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

RESEARCH ARTICLE D-amino acids reduce Enterococcus faecalis biofilms in vitro and in the presence of antimicrobials used for root canal treatment Peter S. Zilm1☯*, Victor Butnejski1☯, Giampiero Rossi-Fedele1, Stephen P. Kidd2, Suzanne Edwards3, Krasimir Vasilev4 1 Microbiology laboratory, The School of Dentistry, The University of Adelaide, Adelaide, South Australia, Australia, 2 Australian Centre for Antimicrobial Resistance Ecology, Research Centre for Infectious Disease, School of Biological Science, The University of Adelaide, Adelaide, South Australia, Australia, 3 School of Public Health, The University of Adelaide, Adelaide, South Australia, Australia, 4 School of Engineering, University of South Australia, Adelaide, South Australia, Australia a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Zilm PS, Butnejski V, Rossi-Fedele G, Kidd SP, Edwards S, Vasilev K (2017) D-amino acids reduce Enterococcus faecalis biofilms in vitro and in the presence of antimicrobials used for root canal treatment. PLoS ONE 12(2): e0170670. doi:10.1371/journal.pone.0170670 Editor: Riccardo Manganelli, University of Padova, Medical School, ITALY Received: September 5, 2016 Accepted: January 9, 2017 Published: February 2, 2017 Copyright: © 2017 Zilm et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. ☯ These authors contributed equally to this work. * Abstract 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, Dtyrosine 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 (NaOCl0.031%) 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. Funding: The authors received no specific funding for this work. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0170670 February 2, 2017 1 / 14 D-amino acids reduce Enterococcus faecalis biofilms Introduction 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]. The chemo-mechanical preparation of a root canal is not sufficient to completely eliminate bacteria from the root canal. The use of an intra-canal medicament placed in-between appointments has been suggested to achieve the maximum reduction of bacterial load prior to obturation [3]. The most common reason for root canal treatment failure and the persistence of apical periodontitis is the presence of intra-radicular bacteria that had not been eliminated during endodontic therapy [2]. The use of calcium hydroxide as an intra-canal medication has been associated with periradicular healing and high antibacterial efficacy [4]. Antimicrobial agents within medicaments must be able to penetrate into the many dentinal tubules, apical deltas and accessory canals and reach a concentration that will eliminate disease causing bacteria. Calcium hydroxide has an inherently high pH and acts by damaging cytoplasmic membranes, interfering with enzymatic reactions and denaturing DNA [4, 5]. Calcium hydroxide increases the pH of the main root canal to about 12.2, however a pH of only 7.4 to 9.6 is shown to be achieved in peripheral dentine [6]. This is supported by other studies have shown that hydroxyl ions take 1–7 days to reach the outer root dentine and 3–4 weeks to reach peak pH levels [4]. Within this time, bacteria may respond by eliciting a stress response which promotes survival and persistence. One such example is an increase in biofilms. Enterococcus faecalis is the most frequent species isolated from obturated root canals, with prevalence values reaching up to 90% [7–9]. Importantly its ability to penetrate deep within dentinal tubules enables the organism to escape the effects of endodontic medicaments, irrigants and instruments [6]. E. faecalis is also resistant to high pH and is extremely proficient at invading dentinal tubules where the pH increases over an extended period of time because of the buffering effect of organic matter [6]. The persistence of E. faecalis can also be attributed to the biofilm which acts as a defensive mechanism when exposed to environmental stresses [10, 11]. Thus the development of biological anti-biofilm strategies has been attracting considerable interest in endodontology [12]. Kolodkin-Gal et al. (2010) [13] demonstrated that four D-amino acids, (DAAs) D-leucine, D-methionine, D-tryptophan, and D-tyrosine were produced at late stages of biofilm growth and could disrupt axenic Bacillus subtilis biofilms. It was concluded that biofilm disruption resulted from the displacement of D-alanine in the peptide side chains of peptidoglycan. This is thought to trigger the release of TasA fibres from the cell wall releasing the bacteria from the biofilm. Leiman et al. (2013) [14] also investigated the mechanism by which DAAs disrupted biofilms in B. subtilis but concluded that DAAs had a toxic effect on protein synthesis which re (...truncated)