Characterization of hemolytic bacteria in subgingival plaque

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Abstract
Three‐quarters of the patients with periodontal diseases surveyed in this study had one or more distinct types of hemolytic bacteria in their subgingival plaque. Twelve different species of bacteria were identified, belonging to five genera (Actinomyces, Streptococcus, Staphylococcus, Prevotella, and Actinobacillus). Nine hemolytic isolates, consisting of four Prevotella denticola strains, two Actinomyces naeslundii genospecies 2 strains, and one each of P. melaninogenica, Streptococcus constellatus, and A. naeslundii genospecies 1 strains were characterized. Incorporation of pronase into blood agar medium inhibited hemolysis by all of the isolates, suggesting a proteinaceous component for each of their hemolysins. With one exception, hemolysin production appeared to be regulated by the concentration of environmental iron: exogenous hemin was found to inhibit hemolysin production, and the iron scavenging compound, 2,2′‐dipyridyl, was found to promote hemolysin production by all of the strains except for the S. constellatus isolate. Genomic libraries of each of the hemolytic plaque isolates were prepared in Escherichia coli using pBR322. Hemolytic clones were isolated on blood agar medium containing ampicillin at frequencies ranging from 1–6.7 × 10−4. Extensive restriction mapping revealed regions of homology in the case of clones derived from three P. denticola strains isolated from the same subject. Two of the P. denticola‐derived clones were virtually 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 even though all three P. denticola isolates were shown to be clonally related by ribotyping. The results indicate that hemolytic bacteria are frequently recovered from active disease sites of subjects with periodontal diseases. The hemolytic phenotype appears to be restricted to a small proportion of the total number of species normally resident in subgingival plaque. Restriction mapping suggested that a variety of hemolysin genes may be involved and that, at least in certain cases, they may be on mobile genetic elements.

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