This proposal is designed to provide information concerning the development of bacterial resistance to antibiotics that are frequently used as adjuncts in the treatment of destructive periodontal disease and to elicit the molecular mechanisms involved. Patients have been encountered whose disease condition does not respond to conventional therapy combined with the adjunctive use of tetracycline antibiotics. The microbiotas associated with sites undergoing active destruction were initially susceptible to tetracyclines prior to therapy but demonstrated significant resistance following therapy. The post-therapy microflora closely resembles that associated with refractory periodontitis. The predominant cultivable bacteria present before and after therapy have been characterized and identified. Minimal inhibitory concentrations (MICs) will be determined by agar dilution technique for these bacteria to determine if significant differences exist within species present both before and after therapy. Mechanisms of bacterial resistance which may account for the failure of a patient to favorably respond to antimicrobial therapy will be investigated both functionally and at the genetic level. B-lactamase enzymes produced by periodontal strains of Bacteroides intermedius and Eikenella corrodens will be characterized to determine if differences exist in the enzymes produced within the species and if such enzymes are chromosomal or plasmid mediated. The in vivo effect of bacterial B-lactamases on B-lactam antibiotics in gingival crevicular fluid will be determined. Gingival fluid concentrations of amoxicillin, a B-lactamase susceptible penicillin, and Augmentin, which contains amoxicillin and a B-lactamase blocker, will be individually monitored over time in sites with and without B-lactamase activity. Comparisons will be made of the antibiotic levels maintained to determine the in vivo effect of B-lactamase on these antibiotics. Plasmid carriage, an important mechanism which often accounts for acquired antibiotic resistance in bacteria, will be investigated in strains of B. intermedius and E. corrodens to determine if plasmids encode for resistance in these organisms. Experiments will be conducted t determine if these plasmids encode for single or multiple antibiotic resistance by curing the strain of its plasmid(s) and determining the effect of the loss of plasmid carriage on susceptibilities to specific antibiotics. Confirmation of plasmid-encoded resistance will be performed by transferring the plasmid into a susceptible donor either by conjugation or electroporation. Plasmid transmission will be investigated to determine if antibiotic resistance may be transferred between strains of a species as well as between species. Plasmid transfer will be tested by conjugation using a membrane filter mating technique. DNA probes will be constructed of the portion of the plasmid or chromosomal sequence which encodes for resistance to an antibiotic. These probes will be used to determine if similar DNA sequences are present in strains within the same species and in different species.
