Objective: The aim of this investigation was to study the biochemical mechanisms employed by the endodontic pathogen Enterococcus faecalis to survive alkaline environment during biofilm formation.
Design: E. faecalis ATCC33186 was inoculated in media at pH 7, 9, 10 and 11 for biofilm formation. The morphology and ultrastructure of biofilm cells were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The physiochemical properties of the cell wall were investigated by measuring the hydrophobicity and Na(+)K(+)-ATPase and H(+)K(+)-ATPase activity. The expression of stress and virulence genes was quantified by real-time quantitative polymerase chain reaction.
Results: E. faecalis grown in alkaline medium developed an irregular shape and asymmetrical septation. The activity of Na(+)K(+)-ATPase increased dramatically with rising pH, whereas the activity of H(+)K(+)-ATPase exhibited no increase, except at pH 10. A marked increase in cell surface hydrophobicity was also observed with increased pH and time. In addition, transcription of most of the genes tested increased 2- to 15-fold at pH 9 or 10 compared with pH 7 and increased more than 50-fold at pH 11, which is generally recognised as nearly lethal stress.
Conclusions: E. faecalis survival and biofilm formation under alkaline stress was unrelated to H(+)K(+)-ATPase but was correlated with an increase in Na(+)K(+)-ATPase activity and cell-surface hydrophobicity in addition to the up-regulation of genes involved in stress response and biofilm formation. These characteristics may explain why E. faecalis resists alkaline root canal medications.
Keywords: ATPase activity; Alkaline pH; Biofilm; Enterococcus faecalis; Gene expression; Hydrophobicity.
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