doi:10.1111/iej.12465
Antimicrobial 2-hydroxyisocaproic acid and chlorhexidine resist inactivation by dentine
€derhane1,2,3, T. Sorsa2,4, P. Hietala5 & R. Rautemaa6 M. Sakko1,2, L. Tja 1 Institute of Dentistry, University of Oulu, Oulu; 2Department of Oral and Maxillofacial Diseases, Helsinki University and Helsinki University Hospital, Helsinki; 3Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Oulu, Finland; 4Division of Periodontology, Department of Dental Medicine, Karolinska Institutet, Huddinge, Sweden; 5Extracta Ltd, Vantaa, Finland; and 6Manchester Academic Health Science Centre, Institute of Inflammation and Repair, University Hospital of South Manchester, University of Manchester, Manchester, UK
Abstract €derhane L, Sorsa T, Hietala P, Sakko M, Tja Rautemaa R. Antimicrobial 2-hydroxyisocaproic acid and chlorhexidine resist inactivation by dentine. International Endodontic Journal, 49, 352–360, 2016.
Aim To compare the antibacterial activity of 2-hydroxyisocaproic acid (HICA) with currently used root canal medicaments and to examine their interactions with potential inhibitors in nutrient-deficient and nutrient-rich conditions. Methodology First, the antibacterial activity of single concentrations of HICA, calcium hydroxide solution or slurry, chlorhexidine digluconate or acetate was tested against Enterococcus faecalis with and without potential inhibitors: dentine powder (DP), hydroxyapatite or bovine serum albumin, in a low concentration of peptone water. Relative viable counts were determined by culture at 1, 24 and 48 h. In the second set of experiments, the activity of three concentrations of HICA was evaluated against two isolates of E. faecalis with and without potential inhibitors in nutrient-rich thioglycollate broth using a modification of a standard microdilution method. The minimum bactericidal concentration was determined by culture at 1, 24 and 48 h.
Results Concentrations of ≥33 mg mL 1 of HICA were found to be bactericidal against E. faecalis in both nutrient-deficient and nutrient-rich environments at 24- to 48-h incubation, whereas the initial activity of Ca(OH)2 slurry was lost at 48-h incubation. HICA tolerated well all tested potential inhibitors up to 19 mg mL 1. DP concentrations higher than this inhibited its activity in a dose-dependent manner in both environments. DP demonstrated moderate antibacterial activity, and it enhanced the otherwise limited activity of Ca(OH)2 slurry and solution. DP did not impact on the activity of chlorhexidine. Conclusions These results support the long-term antibacterial activity of HICA and indicate its tolerance to clinically relevant concentrations of dentine and other inhibitors commonly present in the root canal system. Therefore, HICA may have potential as an interappointment medication in the treatment of root canal infections. Keywords: calcium hydroxide, chlorhexidine, Enterococcus faecalis, hydroxyapatite, root canal medication, serum albumin. Received 2 January 2015; accepted 30 April 2015
Introduction
Correspondence: Marjut Sakko, Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Haartmaninkatu 3, P.O. Box 21, FIN-00014 Helsinki, Finland (e-mail: [email protected]).
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Topically used antiseptics are the first-line medicaments in the treatment of root canal infections. Sodium hypochlorite (NaOCl) has good activity against bacteria in biofilms, but it rapidly loses its activity in root canals and cannot be used as an in-
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terappointment medicament (Haapasalo et al. 2010). Chlorhexidine is another routinely used irrigant with rapid microbicidal effect (Mohammadi & Abbott 2009, Carrilho et al. 2010, Haapasalo et al. 2010, Salim et al. 2013). Antimicrobial activity of calcium hydroxide (Ca (OH)2) slurry (paste) is believed to be due to its ability to slowly release hydroxyl ions. Resistance to Ca(OH)2 has been reported in many bacteria, and this is a clinically significant problem especially with enterococci (Portenier et al. 2003, Nakajo et al. 2004, Sathorn et al. 2007). Chlorhexidine has a broader spectrum of activity including Ca(OH)2-resistant bacteria (Haapasalo & Shen 2012). However, the risk of allergic reactions and tissue toxicity caused by chlorhexidine limit its wider use (Guleri et al. 2012). 2-Hydroxyisocaproic acid (HICA) is a by-product of the leucine metabolic pathway and can be produced and metabolized by human cells (Hoffer et al. 1993). HICA has broad-spectrum activity against bacteria (Hietala et al. 1979, Sakko et al. 2012) and fungi (Nieminen et al. 2014a, Sakko et al. 2014). Only a few microbes are known to express enzymes needed for HICA elimination (Hummel et al. 1985, Bossow & Wandrey 1987, Lerch et al. 1989, Kallwass 1992, Bernard et al. 1994). The bactericidal concentration of HICA for E. faecalis is 36 mg mL 1, and 90% of growth is inhibited already at 9 mg mL 1 concentration (Sakko et al. 2012). HICA is approved by the European Food Safety Authority as a flavouring substance in nutrition with theoretical maximum daily intake (mTAMDI) of 3800 lg capita 1 day 1 (European Food Safety Authority 2012). The presence of dentine debris has an impact on the success of root canal treatment (Haapasalo et al. 2007). Dentine components may react with antimicrobial agents and inhibit their activity (Haapasalo et al. 2000, Portenier et al. 2001, 2002, 2006). On the other hand, extracellular matrix extracts of dentine have been reported to have antimicrobial properties (Smith et al. 2012). There is a clinical need for new, safe and broadspectrum antimicrobial agents for the treatment of root canal infections. The long-lasting activity of HICA against a broad spectrum of microorganisms including many common endodontic pathogens such as E. faecalis (Sakko et al. 2012) indicates that HICA could have potential as an alternative interappointment medicament, especially as it also exerts antiinflammatory effects (Nieminen et al. 2014b). However, nothing is known about the possible interactions
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between HICA and substances such as dentine and hydroxyapatite. The first aim of this study was to compare the antibacterial activity of HICA against E. faecalis with currently used root canal medicaments in vitro. The second aim was to test the potential interactions of dentine powder, hydroxyapatite and albumin with these agents. The hypothesis was that HICA has potential as an interappointment medicament in endodontics. As microbiology laboratory standards recommend the use of nutritionally rich conditions for testing of antimicrobial activity (Jorgensen et al. 2007), the hypothesis was tested in both nutrient-deficient and nutrient-rich conditions.
Materials and methods Study design In the first set of experiments, an Enterococcus faecalis clinical isolate was incubated with single concentrations of either DL-HICA solution or one of the positive controls chlorhexidine acetate (CHA) solution, chlorhexidine digluconate (CHG) solution or one of the comparators calcium hydroxide (Ca(OH)2) solution or slurry in the presence of potential inhibitors: three concentrations of dentine powder (DP), one concentration of hydroxyapatite (HA) or bovine serum albumin (BSA) in nutrient-deficient conditions for up to 48 h at 37°C (Haapasalo et al. 2000). Viability of the cells was tested at 1, 24 and 48 h by culture on agar, and relative viable counts (cfu%) were calculated. The drug and inhibitor solutions and suspensions were prepared in deionized water (sterile), and the bacterial suspensions were prepared in peptone water (final concentration 1.7 mg mL 1: negative control). In the second set of experiments, the E. faecalis clinical isolate or a culture collection strain was incubated with three concentrations of DL-HICA in the presence of three different concentrations of DP, HA or BSA in nutrient-rich conditions for up to 48 h at 37°C using a modification of a standard microdilution method (Jorgensen et al. 2007). CHG solution was used as positive control. Minimum bactericidal concentration (MBC) was determined by culture on agar. The solutions and suspensions of antimicrobials, potential inhibitors and microbes were prepared in thioglycollate broth. Drug- and inhibitor-free controls were included for both sets of experiments. The test conditions and antimicrobial concentrations used in this study were selected based on previous studies (Haapasalo et al.
