Objectives:Rough type strains of Actinobacillus actinomycetemcomitans form biofilms on the glass surface of a test tube when cultured in broth. Repeating subculture results in a conversion of the rough morphological type to a smooth type. The smooth type exhibits turbid growth in broth. It has been reported that susceptibility to antibiotics differs between bacteria in biofilms and planktonic cells. This study aimed to examine the relationship between A. actinomycetemcomitans biofilm formation and antibiotic susceptibility. Methods:Rough and smooth type strains of A. actinomycetemcomitans 310a and AB55 were subjected. We determined their antibiotic susceptibility to 0.1, 1.0 or 10mg of erythromycin(EM), ofloxacin(OFX), and ampicillin(AMP). A. actinomycetemcomitans cells precultured for 40-h were inoculated into the wells of a 96-well microtiterplate. Antibiotics were added at the culture starting time or after 24-h of culture. After 48-h of incubation under anaerobic conditions, cell growth was evaluated by measuring the absorbance at 595 nm using a microplate reader. Then free-floating bacteria were removed by aspiration. The biofilms were stained with 0.1% crystal violet, and the absorbances were measured at 595 nm. Results:The rough type of A. actinomycetemcomitans strains formed microcolonies at the bottoms of the wells. In contrast, the smooth type attached slightly and uniformly. When antibiotics were added at the starting time, the cell growth of rough type was not much inhibited by any of the used antibiotics at 0.1-10mg/ml, but the smooth type was significantly inhibited by all the used antibiotics at 1.0-10mg/ml, especially by EM and AMP in a dose-dependent manner. When OFX was added to 24-h biofilm cultures, cell growth and biofilm formation of the rough type were not reduced, but those of the smooth type were reduced about 50%(P<0.05). Conclusion:This study showed that rough type A. actinomycetemcomitans can form such a stable biofilm that the antibiotic susceptibility declines.

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