CHARACTERIZATION OF HEMOLYTIC BACTERIA IN SUBGINGIVAL PLAQUE

Three-quarters of the patients with periodontal diseases surveyed in t his study had one or more distinct types of hemolytic bacteria in thei r subgingival plaque. Twelve different species of bacteria were identi fied, belonging to five genera (Actinomyces, Streptococcus, Staphyloco ccus, Prevotella, and Actinobacillus). Nine hemolytic isolates, consis ting of four Prevotella denticola strains, two Actinomyces naeslundii genospecies 2 strains, and one each of P. melaninogenica, Streptococcu s constellatus, and A. naeslundii genospecies 1 strains were character ized. Incorporation of pronase into blood agar medium inhibited hemoly sis by all of the isolates, suggesting a proteinaceous component for e ach of their hemolysins. With one exception, hemolysin production appe ared to be regulated by the concentration of environmental iron: exoge nous hemin was found to inhibit hemolysin production, and the iron sca venging compound, 2,2'-dipyridyl, was found to promote hemolysin produ ction by all of the strains except for the S. constellatus isolate. Ge nomic libraries of each of the hemolytic plaque isolates were prepared in Escherichia coli using pBR322. Hemolytic clones were isolated on b lood agar medium containing ampicillin at frequencies ranging from 1-6 .7 x 10(-4). Extensive restriction mapping revealed regions of homolog y in the case of clones derived from three P denticola strains isolate d from the same subject. Two of the P denticola-derived clones were vi rtually identical throughout the entirety of their > 5 Kb inserts. The clone derived from the third strain showed good homology to the other two within a 1.3 Kb region, but the flanking DNA showed no homology e ven though all three P denticola isolates were shown to be clonally re lated by ribotyping. The results indicate that hemolytic bacteria are frequently recovered from active disease sites of subjects with period ontal diseases. The hemolytic phenotype appears to be restricted to a small proportion of the total number of species normally resident in s ubgingival plaque. Restriction mapping suggested that a variety of hem olysin genes may be involved and that, at least in certain cases, they may be on mobile genetic elements.