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This study sought to develop an immunolabelling technique to identify specific bacteria by scanning electron microscopy. Bacterial suspensions were prepared of Actinobacillus actinomycetemcomitans, Porphyromonas (Bacteroides) gingivalis and mixtures of these species with other genera of common oral microorganisms. This method was also used to examine subgingival plaques from 6 subjects with chronic adult periodontitis. Sample preparation consisted of prefixation of the bacterial suspensions with 0.2% glutaraldehyde, incubation with species-specific rabbit antisera and goat anti-rabbit IgG conjugated with colloidal gold particles, postfixation in 2% glutaraldehyde and dehydration in ethanol. Finally, the samples were dried, coated with evaporated carbon and examined by scanning electron microscopy. Pure cultures and artificial mixtures of A. actinomycetemcomitans and Bact. gingivalis were specifically labelled by gold probes as demonstrated by both secondary and back-scattered imaging. These species were also evident in samples of subgingival plaque. The findings indicate that this new technique can be used to identify specific microorganisms both in plaque samples and on the root surface of extracted teeth.

In order to study the role of membrane proteins in mucosal colonization by Actinobacillus actinomycetemcomitans, a plasmid library was constructed by ligating EcoRI-digested genomic DNA from A. actinomycetemcomitans strain Y4 into EcoRI-digested and dephosphorylated pUC13 DNA. A second library was constructed by ligating Sau3A-digested and size-fractionated A. actinomycetemcomitans strain Y4 DNA into BamHI-cleaved and dephosphorylated pUC13 DNA. The DNA was transformed into Escherichia coli strain MC1022 and plated onto Luria-Bertani agar containing 100 micrograms/ml ampicillin and X-gal. Recombinant colonies were examined for the expression of A. actinomycetemcomitans antigens by colony immunoscreening using rabbit antiserum to strain Y4. Nine positive clones were isolated. Three of these clones produced proteins that reacted with rabbit antiserum to strain Y4 on a Western transfer, and seven of these clones reacted with rabbit antiserum to strain Y4 as measured by indirect immunofluorescence. One reactive clone contained an 8.4 kb plasmid, which directed the synthesis of a peptide with an Mr of 20,000 as measured by SDS-PAGE. Partial DNA sequence analysis revealed the presence of a signal-peptide leader sequence at the amino terminus suggesting that the native protein is a membrane component of A. actinomycetemcomitans. Thus A. actinomycetemcomitans antigens can be expressed in E. coli, and some of these proteins may be expressed on the E. coli cell surface.

Actinobacillus actinomycetemcomitans and the genetically-related oral haemophili ( Hae mophilus segnis , Haemophilus aprhophilus and Haemophilus paraphrophilus ) exhibit a range of sensitivities to the lethal effect of hydrogen peroxide (H 2 O 2 ), A. actinomycetemcomitans being the most resistant. To extend this information, susceptibility to a range of H 2 O 2 concentrations (10 −6 -10 −3 M) was assessed by incubating bacterial suspensions for l h at 37°C in the presence of H 2 O 2 and spreading the suspensions on chocolate agar plates to determine the concentration of H 2 O 2 producing a 50 per cent reduction in colony-forming units (LD 50 ). Catalase activity was quantified with a dark-type oxygen electrode, which polarographically monitored the formation of dissolved oxygen in bacterial suspensions or sonicates following addition of reagent H 2 O 2 . Sensitivity to H 2 O 2 did not correlate with catalase activity, either in intact cells or in bacterial sonicates. Specifically, some bacterial strains with undetectable catalase activity were highly resistant to H 2 O 2 . Micromolar concentrations of sodium azide which completely inhibited cell-associated catalase activity did not affect the resistance of A. actinomycetemcomitans to H 2 O 2 . Thus, the endogenous catalase activity of A. actinomycetemcomitans and certain oral haemophili is not an important determinant of resistance to the bactericidal effects of H 2 O 2 .