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2000, Sakko et al. 2012) (for details, see below). The experiments were performed in triplicate.
Microbial isolates The clinical isolate of E. faecalis (T-75359) had been isolated from a root canal and identified using conventional biochemical methods and 16S sequencing (HUSLAB Clinical Microbiology Reference Laboratory of the Helsinki University Hospital, Helsinki, Finland). ATCC 29212 (urine isolate) was used as a reference strain. Purity of the cultures was confirmed prior to use. For the nutrient-deficient conditions, bacterial isolates were cultured on Tryptic soya agar (Difco Laboratories, Detroit, MI, USA) and the bacterial suspension was prepared to 5 mg mL 1 of peptone water (Bacto Peptone; Difco). For the nutrient-rich conditions, bacterial isolates were cultured on Blood agar (Oxoid, Basingstoke, UK) and the bacterial suspensions were prepared to thioglycollate broth (CM0173; Oxoid) supplemented with haemin (5 lg L 1) and vitamin K1 (0.01 lg L 1). The bacterial suspensions were prepared from 24-h cultures in 37°C and 5% CO2 and adjusted to 3 9 108 cfu mL 1 for both experiments.
Antimicrobials For the experiments in nutrient-deficient conditions, a 100 mg mL 1 solution of DL-HICA (TCI Europe nv, Zwijndrecht, Belgium) was prepared in deionized water and the pH was adjusted to 5.2 (Hietala et al. 1979). Saturated Ca(OH)2 solution and slurry (pH 12.5; Sigma-Aldrich, Gillingham, UK) were prepared by adding 300 mg mL 1 of Ca(OH)2 in deionized water resulting in 1.65 mg mL 1 hydrolysed ions in the saturated solution at 20°C (Dean 1973). A stock solution of 20 mg mL 1 of CHA and a stock solution of 200 mg mL 1 of CHG were prepared in purified water (pH 6; University Pharmacy, Helsinki, Finland) and further diluted to 0.5 mg mL 1 in deionized water for the experiment. For the experiments in nutrient-rich conditions, three concentrations of DL-HICA (40, 80 and 120 mg mL 1) were prepared in thioglycollate broth from a 200 mg mL 1 stock solution prepared in deionized water (pH 5.2). Water solutions of CHA and CHG formed an insoluble precipitate in broth. Therefore, a commercial CHG solution (2 mg mL 1, pH 6; Curaseptâ, Curaprox UK, Kimbolton, UK), which could be diluted into thioglycollate broth, was used.
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Potential inhibitors of antimicrobials DP was a kind gift from Dr. I Portenier (University of Oslo) and prepared as described previously (Haapasalo et al. 2000). The roots of extracted human third molars had been cleaned and autoclaved before crushing into 0.2–20 lm particles. DP was stored dry at room temperature until use. HA (Bio-Rad, M€ unchen, Germany) or BSA (Sigma-Aldrich) were used as comparators. In the nutrient-deficient conditions, the inhibitor solutions and suspensions were prepared into deionized water (DP 5.6, 56 and 560 mg mL 1; HA and BSA 560 mg mL 1). In the nutrient-rich conditions, the inhibitor solutions and suspensions were prepared into thioglycollate broth (6.2, 62 and 620 mg mL 1). For the co-incubations with antimicrobials, the antimicrobials were mixed with the test solutions prior to adding the inhibitors.
Antibacterial activity in nutrient-deficient conditions In nutrient-deficient conditions, 50 lL of each antimicrobial agent was first pre-incubated with 50 lL of one of the potential inhibitors or controls in sealed Eppendorf tubes at 37°C and 5% CO2 for 1 h. Then, 50 lL of E. faecalis suspension in 5 mg mL 1 of peptone water (Bacto Peptone; Difco) was added, the mixture was vortexed and incubated for 48 h. The final bacterial density of the test suspensions was 107 cfu mL 1. Final concentrations of antimicrobials were 33.3 mg mL 1 of HICA, 0.55 mg mL 1 of Ca (OH)2 solution, 100 mg mL 1 of Ca(OH)2 slurry, 0.17 mg mL 1 of CHA and 0.17 mg mL 1 of CHG in the incubation volume (150 lL). Potential inhibitors were tested in final concentrations of 1.87, 18.7 and 187 mg mL 1 of DP, 187 mg mL 1 of HA and 187 mg mL 1 of BSA. Drug- and inhibitor-free controls were included. All antimicrobials except Ca(OH)2 slurry stayed in solution in all incubations. Ca(OH)2 slurry, HA and DP suspensions could be homogenized. BSA dissolved fully into solution, but in the presence of Ca(OH)2 slurry, it formed an insoluble precipitate, which could not be homogenized. Therefore, BSA-Ca(OH)2 slurry results were excluded from the final analysis. After 0-, 1-, 24- and 48-h incubation, the solutions and suspensions were vortexed and 10 lL was used to prepare ten-fold serial dilutions in peptone water to 10 4. Then, 20 lL from each dilution was cultured on tryptic soya agar plates (Difco Laboratories) and
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incubated for 24 h. Colonies were then counted, and purity of the cultures was checked with a stereomicroscope. The results were expressed either as relative viable counts (cfu%) with standard deviations of the initial bacterial inoculum (impact of time on growth, Figures) or relative viable counts (cfu%) of the antimicrobialand inhibitor-free peptone water control at each timepoint (impact of antimicrobials on growth, text).
Antibacterial activity in nutrient-rich conditions A modification of a standard microdilution susceptibility testing method was used to test the activity of DLHICA in nutrient-rich conditions (Jorgensen et al. 2007). First, three concentrations of DL-HICA were prepared into thioglycollate broth (pH 5.2). Colonies of E. faecalis T-75359 and ATCC 29212 from 24-h cultures on blood agar (Oxoid) were suspended into thioglycollate broth. Then, 180 lL of test solutions and suspensions (HICA in the presence of DP, HA or BSA) were incubated with 20 lL of microbial suspensions in 96-well microtiter plates in 37°C in 5% CO2 for 48 h. The final density of bacterial suspension in the wells was 106 cfu mL 1. Final concentrations of DL-HICA used were 36, 72 and 108 mg mL 1 and DP, HA or BSA 1, 10 and 100 mg mL 1 in the incubation volume (200 lL). Drug- and inhibitor-free controls were included. CHG 0.9 mg mL 1 was used as a positive control. Minimum bactericidal concentration (MBC) for DL-HICA was determined by culture (20 lL) on blood agar plates at 1, 24 and 48 h. The purity of the cultures was checked in the beginning and at the end of the experiment.
