucial in formulating preventive strategies. Recently, a technique from molecular biology, restriction endonuclease analysis, has been used to track bacterial infections. In the present study, this method was used to investigate the epidemiology of A. actinomycetemcomitans infection. One hundred twenty-four human subgingival plaque isolates of A. actinomycetemcomitans were examined including bacterial strains from the United States, Korea, and Norway as well as 15 strains from cynomolgus (Macaca fasicularis) and spider monkeys {Macaca iris) and 4 reference strains. The genomic DNA from each strain was purified, digested with each of 16 restriction endonucleases, and the DNA digests were resolved by electrophoresis. The resulting patterns of DNA fragments were compared and also correlated with the A. actinomycetemcomitans serotype determined using serotype-specific antisera in immunofluorescence. Human isolates of A. actinomycetemcomitans even from disparate geographic sources showed little diversity by restriction endonuclease analysis. Three major restriction patterns were found. Restriction pattern I was common to all 20 of the serotype a isolates, restriction pattern II was associated with 58% of the 73 serotype b isolates examined, while restriction pattern III was associated with the remaining serotype b strains and with all 15 of the serotype c strains. All A. actinomycetemcomitans isolates from a single site, or from several sites in the same patient, or from a single patient over time showed the same restriction pattern suggesting a clonai infection by this microorganism. By contrast, strains of A. actinomycetemcomitans isolated from non-human primates revealed numerous and distinct restriction endonuclease patterns none of which resembled human strains. These results are consistent with the idea that human oral infection by A. actinomycetemcomitans is clonal in any one patient and that human oral infection is associated with a limited number of possible genotypes. Restriction endonuclease analysis of genomic DNA from A actinomycetemcomitans and other periodontal pathogens provides a powerful tool for analyzing the microbial ecology of subgingival plaque as well as determining the mode of transmission and acquisition for individual species. J Periodontol 1990;61:75–80.