D-amino acids reduce Enterococcus faecalis biofilms in vitro and in the presence of antimicrobials used for root canal treatment.

Enterococcus faecalis is the most frequent species present in post-treatment disease and plays a significant role in persistent periapical infections following root canal treatment. Its ability to persist in stressful environments is inter alia, due to its ability to form biofilms. The presence of certain D-amino acids (DAAs) has previously been shown to reduce formation of Bacillus subtilis biofilms. The aims of this investigation were to determine if DAAs disrupt biofilms in early and late growth stages for clinical E. faecalis strains and to test their efficacy in disrupting E. faecalis biofilms grown in sub-minimum inhibitory concentrations of commonly used endodontic biocides. From thirty-seven E. faecalis strains, the ten "best" biofilm producers were used to test the ability of a mixture containing D-leucine, D-methionine, D-tyrosine and D-tryptophan to reduce biofilm growth over a period of 24, 72 and 144 hours and when compared to their cognate L-Amino Acids (LAAs). We have previously shown that sub-MIC levels of tetracycline and sodium hypochlorite promotes biofilm growth in clinical strains of E. faecalis. DAAs were therefore tested for their effectiveness to reduce biofilm growth in the presence of sub-minimal concentrations of sodium hypochlorite (NaOCl-0.031%) and Odontocide™ (0.25% w/v), and in the presence of Odontopaste™ (0.25% w/v). DAAs significantly reduced biofilm formation for all strains tested in vitro, while DAAs significantly reduced biofilm formation compared to LAAs. The inhibitory effect of DAAs on biofilm formation was concentration dependent. DAAs were also shown to be effective in reducing E. faecalis biofilms in the presence of Odontopaste™ and sub-MIC levels of NaOCl and Odontocide™. The results suggest that the inclusion of DAAs into current endodontic procedures may reduce E. faecalis biofilms

Mean optical density of the Planktonic cultures during biofilm growth.

<p>The mean optical density (570nm) of the planktonic culture removed after 1, 3 and 7 days incubation in the presence of 2.5mM (A) or 25mM (B) L-Leu, L-Met and L-Trp (0.25mM D- Tyr in both treatments). Asterisk denotes a significant reduction (p<0.05) in biofilm compared to the control (no DAAs).</p

<i>E</i>. <i>faecalis</i> strains supplied by the Microbiology and Infectious Disease Laboratory, SA Health, South Australia.

<p><i>E</i>. <i>faecalis</i> strains supplied by the Microbiology and Infectious Disease Laboratory, SA Health, South Australia.</p

Mean optical density of each E. <i>faecalis strain</i>.

<p>Mean optical density of the ten strains used in the study. Optical density (570nm) was proportional to biofilm growth as determined by the level of crystal violet staining of the biofilm. The x-axis contains the identification of each <i>E</i>. <i>faecalis</i> strain.</p

Mean density (570nm) of Control group (no D-amino acids) versus treatment groups.

<p>Biofilm growth following staining of E. faecalis biofilms with crystal violet. The medicaments Odontopaste^™ (Odont), Odontocide^™ (CH) and the irrigant, sodium hypochlorite (NaOCl) were added separately with D-Leu, D-Met and D-Trp (25mM) and 0.25mM D-Tyr at the time of inoculation for the 1day biofilm, then 1 day and 3 days for the 3 and 6 day biofilm respectively. Asterisk denotes a significant reduction in biofilm growth compared to controls (medicament or irrigant only).</p