Results Activity of HICA in nutrient-deficient conditions In the nutrient-deficient conditions, HICA was highly bactericidal against E. faecalis in 24- and 48-h incubation although it demonstrated only minimal bactericidal effect at 1 h (Fig. 1). In contrast, Ca(OH)2 slurry reduced the E. faecalis viability by >93% at 1 and 24 h, but the effect was lost in 48-h incubation. Ca(OH)2 solution showed 74% inhibition of bacterial growth at 1 h but no inhibition at 24 or 48 h. CHA and CHG were bactericidal at all time-points (Fig. 1). In the drug- and inhibitor-free peptone water control (1.7 mg mL 1), bacterial viability was reduced from the initial counts to 99% at 1 h, 30% at 24 h and 23% at 48 h (Figs 1–4).
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Figure 1 The impact of HICA and comparator antimicrobials on the growth of Enterococcus faecalis in nutritionally deficient conditions in the absence of inhibitors. Percentage of relative viable counts at 1, 24 or 48 h was depicted with standard deviations. Control, 1.7 mg mL 1 of peptone water; Ca(OH)so, calcium hydroxide solution; Ca(OH)sl, calcium hydroxide slurry; HICA, 2-hydroxyisocaproic acid solution; CHA, chlorhexidine acetate solution; CHG, chlorhexidine digluconate solution.
DP reduced the antimicrobial activity of HICA in a time- and dose-dependent manner (Fig. 2a). The highest concentration of DP (187 mg mL 1) inhibited the antimicrobial effect of HICA fully. However, 18.7 mg mL 1 of DP reduced the activity of HICA only by 3%, and 1.87 mg mL 1 of DP did not inhibit the activity of HICA at 48 h. DP did not inhibit the activity of the positive controls, CHA (≤1%) and CHG (0%) (Fig. 2d,e). Higher concentrations of DP (18.7 and 187 mg mL 1) enhanced the antibacterial activity of Ca(OH)2 slurry (Fig. 2b) and the highest concentration that of the Ca(OH)2 solution (Fig. 2c). In 1-h incubation, the antibacterial activity of Ca(OH)2 slurry was increased to 100% in the presence of all concentrations of DP. With 18.7 and 187 mg mL 1 of DP, the antibacterial activity of Ca(OH)2 slurry remained high up to 48-h incubation. However, 1.87 mg mL 1 of DP improved the antibacterial activity of Ca(OH)2 slurry only in 1-h incubation. In the absence of any antimicrobials but in the presence of 187 mg mL 1 of DP, there was >86% reduction in the proportion of viable E. faecalis cells in comparison with negative control in 24- and 48-h incubation. Lower concentrations (DP 1.87 and 18.7 mg mL 1) did not show any antibacterial effect (Fig. 3). BSA and HA inhibited the activity of all antimicrobials tested. The antibacterial activity of HICA was lost in the presence of 187 mg mL 1 of BSA in 1-, 24- and 48-h incubation and in the presence of 187 mg mL 1 of HA in 1- and 24-h incubation. In 48-h incubation, HICA reduced the viable counts of
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(a)
(c)
(b)
(d)
(e)
Figure 2 The impact of dentine powder on the activity of HICA solution (a) and comparator antimicrobials: calcium hydroxide slurry (b) or solution (c) chlorhexidine acetate solution (d) or chlorhexidine digluconate solution (e) on the growth of Enterococcus faecalis in nutritionally deficient conditions. Percentage of relative viable counts (%) with standard deviations at 1, 24 or 48 h are depicted.
E. faecalis by 95% also in the presence of 187 mg mL 1 HA. The antibacterial activity of Ca (OH)2 slurry was also completely lost in the presence of HA and BSA, and Ca(OH)2 solution was inactive both in the presence and absence of these inhibitors. HA and BSA inhibited CHA at 1 h, but had no effect at 24 or 48 h. BSA reduced the antibacterial activity of CHG by 56% at 1 h and by 11% at 24 h (Fig. 4a, b).
Activity of HICA in nutrient-rich conditions Concentration of 36 mg mL 1 of HICA was bactericidal for both isolates of E. faecalis at 48 h in the absence of potential inhibitors (Fig. 5). Positive control (0.9 mg mL 1 CHG) without inhibitors was
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bactericidal. DP, HA and BSA inhibited the antibacterial activity of HICA in a dose-dependent manner. In the presence of 100 mg mL 1 of DP, or 10 or 100 mg mL 1 of HA, even the highest tested concentration of HICA (108 mg mL 1) was inactive. In the presence of 1 mg mL 1 HA or 10 mg mL 1 DP, the MBC for HICA was 72 mg mL 1. The MBC of HICA was reduced marginally from 36 mg mL 1 to 72 mg mL 1 by 10 and 100 mg mL 1 of BSA. Low concentrations of DP or BSA (1 mg mL 1) did not have any effect on the antibacterial activity of HICA.
Discussion The results of the present study show that HICA is bactericidal against E. faecalis in both nutrient-defi-
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(a)
Figure 3 The impact of dentine powder on the growth of
(b)
Enterococcus faecalis in nutritionally deficient conditions. Percentage of relative viable counts (%) at 1, 24 and 48 h were depicted with standard deviations.
cient and nutrient-rich environments. Over longer (24–48 h) incubation times, concentrations of 33 mg mL 1 had better activity than Ca(OH)2 slurry. The presence of organic and inorganic debris in the root canal system is a major challenge in endodontics as they have been shown to inhibit the activity of most medicaments (Haapasalo et al. 2000, 2007, Portenier et al. 2001, 2002, 2006, Oliveira et al. 2010). Therefore, it is of importance that small amounts of potential inhibitors including DP, HA and BSA did not notably inhibit the activity of HICA at 48-h incubation, whereas the activity of both Ca (OH)2 solution and slurry was short-lived and completely lost in their presence. Although low concentrations of DP did not inhibit the activity of HICA against E. faecalis, higher concentrations did inhibit its activity in a dose-dependent manner in both nutrient-deficient and nutrient-rich environments. The highest concentration of DP (187 mg mL 1) resulted in complete loss of HICA’s activity. This is in line with what has been reported for other root canal medicaments previously (Haapasalo et al. 2000, Portenier et al. 2001) and also presented here for Ca(OH)2 slurry with low concentrations of DP. However, the finding of high concentrations of DP having a synergistic impact on the long-term (24 and 48 h) activity of Ca(OH)2 slurry is the opposite to the findings by Portenier et al. (2001) using Ca(OH)2 solution. The improved antibacterial effect may be a result of the antibacterial activity of high concentrations of DP over longer incubation time, as seen in the present study and also reported before (Smith et al. 2012). Also, Ca(OH)2 slurry provides a reservoir for maintaining a saturated solution and is likely to be more resistant to the
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Figure 4 The impact of hydroxyapatite 187 mg mL 1 suspension (a) and bovine serum albumin 187 mg mL 1 solution (b) on the antibacterial activity of HICA and comparator antimicrobials against Enterococcus faecalis in nutritionally deficient conditions. Percentage of relative viable counts (%) with standard deviations at 1, 24 and 48 h are depicted. Control, 1.7 mg mL 1 of peptone water; HICA, 2-hydroxyisocaproic acid solution; Ca(OH)so, calcium hydroxide solution; Ca(OH)sl, calcium hydroxide slurry; CHA, chlorhexidine acetate solution; CHG, chlorhexidine digluconate solution.
