A sensitive enzymatic method (SK‐013) for detection and quantification of specific periodontopathogens

Abstract

Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola have been found to predominate in periodontal pockets of patients with adult periodontitis. These microorganisms hydrolyze the synthetic peptide N-benzoyl-DL-arginine-2-naphthylamide (BANA). In this study, we developed an enzymatic method, designated SK-013, to detect the existence of these microorganisms in subgingival plaque bacteria. This enzymatic method was based on the observation of the hydrolysis of N-carbobenzoxy-glycyl-glycyl-arginyl-3,5-dibromo-4-hydroxyaniline (N-CBz-Gly-Gly-Arg-DBHA) and made more sensitive by adding an enhancing system. The SK-013 was specifically positive for P. gingivalis, B. forsythus, T. denticola, and some strains of Capnocytophaga species, but was not specific for any of the other bacterial strains tested. This SK-013 system may be valuable for detection and quantification of periodontal disease-associated bacteria in subgingival plaque and thus for diagnosis of periodontal infections.

Investigation of Subgingival Profile of Periodontopathic Bacteria Using Polymerase Chain Reaction

“…have been detected by several methods: anaerobic culture 15) , microscopy 11) , enzyme reaction 6) , immunohistology 8) , DNA probe 9) and polymerase chain reaction (PCR) 2) . The culture method is time-consuming and requires specific techniques.…”

Expression of trypsin-like activity by the genera Corynebacterium and Actinomyces in canine periodontitis

“…The three species, Por. gingivalis, B. forsythus and T. denticola, are always positive for BANA hydrolysis [6,7,9]. Other BANA hydrolytic species in man were reported to include some Capnocytophaga spp.…”

“…The accumulation of plaque and calculus on tooth surfaces, particularly at the gingival margin in dogs, causes an increase in the number of micro-organisms within the gingival pocket and progression and development to periodontitis. Diagnostic tests for human periodontal infections have been developed based on the hydrolysis of trypsin-like enzyme substrates BANA and DBHA by micro-organisms in subgingival plaque and crevicular¯uid [7,15,16]. However, there are very few BANA-positive species among the cultivable plaque¯ora [17].…”

“…TLA was measured by the SK-013 method [7], now commercially developed as the Periocheck periodontal diagnostic kit (Sunstar, Takatsuki, Japan). Brie¯y, three paper points were placed in the pocket for 30 s, then placed into a small vial containing substrate (DBHA), chromogen and ascorbic oxidase solution, followed by agitation for 10 s. The mixture was then incubated at 378C for 15 min.…”

“…These bacteria hydrolyse the synthetic trypsin substrates Nbenzoyl-DL-arginine-2-naphthylamide (BANA) and N-carbobenzoxy-glycyl-glycyl-arginyl-3,5-dibromo-4-hydroxyaniline (DBHA). BANA and DBHA have been used to develop rapid and simple methods for diagnosis and monitoring of periodontal therapy in human periodontitis [6,7].…”