Effects of triclosan and triclosan monophosphate on maximum specific growth rates, biomass and hydrolytic enzyme production of Streptococcus sanguis and Capnocytophaga gingivalis in continuous culture

Dental plaque species, Streptococcus sanguis and Capnocytophaga gingivalis, were grown in continuous culture with progressively increasing concentrations of triclosan or its phosphorylated derivative, triclosan monophosphate (TMP). For both organisms, the maximum specific growth rates decreased with increasing concentrations of triclosan or TMP until complete inhibition of growth occurred, which for S. sanguis was at 20 mg/L and 50 mg/L, and for C. gingivalis was at 10 mg/L and 5 mg/L for triclosan and TMP respectively. For both species, biomass levels remained approximately constant or, in some cases, increased slightly at low levels of triclosan or TMP. However, biomass levels then decreased significantly as the triclosan or TMP concentrations approached lethal levels. For S. sanguis, levels of hydrolytic enzymes (acid phosphatase, leucine aminopeptidase and esterase) generally remained approximately constant or increased with increasing concentrations of triclosan or TMP until close to inhibitory levels where enzyme levels were reduced. The ratio of extracellular soluble enzymes to cell-bound enzymes remained constant or increased slightly with increasing levels of triclosan or TMP. For C. gingivalis, production of hydrolytic enzymes (neutral phosphatase, leucine aminopeptidase and trypsin-like protease) remained constant or were reduced when grown with low levels of triclosan and TMP but in some cases increased with higher levels of agents. The proportion of extracellular soluble activity increased significantly when concentrations of agent neared inhibitory levels. The results taken together show that the physiology of cells is significantly altered and that hydrolytic enzymes are released from the cells when these are grown in the presence of increasing concentrations of triclosan or TMP. Enzyme release is more pronounced in the Gram-negative C. gingivalis and indicates that triclosan or TMP can cause membrane perturbation with subsequent release of membrane-located (S. sanguis) or periplasmic (C. gingivalis) hydrolytic enzymes. S. sanguis was more sensitive to triclosan than TMP while C. gingivalis was more sensitive to TMP. This suggests that, in C. gingivalis, TMP may diffuse into the cell wall more easily than triclosan and then be converted to triclosan by phosphatase activity within the cell wall complex, where it may give rise to high localized concentrations and subsequent cell damage.