buffering capacity of dentine than Ca(OH)2 solution (Haapasalo et al. 2007). Interpreting the clinical relevance of the in vitro DP experiments requires caution. Concentrations as high as those used here are unlikely to be present in wellprepared root canals. Also, finely ground powder has a much larger surface area than root canal walls, which increases the potential for interactions. It is likely that the lower DP concentrations would better reflect the clinical reality. In this case, unlike Ca(OH)2 slurry HICA appears to be able to retain its antimicrobial activity in the presence of clinically relevant concentrations of DP. Previous studies have indicated that HA would be the main dentine component responsible for the loss of Ca(OH)2 antimicrobial effect in the root canal (Haapasalo et al. 2007). In the present study, HA inhibited the antibacterial activity of HICA in a dose- and time-dependent manner: increasing concentrations of HA resulted in an increase in MBCs, and 48-h incubation instead of 24 h was required
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(a)
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Figure 5 Minimum bactericidal concentrations (MBC; mg mL 1) of HICA for two Enterococcus faecalis isolates (T-75359 and ATCC 29212) in the presence of 0–100 mg mL 1 of potential inhibitors in (a–c) nutritionally rich conditions at 24 and 48 h. (a) dentine powder, (b) hydroxyapatite, (c) bovine serum albumin.
for >95% killing in the presence of high concentration of HA (187 mg mL 1). The impact of HA appeared to be more distinct in the nutrient-rich environment. In 48-h incubation, 10 mg mL 1 of HA eliminated the antimicrobial activity of HICA, but in nutrient-deficient conditions, 187 mg mL 1 of HA inhibited the activity of HICA only by 5%. This may be due to large amounts of proteins in the thioglycollate broth used in the nutrient-rich experiment, providing a high chance for protein binding and other interactions. This is supported by the inhibitory effect of BSA on HICA: high concentration of BSA (100 mg mL 1) eliminated the activity of HICA, but lower concentrations had only a marginal impact on the activity. Therefore, the results of tests performed in nutrient-rich conditions are likely to reflect the summative impact of tested inhibitors and broth proteins such as albumin. Importantly, in nutrient-deficient conditions, HICA resisted the inhibition by HA better than Ca(OH)2 slurry, the antimicrobial activity of which was completely eliminated with HA, in line with a previous report (Portenier et al. 2001). The main source for albumin and other serum proteins in infected root is the inflammatory exudate (Haapasalo et al. 2007). The levels of serum proteins in infected root canals have not been studied. However, the finding that 10–100 mg mL 1 of BSA
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increased MBC of HICA by one dilution only strongly indicates that HICA would retain its antimicrobial activity also in cases of exudate flow into the root canal. Therefore, the fact that 10–100 mg mL 1 of BSA increased MBC of HICA by one dilution only supports the suitability of HICA as an interappointment medicament. However, in the present study, BSA was used instead of human serum albumin or other serum proteins. Although the two proteins are highly homologous and BSA is commonly used in similar experiments (Portenier et al. 2001, Sassone et al. 2003, Pappen et al. 2010), it is possible that the results of the present study may not fully reflect the situation in the root canal in clinical infections. Interestingly, the activity of HICA in nutrient-rich conditions was comparable to that seen in nutrientdeficient conditions even though the presence of proteins is known to inhibit most disinfectants and root canal medicaments (Haapasalo et al. 2007, Oliveira et al. 2010). The two chlorhexidine solutions used as controls were highly active against E. faecalis in both conditions, and their activities were not substantially affected by the presence of DP or HA, which is in line with previous reports (Haapasalo et al. 2000, Portenier et al. 2001, 2002, 2006). BSA weakly inhibited CHG but not CHA in nutrient-deficient conditions, also confirming previous results by Portenier et al. (2006).
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Conclusion
References
HICA was more active against E. faecalis than Ca (OH)2 in nutrient-deficient conditions at 24 h and as active as chlorhexidine in both conditions at 48 h. Small amounts of potential inhibitors including DP, HA and BSA did not substantially inhibit the activity of HICA at 48-h incubation, whereas the activity of both Ca(OH)2 solution and slurry was abolished in their presence. HICA required a longer exposure time than chlorhexidine but was equally bactericidal at 48 h in both test conditions. Due to its biocompatibility, low toxicity, anti-inflammatory properties (Nieminen et al. 2014b), broad spectrum of activity including most important endodontic bacterial and fungal pathogens and tolerance to common inhibitors present in the root canal system HICA may have potential as an interappointment medicament in the treatment of root canal infections. Further studies using infected root canal models and clinical trials are required before clinical recommendations on the use of HICA can be made.
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Acknowledgements This work was supported by Finnish Doctoral Program in Oral Sciences (FINDOS), Medical Research Center Oulu and by grants from Finnish Dental Foundation, Finnish Dental Society Apollonia, Finnish Association of Female Dentists, University of Helsinki and University Hospital of Helsinki Research Funds (EVO), Academy of Finland and the UK National Aspergillosis Centre.
Conflict of interest Marjut Sakko, Leo Tj€ aderhane and Riina RautemaaRichardson have none to declare. Timo Sorsa and Pentti Hietala are inventors of patent EP 0871438 B1, and Pentti Hietala is a shareholder of Extracta Ltd.
Disclaimer This report is independent research supported by the National Institute for Health Research Clinical Research Facility at University Hospital of South Manchester NHS Foundation Trust. The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the National Institute of Health Research or the Department of Health.
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Clinical Microbiology, 9th edn. Washington DC: ASM press, pp. 1075–1267. Kallwass H (1992) Potential of R-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei for stereospecific reductions. Enzyme and Microbial Technology 14, 28–35. Lerch H, Bl€ ocker H, Kallwass H, Hoppe J, Tsai H, Collins J (1989) Cloning, sequencing and expression in Escherichia coli of the D-2-hydroxyisocaproate dehydrogenase gene of Lactobacillus casei. Gene 78, 47–57. Mohammadi Z, Abbott P (2009) The properties and applications of chlorhexidine in endodontics. International Endodontic Journal 42, 288–302. Nakajo K, Nakazawa F, Iwaku M, Hashino E (2004) Alkaliresistant bacteria in root canal system. Oral Microbiology and Immunology 19, 390–394. Nieminen MT, Novak-Frazer L, Rautemaa V et al. (2014a) A novel antifungal is active against Candida albicans biofilms and inhibits mutagenic acetaldehyde production in vitro. PLoS One 9, e97864. Nieminen MT, Hernandez M, Novak-Frazer L et al. (2014b) DL-2-hydroxyisocaproic acid attenuates inflammatory responses in a murine Candida albicans biofilm model. Clinical and Vaccine Immunology 21, 1240–1245. Oliveira J, Alves F, Uzeda M, R^oßcas I, Siqueira J Jr (2010) Influence of serum and necrotic soft tissue on the antimicrobial effects of intracanal medicaments. Brazilian Dental Journal 21, 295–300. Pappen F, Qian W, Aleksej uniene J, de Toledo Leonardo R, Leonardo M, Haapasalo M (2010) Inhibition of sodium hypochlorite antimicrobial activity in the presence of bovine serum albumin. Journal of Endodontics 36, 268– 271. Portenier I, Haapasalo H, Rye A, Waltimo T, Ørstavik D, Haapasalo M (2001) Inactivation of root canal medicaments by dentine, hydroxylapatite and bovine serum albumin. International Endodontic Journal 34, 184–188.
