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.

Actinobacillus actinomycetemcomitans is a key microorganism in the pathogenesis of several different forms of periodontal diseases. Identification of this bacterium from clinical specimens may often be complicated by the fact that the colony morphology on TSBV selective medium closely resembles that of Haemophilus aphrophilus and a key differentiating characteristic, catalase reaction, may be variable. Recent genetic studies have shown that the 23S ribosomal RNA molecule is split into two smaller forms in A. actinomycetemcomitans, but is intact in H. aphrophilus. Based on this finding, we describe a new, rapid method for identifying A. actinomycetemcomitans in which single colonies isolated from culture on TSBV agar in 5% CO2 in air are lysed, electrophoresed on 1.5% submarine agarose gels and visualized by staining with ethidium bromide. Using this assay, A. actinomycetemcomitans can be easily distinguished from morphologically similar colonies such as H. aphrophilus strains by differences in 23S rRNA within 2 h.

Eikenella corrodens is a gram-negative facultative bacillus commonly found in the oral cavity. Although the role of E. corrodens in periodontitis is not clear, its isolation from extraoral infections attests to its pathogenic potential. Previous studies suggested that this species is phenotypically diverse. In the present study, we used restriction endonuclease analysis (REA) to assess the genetic diversity of this species and to explore the applicability of REA in studying the transmission of E. corrodens. Two groups of E. corrodens isolates were used in this study. Group 1 included 47 epidemiologically independent isolates recovered from dental plaques in periodontally healthy subjects and periodontitis patients and from extraoral infections in different geographic areas. Group 2 E. corrodens included 40 isolates recovered from two periodontitis patients and two periodontally healthy subjects. The results indicated that E. corrodens is genetically heterogeneous, as determined by REA. The majority of the group 1 E. corrodens isolates exhibited strain-specific restriction patterns. Forty restriction patterns were distinguishable among the 47 isolates. Analyses of group 2 isolates revealed that three of four subjects harbored more than one clonal type of E. corrodens. In one instance, a periodontitis patient was found to be colonized by six different clones. Furthermore, two different clonal types of E. corrodens were recovered from a single periodontal pocket in this patient. The results indicated that REA may be a useful tool in the epidemiologic investigation of E. corrodens infections.

Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus which can cause certain severe extra-oral infections as well as forms of human periodontal disease such as localized juvenile periodontitis. In contrast to many prokaryotic and eukaryotic species which exhibit an intact 23S ribosomal RNA (rRNA) molecule, examination of six A. actinomycetemcomitans strains--including three serogroup representative strains and two strains from non-human primates--revealed that this micro-organism does not produce an intact 23S ribosomal RNA (rRNA) molecule but, rather, two smaller forms of 1.8 kb and 1.2 kb designated as 23S alpha and 23S beta fragments. On the other hand, 14 other strains of Actinobacillus, Haemophilus, and Pasteurella species demonstrated intact 23S rRNA. The sequence of the region of the 23S rRNA gene in A. actinomycetemcomitans strain ATCC 43718 containing the cleavage site was determined by dideoxynucleotide sequencing, while the location of the 3' and 5' termini of the 23S alpha and 23S beta fragments was resolved by S1 nuclease mapping and cDNA primer-extension. A deletion of 112 bases was noted in comparisons of base sequences from A. actinomycetemcomitans rRNA and rDNA. The DNA intervening sequence was localized to nucleotide 1180 of the Escherichia coli 23S rRNA map. While the primary structure of the gap region showed little homology with the gap regions described in other organisms, the secondary structure was similar to that previously described in the parasitic helminth Schistosoma japonicum. Restriction enzyme and nucleotide sequence analysis of the gap region in eight other A. actinomycetemcomitans strains showed it to be highly conserved.

Ribosomal RNA (rRNA) isolated from Wolinella recta and seven related bacteria was examined by agarose gel electrophoresis. The 23S rRNA molecule could not be detected in W. recta, Wolinella curva, Bacteroides gracilis, or Bacteroides ureolyticus. In place of the 23S molecule, there were three smaller molecules of approximately 1700, 650, and 600 bases designated 23S alpha, 23S beta, and 23S delta, respectively. An intact 23S rRNA molecule could be isolated from Wolinella succinogenes, Campylobacter concisus, and Campylobacter sputorum. The cleavage sites of the W. recta 23S rRNA molecule were located by direct RNA sequence analysis and were found to be in similar locations, nucleotides 546 and 1180, as cleavage sites described in other prokaryotes. The presence or absence of the 23S rRNA molecule may be a useful marker for these micro-organisms.