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

Date Published: February 2, 2017

Publisher: Public Library of Science

Author(s): Peter S. Zilm, Victor Butnejski, Giampiero Rossi-Fedele, Stephen P. Kidd, Suzanne Edwards, Krasimir Vasilev, Riccardo Manganelli.


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.

Partial Text

The invasion by bacteria and their by-products into the pulp and periapical tissues respectively give rise to pulpal necrosis and apical periodontitis [1]. In fact, it is the bacterial biofilms that are the most likely etiological agent of primary and post-treatment apical periodontitis [2].

It has been suggested that contemporary root-canal treatment should progress into “mechano-chemo-biological” procedures [12]. Possible strategies include weakening the microbial biofilm structure per se [12] so that the antimicrobial agent is more effective. The biofilm breaking effects of four DAAs (D-Leu, D-Met, D-Trp, and D-Tyr) has been demonstrated in B. subtilis, Staphylococcus epidermidis isolated form ocular infections [13, 19] and Li et al. (2016) [20] reported that an equimolar concentration of a mixture of DAAs acts as a biocide enhancer against a multispecies biofilm prevalent in industrial pipes. Interestingly the effect could not be reproduced using individual amino acids, so, in the present study, it was decided to only examine the anti-biofilm effect of a mixture of DAAs.




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