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Portenier I, Haapasalo H, Ørstavik D, Yamauchi M, Haapasalo M (2002) Inactivation of the antibacterial activity of iodine potassium iodide and chlorhexidine digluconate against Enterococcus faecalis by dentin, dentin, matrix, type-I collagen, and heat-killed microbial whole cells. Journal of Endodontics 28, 634–637. Portenier I, Waltimo T, Haapasalo M (2003) Enterococcus faecalis – the root canal survivor and ‘star’ in post-treatment disease. Endodontic Topics 6, 135–159. Portenier I, Waltimo T, Ørstavik D, Haapasalo M (2006) Killing of Enterococcus faecalis by MTAD and chlorhexidine digluconate with or without cetrimide in the presence or absence of dentine powder or BSA. Journal of Endodontics 32, 138–141. Sakko M, Tj€ aderhane L, Sorsa T et al. (2012) 2-hydroxyisocaproic acid (HICA): a new potential topical antibacterial agent. International Journal of Antimicrobial Agents 39, 539–540. Sakko M, Moore C, Novak-Frazer L et al. (2014) 2-hydroxyisocaproic acid is fungicidal for Candida and Aspergillus species. Mycoses 57, 214–221. Salim N, Moore C, Silikas N, Satterthwaite J, Rautemaa R (2013) Chlorhexidine is a highly effective topical broadspectrum agent against Candida spp. International Journal of Antimicrobial Agents 41, 65–69. Sassone L, Fidel R, Fidel S, Vieira M, Hirata R Jr (2003) The influence of organic load on the antimicrobial activity of different concentrations of NaOCl and chlorhexidine in vitro. International Endodontic Journal 36, 848–852. Sathorn C, Parashos P, Messer H (2007) Antibacterial efficacy of calcium hydroxide intracanal dressing: a systematic review and meta-analysis. International Endodontic Journal 40, 2–10. Smith J, Smith A, Shelton R, Cooper P (2012) Antibacterial activity of dentine and pulp extracellular matrix extracts. International Endodontic Journal 45, 749–755.
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Antimicrobial 2-hydroxyisocaproic acid and chlorhexidine resist inactivation by dentine
€derhane1,2,3, T. Sorsa2,4, P. Hietala5 & R. Rautemaa6 M. Sakko1,2, L. Tja 1 Institute of Dentistry, University of Oulu, Oulu; 2Department of Oral and Maxillofacial Diseases, Helsinki University and Helsinki University Hospital, Helsinki; 3Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Oulu, Finland; 4Division of Periodontology, Department of Dental Medicine, Karolinska Institutet, Huddinge, Sweden; 5Extracta Ltd, Vantaa, Finland; and 6Manchester Academic Health Science Centre, Institute of Inflammation and Repair, University Hospital of South Manchester, University of Manchester, Manchester, UK
Abstract €derhane L, Sorsa T, Hietala P, Sakko M, Tja Rautemaa R. Antimicrobial 2-hydroxyisocaproic acid and chlorhexidine resist inactivation by dentine. International Endodontic Journal, 49, 352–360, 2016.
Aim To compare the antibacterial activity of 2-hydroxyisocaproic acid (HICA) with currently used root canal medicaments and to examine their interactions with potential inhibitors in nutrient-deficient and nutrient-rich conditions. Methodology First, the antibacterial activity of single concentrations of HICA, calcium hydroxide solution or slurry, chlorhexidine digluconate or acetate was tested against Enterococcus faecalis with and without potential inhibitors: dentine powder (DP), hydroxyapatite or bovine serum albumin, in a low concentration of peptone water. Relative viable counts were determined by culture at 1, 24 and 48 h. In the second set of experiments, the activity of three concentrations of HICA was evaluated against two isolates of E. faecalis with and without potential inhibitors in nutrient-rich thioglycollate broth using a modification of a standard microdilution method. The minimum bactericidal concentration was determined by culture at 1, 24 and 48 h.
Results Concentrations of ≥33 mg mL 1 of HICA were found to be bactericidal against E. faecalis in both nutrient-deficient and nutrient-rich environments at 24- to 48-h incubation, whereas the initial activity of Ca(OH)2 slurry was lost at 48-h incubation. HICA tolerated well all tested potential inhibitors up to 19 mg mL 1. DP concentrations higher than this inhibited its activity in a dose-dependent manner in both environments. DP demonstrated moderate antibacterial activity, and it enhanced the otherwise limited activity of Ca(OH)2 slurry and solution. DP did not impact on the activity of chlorhexidine. Conclusions These results support the long-term antibacterial activity of HICA and indicate its tolerance to clinically relevant concentrations of dentine and other inhibitors commonly present in the root canal system. Therefore, HICA may have potential as an interappointment medication in the treatment of root canal infections. Keywords: calcium hydroxide, chlorhexidine, Enterococcus faecalis, hydroxyapatite, root canal medication, serum albumin. Received 2 January 2015; accepted 30 April 2015
Introduction
Correspondence: Marjut Sakko, Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Haartmaninkatu 3, P.O. Box 21, FIN-00014 Helsinki, Finland (e-mail: [email protected]).
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Topically used antiseptics are the first-line medicaments in the treatment of root canal infections. Sodium hypochlorite (NaOCl) has good activity against bacteria in biofilms, but it rapidly loses its activity in root canals and cannot be used as an in-
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terappointment medicament (Haapasalo et al. 2010). Chlorhexidine is another routinely used irrigant with rapid microbicidal effect (Mohammadi & Abbott 2009, Carrilho et al. 2010, Haapasalo et al. 2010, Salim et al. 2013). Antimicrobial activity of calcium hydroxide (Ca (OH)2) slurry (paste) is believed to be due to its ability to slowly release hydroxyl ions. Resistance to Ca(OH)2 has been reported in many bacteria, and this is a clinically significant problem especially with enterococci (Portenier et al. 2003, Nakajo et al. 2004, Sathorn et al. 2007). Chlorhexidine has a broader spectrum of activity including Ca(OH)2-resistant bacteria (Haapasalo & Shen 2012). However, the risk of allergic reactions and tissue toxicity caused by chlorhexidine limit its wider use (Guleri et al. 2012). 2-Hydroxyisocaproic acid (HICA) is a by-product of the leucine metabolic pathway and can be produced and metabolized by human cells (Hoffer et al. 1993). HICA has broad-spectrum activity against bacteria (Hietala et al. 1979, Sakko et al. 2012) and fungi (Nieminen et al. 2014a, Sakko et al. 2014). Only a few microbes are known to express enzymes needed for HICA elimination (Hummel et al. 1985, Bossow & Wandrey 1987, Lerch et al. 1989, Kallwass 1992, Bernard et al. 1994). The bactericidal concentration of HICA for E. faecalis is 36 mg mL 1, and 90% of growth is inhibited already at 9 mg mL 1 concentration (Sakko et al. 2012). HICA is approved by the European Food Safety Authority as a flavouring substance in nutrition with theoretical maximum daily intake (mTAMDI) of 3800 lg capita 1 day 1 (European Food Safety Authority 2012). The presence of dentine debris has an impact on the success of root canal treatment (Haapasalo et al. 2007). Dentine components may react with antimicrobial agents and inhibit their activity (Haapasalo et al. 2000, Portenier et al. 2001, 2002, 2006). On the other hand, extracellular matrix extracts of dentine have been reported to have antimicrobial properties (Smith et al. 2012). There is a clinical need for new, safe and broadspectrum antimicrobial agents for the treatment of root canal infections. The long-lasting activity of HICA against a broad spectrum of microorganisms including many common endodontic pathogens such as E. faecalis (Sakko et al. 2012) indicates that HICA could have potential as an alternative interappointment medicament, especially as it also exerts antiinflammatory effects (Nieminen et al. 2014b). However, nothing is known about the possible interactions
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between HICA and substances such as dentine and hydroxyapatite. The first aim of this study was to compare the antibacterial activity of HICA against E. faecalis with currently used root canal medicaments in vitro. The second aim was to test the potential interactions of dentine powder, hydroxyapatite and albumin with these agents. The hypothesis was that HICA has potential as an interappointment medicament in endodontics. As microbiology laboratory standards recommend the use of nutritionally rich conditions for testing of antimicrobial activity (Jorgensen et al. 2007), the hypothesis was tested in both nutrient-deficient and nutrient-rich conditions.
Materials and methods Study design In the first set of experiments, an Enterococcus faecalis clinical isolate was incubated with single concentrations of either DL-HICA solution or one of the positive controls chlorhexidine acetate (CHA) solution, chlorhexidine digluconate (CHG) solution or one of the comparators calcium hydroxide (Ca(OH)2) solution or slurry in the presence of potential inhibitors: three concentrations of dentine powder (DP), one concentration of hydroxyapatite (HA) or bovine serum albumin (BSA) in nutrient-deficient conditions for up to 48 h at 37°C (Haapasalo et al. 2000). Viability of the cells was tested at 1, 24 and 48 h by culture on agar, and relative viable counts (cfu%) were calculated. The drug and inhibitor solutions and suspensions were prepared in deionized water (sterile), and the bacterial suspensions were prepared in peptone water (final concentration 1.7 mg mL 1: negative control). In the second set of experiments, the E. faecalis clinical isolate or a culture collection strain was incubated with three concentrations of DL-HICA in the presence of three different concentrations of DP, HA or BSA in nutrient-rich conditions for up to 48 h at 37°C using a modification of a standard microdilution method (Jorgensen et al. 2007). CHG solution was used as positive control. Minimum bactericidal concentration (MBC) was determined by culture on agar. The solutions and suspensions of antimicrobials, potential inhibitors and microbes were prepared in thioglycollate broth. Drug- and inhibitor-free controls were included for both sets of experiments. The test conditions and antimicrobial concentrations used in this study were selected based on previous studies (Haapasalo et al.
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2000, Sakko et al. 2012) (for details, see below). The experiments were performed in triplicate.
Microbial isolates The clinical isolate of E. faecalis (T-75359) had been isolated from a root canal and identified using conventional biochemical methods and 16S sequencing (HUSLAB Clinical Microbiology Reference Laboratory of the Helsinki University Hospital, Helsinki, Finland). ATCC 29212 (urine isolate) was used as a reference strain. Purity of the cultures was confirmed prior to use. For the nutrient-deficient conditions, bacterial isolates were cultured on Tryptic soya agar (Difco Laboratories, Detroit, MI, USA) and the bacterial suspension was prepared to 5 mg mL 1 of peptone water (Bacto Peptone; Difco). For the nutrient-rich conditions, bacterial isolates were cultured on Blood agar (Oxoid, Basingstoke, UK) and the bacterial suspensions were prepared to thioglycollate broth (CM0173; Oxoid) supplemented with haemin (5 lg L 1) and vitamin K1 (0.01 lg L 1). The bacterial suspensions were prepared from 24-h cultures in 37°C and 5% CO2 and adjusted to 3 9 108 cfu mL 1 for both experiments.
Antimicrobials For the experiments in nutrient-deficient conditions, a 100 mg mL 1 solution of DL-HICA (TCI Europe nv, Zwijndrecht, Belgium) was prepared in deionized water and the pH was adjusted to 5.2 (Hietala et al. 1979). Saturated Ca(OH)2 solution and slurry (pH 12.5; Sigma-Aldrich, Gillingham, UK) were prepared by adding 300 mg mL 1 of Ca(OH)2 in deionized water resulting in 1.65 mg mL 1 hydrolysed ions in the saturated solution at 20°C (Dean 1973). A stock solution of 20 mg mL 1 of CHA and a stock solution of 200 mg mL 1 of CHG were prepared in purified water (pH 6; University Pharmacy, Helsinki, Finland) and further diluted to 0.5 mg mL 1 in deionized water for the experiment. For the experiments in nutrient-rich conditions, three concentrations of DL-HICA (40, 80 and 120 mg mL 1) were prepared in thioglycollate broth from a 200 mg mL 1 stock solution prepared in deionized water (pH 5.2). Water solutions of CHA and CHG formed an insoluble precipitate in broth. Therefore, a commercial CHG solution (2 mg mL 1, pH 6; Curaseptâ, Curaprox UK, Kimbolton, UK), which could be diluted into thioglycollate broth, was used.
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Potential inhibitors of antimicrobials DP was a kind gift from Dr. I Portenier (University of Oslo) and prepared as described previously (Haapasalo et al. 2000). The roots of extracted human third molars had been cleaned and autoclaved before crushing into 0.2–20 lm particles. DP was stored dry at room temperature until use. HA (Bio-Rad, M€ unchen, Germany) or BSA (Sigma-Aldrich) were used as comparators. In the nutrient-deficient conditions, the inhibitor solutions and suspensions were prepared into deionized water (DP 5.6, 56 and 560 mg mL 1; HA and BSA 560 mg mL 1). In the nutrient-rich conditions, the inhibitor solutions and suspensions were prepared into thioglycollate broth (6.2, 62 and 620 mg mL 1). For the co-incubations with antimicrobials, the antimicrobials were mixed with the test solutions prior to adding the inhibitors.
Antibacterial activity in nutrient-deficient conditions In nutrient-deficient conditions, 50 lL of each antimicrobial agent was first pre-incubated with 50 lL of one of the potential inhibitors or controls in sealed Eppendorf tubes at 37°C and 5% CO2 for 1 h. Then, 50 lL of E. faecalis suspension in 5 mg mL 1 of peptone water (Bacto Peptone; Difco) was added, the mixture was vortexed and incubated for 48 h. The final bacterial density of the test suspensions was 107 cfu mL 1. Final concentrations of antimicrobials were 33.3 mg mL 1 of HICA, 0.55 mg mL 1 of Ca (OH)2 solution, 100 mg mL 1 of Ca(OH)2 slurry, 0.17 mg mL 1 of CHA and 0.17 mg mL 1 of CHG in the incubation volume (150 lL). Potential inhibitors were tested in final concentrations of 1.87, 18.7 and 187 mg mL 1 of DP, 187 mg mL 1 of HA and 187 mg mL 1 of BSA. Drug- and inhibitor-free controls were included. All antimicrobials except Ca(OH)2 slurry stayed in solution in all incubations. Ca(OH)2 slurry, HA and DP suspensions could be homogenized. BSA dissolved fully into solution, but in the presence of Ca(OH)2 slurry, it formed an insoluble precipitate, which could not be homogenized. Therefore, BSA-Ca(OH)2 slurry results were excluded from the final analysis. After 0-, 1-, 24- and 48-h incubation, the solutions and suspensions were vortexed and 10 lL was used to prepare ten-fold serial dilutions in peptone water to 10 4. Then, 20 lL from each dilution was cultured on tryptic soya agar plates (Difco Laboratories) and
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incubated for 24 h. Colonies were then counted, and purity of the cultures was checked with a stereomicroscope. The results were expressed either as relative viable counts (cfu%) with standard deviations of the initial bacterial inoculum (impact of time on growth, Figures) or relative viable counts (cfu%) of the antimicrobialand inhibitor-free peptone water control at each timepoint (impact of antimicrobials on growth, text).
Antibacterial activity in nutrient-rich conditions A modification of a standard microdilution susceptibility testing method was used to test the activity of DLHICA in nutrient-rich conditions (Jorgensen et al. 2007). First, three concentrations of DL-HICA were prepared into thioglycollate broth (pH 5.2). Colonies of E. faecalis T-75359 and ATCC 29212 from 24-h cultures on blood agar (Oxoid) were suspended into thioglycollate broth. Then, 180 lL of test solutions and suspensions (HICA in the presence of DP, HA or BSA) were incubated with 20 lL of microbial suspensions in 96-well microtiter plates in 37°C in 5% CO2 for 48 h. The final density of bacterial suspension in the wells was 106 cfu mL 1. Final concentrations of DL-HICA used were 36, 72 and 108 mg mL 1 and DP, HA or BSA 1, 10 and 100 mg mL 1 in the incubation volume (200 lL). Drug- and inhibitor-free controls were included. CHG 0.9 mg mL 1 was used as a positive control. Minimum bactericidal concentration (MBC) for DL-HICA was determined by culture (20 lL) on blood agar plates at 1, 24 and 48 h. The purity of the cultures was checked in the beginning and at the end of the experiment.
Results Activity of HICA in nutrient-deficient conditions In the nutrient-deficient conditions, HICA was highly bactericidal against E. faecalis in 24- and 48-h incubation although it demonstrated only minimal bactericidal effect at 1 h (Fig. 1). In contrast, Ca(OH)2 slurry reduced the E. faecalis viability by >93% at 1 and 24 h, but the effect was lost in 48-h incubation. Ca(OH)2 solution showed 74% inhibition of bacterial growth at 1 h but no inhibition at 24 or 48 h. CHA and CHG were bactericidal at all time-points (Fig. 1). In the drug- and inhibitor-free peptone water control (1.7 mg mL 1), bacterial viability was reduced from the initial counts to 99% at 1 h, 30% at 24 h and 23% at 48 h (Figs 1–4).
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Figure 1 The impact of HICA and comparator antimicrobials on the growth of Enterococcus faecalis in nutritionally deficient conditions in the absence of inhibitors. Percentage of relative viable counts at 1, 24 or 48 h was depicted with standard deviations. Control, 1.7 mg mL 1 of peptone water; Ca(OH)so, calcium hydroxide solution; Ca(OH)sl, calcium hydroxide slurry; HICA, 2-hydroxyisocaproic acid solution; CHA, chlorhexidine acetate solution; CHG, chlorhexidine digluconate solution.
DP reduced the antimicrobial activity of HICA in a time- and dose-dependent manner (Fig. 2a). The highest concentration of DP (187 mg mL 1) inhibited the antimicrobial effect of HICA fully. However, 18.7 mg mL 1 of DP reduced the activity of HICA only by 3%, and 1.87 mg mL 1 of DP did not inhibit the activity of HICA at 48 h. DP did not inhibit the activity of the positive controls, CHA (≤1%) and CHG (0%) (Fig. 2d,e). Higher concentrations of DP (18.7 and 187 mg mL 1) enhanced the antibacterial activity of Ca(OH)2 slurry (Fig. 2b) and the highest concentration that of the Ca(OH)2 solution (Fig. 2c). In 1-h incubation, the antibacterial activity of Ca(OH)2 slurry was increased to 100% in the presence of all concentrations of DP. With 18.7 and 187 mg mL 1 of DP, the antibacterial activity of Ca(OH)2 slurry remained high up to 48-h incubation. However, 1.87 mg mL 1 of DP improved the antibacterial activity of Ca(OH)2 slurry only in 1-h incubation. In the absence of any antimicrobials but in the presence of 187 mg mL 1 of DP, there was >86% reduction in the proportion of viable E. faecalis cells in comparison with negative control in 24- and 48-h incubation. Lower concentrations (DP 1.87 and 18.7 mg mL 1) did not show any antibacterial effect (Fig. 3). BSA and HA inhibited the activity of all antimicrobials tested. The antibacterial activity of HICA was lost in the presence of 187 mg mL 1 of BSA in 1-, 24- and 48-h incubation and in the presence of 187 mg mL 1 of HA in 1- and 24-h incubation. In 48-h incubation, HICA reduced the viable counts of
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(a)
(c)
(b)
(d)
(e)
Figure 2 The impact of dentine powder on the activity of HICA solution (a) and comparator antimicrobials: calcium hydroxide slurry (b) or solution (c) chlorhexidine acetate solution (d) or chlorhexidine digluconate solution (e) on the growth of Enterococcus faecalis in nutritionally deficient conditions. Percentage of relative viable counts (%) with standard deviations at 1, 24 or 48 h are depicted.
E. faecalis by 95% also in the presence of 187 mg mL 1 HA. The antibacterial activity of Ca (OH)2 slurry was also completely lost in the presence of HA and BSA, and Ca(OH)2 solution was inactive both in the presence and absence of these inhibitors. HA and BSA inhibited CHA at 1 h, but had no effect at 24 or 48 h. BSA reduced the antibacterial activity of CHG by 56% at 1 h and by 11% at 24 h (Fig. 4a, b).
Activity of HICA in nutrient-rich conditions Concentration of 36 mg mL 1 of HICA was bactericidal for both isolates of E. faecalis at 48 h in the absence of potential inhibitors (Fig. 5). Positive control (0.9 mg mL 1 CHG) without inhibitors was
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bactericidal. DP, HA and BSA inhibited the antibacterial activity of HICA in a dose-dependent manner. In the presence of 100 mg mL 1 of DP, or 10 or 100 mg mL 1 of HA, even the highest tested concentration of HICA (108 mg mL 1) was inactive. In the presence of 1 mg mL 1 HA or 10 mg mL 1 DP, the MBC for HICA was 72 mg mL 1. The MBC of HICA was reduced marginally from 36 mg mL 1 to 72 mg mL 1 by 10 and 100 mg mL 1 of BSA. Low concentrations of DP or BSA (1 mg mL 1) did not have any effect on the antibacterial activity of HICA.
Discussion The results of the present study show that HICA is bactericidal against E. faecalis in both nutrient-defi-
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(a)
Figure 3 The impact of dentine powder on the growth of
(b)
Enterococcus faecalis in nutritionally deficient conditions. Percentage of relative viable counts (%) at 1, 24 and 48 h were depicted with standard deviations.
cient and nutrient-rich environments. Over longer (24–48 h) incubation times, concentrations of 33 mg mL 1 had better activity than Ca(OH)2 slurry. The presence of organic and inorganic debris in the root canal system is a major challenge in endodontics as they have been shown to inhibit the activity of most medicaments (Haapasalo et al. 2000, 2007, Portenier et al. 2001, 2002, 2006, Oliveira et al. 2010). Therefore, it is of importance that small amounts of potential inhibitors including DP, HA and BSA did not notably inhibit the activity of HICA at 48-h incubation, whereas the activity of both Ca (OH)2 solution and slurry was short-lived and completely lost in their presence. Although low concentrations of DP did not inhibit the activity of HICA against E. faecalis, higher concentrations did inhibit its activity in a dose-dependent manner in both nutrient-deficient and nutrient-rich environments. The highest concentration of DP (187 mg mL 1) resulted in complete loss of HICA’s activity. This is in line with what has been reported for other root canal medicaments previously (Haapasalo et al. 2000, Portenier et al. 2001) and also presented here for Ca(OH)2 slurry with low concentrations of DP. However, the finding of high concentrations of DP having a synergistic impact on the long-term (24 and 48 h) activity of Ca(OH)2 slurry is the opposite to the findings by Portenier et al. (2001) using Ca(OH)2 solution. The improved antibacterial effect may be a result of the antibacterial activity of high concentrations of DP over longer incubation time, as seen in the present study and also reported before (Smith et al. 2012). Also, Ca(OH)2 slurry provides a reservoir for maintaining a saturated solution and is likely to be more resistant to the
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Figure 4 The impact of hydroxyapatite 187 mg mL 1 suspension (a) and bovine serum albumin 187 mg mL 1 solution (b) on the antibacterial activity of HICA and comparator antimicrobials against Enterococcus faecalis in nutritionally deficient conditions. Percentage of relative viable counts (%) with standard deviations at 1, 24 and 48 h are depicted. Control, 1.7 mg mL 1 of peptone water; HICA, 2-hydroxyisocaproic acid solution; Ca(OH)so, calcium hydroxide solution; Ca(OH)sl, calcium hydroxide slurry; CHA, chlorhexidine acetate solution; CHG, chlorhexidine digluconate solution.
buffering capacity of dentine than Ca(OH)2 solution (Haapasalo et al. 2007). Interpreting the clinical relevance of the in vitro DP experiments requires caution. Concentrations as high as those used here are unlikely to be present in wellprepared root canals. Also, finely ground powder has a much larger surface area than root canal walls, which increases the potential for interactions. It is likely that the lower DP concentrations would better reflect the clinical reality. In this case, unlike Ca(OH)2 slurry HICA appears to be able to retain its antimicrobial activity in the presence of clinically relevant concentrations of DP. Previous studies have indicated that HA would be the main dentine component responsible for the loss of Ca(OH)2 antimicrobial effect in the root canal (Haapasalo et al. 2007). In the present study, HA inhibited the antibacterial activity of HICA in a dose- and time-dependent manner: increasing concentrations of HA resulted in an increase in MBCs, and 48-h incubation instead of 24 h was required
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Figure 5 Minimum bactericidal concentrations (MBC; mg mL 1) of HICA for two Enterococcus faecalis isolates (T-75359 and ATCC 29212) in the presence of 0–100 mg mL 1 of potential inhibitors in (a–c) nutritionally rich conditions at 24 and 48 h. (a) dentine powder, (b) hydroxyapatite, (c) bovine serum albumin.
for >95% killing in the presence of high concentration of HA (187 mg mL 1). The impact of HA appeared to be more distinct in the nutrient-rich environment. In 48-h incubation, 10 mg mL 1 of HA eliminated the antimicrobial activity of HICA, but in nutrient-deficient conditions, 187 mg mL 1 of HA inhibited the activity of HICA only by 5%. This may be due to large amounts of proteins in the thioglycollate broth used in the nutrient-rich experiment, providing a high chance for protein binding and other interactions. This is supported by the inhibitory effect of BSA on HICA: high concentration of BSA (100 mg mL 1) eliminated the activity of HICA, but lower concentrations had only a marginal impact on the activity. Therefore, the results of tests performed in nutrient-rich conditions are likely to reflect the summative impact of tested inhibitors and broth proteins such as albumin. Importantly, in nutrient-deficient conditions, HICA resisted the inhibition by HA better than Ca(OH)2 slurry, the antimicrobial activity of which was completely eliminated with HA, in line with a previous report (Portenier et al. 2001). The main source for albumin and other serum proteins in infected root is the inflammatory exudate (Haapasalo et al. 2007). The levels of serum proteins in infected root canals have not been studied. However, the finding that 10–100 mg mL 1 of BSA
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increased MBC of HICA by one dilution only strongly indicates that HICA would retain its antimicrobial activity also in cases of exudate flow into the root canal. Therefore, the fact that 10–100 mg mL 1 of BSA increased MBC of HICA by one dilution only supports the suitability of HICA as an interappointment medicament. However, in the present study, BSA was used instead of human serum albumin or other serum proteins. Although the two proteins are highly homologous and BSA is commonly used in similar experiments (Portenier et al. 2001, Sassone et al. 2003, Pappen et al. 2010), it is possible that the results of the present study may not fully reflect the situation in the root canal in clinical infections. Interestingly, the activity of HICA in nutrient-rich conditions was comparable to that seen in nutrientdeficient conditions even though the presence of proteins is known to inhibit most disinfectants and root canal medicaments (Haapasalo et al. 2007, Oliveira et al. 2010). The two chlorhexidine solutions used as controls were highly active against E. faecalis in both conditions, and their activities were not substantially affected by the presence of DP or HA, which is in line with previous reports (Haapasalo et al. 2000, Portenier et al. 2001, 2002, 2006). BSA weakly inhibited CHG but not CHA in nutrient-deficient conditions, also confirming previous results by Portenier et al. (2006).
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Conclusion
References
HICA was more active against E. faecalis than Ca (OH)2 in nutrient-deficient conditions at 24 h and as active as chlorhexidine in both conditions at 48 h. Small amounts of potential inhibitors including DP, HA and BSA did not substantially inhibit the activity of HICA at 48-h incubation, whereas the activity of both Ca(OH)2 solution and slurry was abolished in their presence. HICA required a longer exposure time than chlorhexidine but was equally bactericidal at 48 h in both test conditions. Due to its biocompatibility, low toxicity, anti-inflammatory properties (Nieminen et al. 2014b), broad spectrum of activity including most important endodontic bacterial and fungal pathogens and tolerance to common inhibitors present in the root canal system HICA may have potential as an interappointment medicament in the treatment of root canal infections. Further studies using infected root canal models and clinical trials are required before clinical recommendations on the use of HICA can be made.
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Acknowledgements This work was supported by Finnish Doctoral Program in Oral Sciences (FINDOS), Medical Research Center Oulu and by grants from Finnish Dental Foundation, Finnish Dental Society Apollonia, Finnish Association of Female Dentists, University of Helsinki and University Hospital of Helsinki Research Funds (EVO), Academy of Finland and the UK National Aspergillosis Centre.
Conflict of interest Marjut Sakko, Leo Tj€ aderhane and Riina RautemaaRichardson have none to declare. Timo Sorsa and Pentti Hietala are inventors of patent EP 0871438 B1, and Pentti Hietala is a shareholder of Extracta Ltd.
Disclaimer This report is independent research supported by the National Institute for Health Research Clinical Research Facility at University Hospital of South Manchester NHS Foundation Trust. The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the National Institute of Health Research or the Department of Health.
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