An in vitro study of the antimicrobial activity of some endodontic medicaments and their bases using an agar well diffusion assay

SCIENTIFIC ARTICLE Australian Dental Journal 2009; 54: 141 146 doi: 10.1111/j.1834-7819.2009.01107.x An in vitro study of the antimicrobial activity of some endodontic medicaments and their bases using an agar well diffusion assay B Athanassiadis,* PV Abbott,* N George, LJ Walshà *School of Dentistry, The University of Western Australia. Division of Microbiology, Pathology Queensland Central Laboratory, Herston Hospitals Complex. àschool of Dentistry, The University of Queensland. ABSTRACT Background: The aim of this study was to determine the in vitro antimicrobial activities of various endodontic medicaments and their bases against selected organisms using an agar diffusion assay. Methods: An agar well diffusion assay was used to test the antimicrobial action of some commonly used endodontic medicaments (Ledermix paste, Pulpdent paste, Ultracal paste, and a 50:50 mix of Ledermix and Pulpdent pastes) and their bases. Three bacterial species (E. faecalis, P. micros, P. intermedia) and one yeast (C. albicans) were selected. The diameters of growth inhibition zones and ph were assessed. Results: P. micros demonstrated the highest level of in vitro resistance. Pulpdent and Ultracal pastes had the highest ph (12.64 and 12.53, respectively). The addition of Pulpdent to Ledermix did not increase the zone sizes significantly. Conclusions: All the commercial products showed some in vitro antimicrobial activity. Ledermix paste and the 50:50 Ledermix Pulpdent mixture being the most effective in this model. The known anti-inflammatory analgesic properties of Ledermix and the results from this agar model suggest that the 50:50 Ledermix Pulpdent combination would be the preferred medicament for clinical use in symptomatic cases, even though the addition of calcium hydroxide to Ledermix did not appear to be synergistic in terms of enhancing the antimicrobial action. Key words: Endodontics, medicaments, antimicrobial action. Abbreviation: PEG = polyethylene glycol. (Accepted for publication 8 August 2008.) INTRODUCTION Micro-organisms play a central role in the development and progression of pulp and periapical diseases. 1 Antimicrobial medicaments placed in the canals between appointments have been shown to significantly reduce the number of organisms, and thereby increase the number of canals that are rendered free of bacteria. 2 Rapid disinfection appears to be impossible to achieve with medicaments alone, as the killing of microorganisms takes time even with powerful antimicrobial agents. 3 The major factor associated with unfavourable outcomes of endodontic treatment is persistence of micro-organisms within the root canal system. 4 When this occurs, the microbial flora usually consists of only one species or a small number of bacterial species. Gram-positive facultative anaerobic species including enterococci, streptococci, lactobacilli and yeast-like organisms have been reported in such cases, and it is noteworthy that these organisms have shown resistance to the commonly-used medicaments such as calcium hydroxide (Ca(OH) 2 ). 4,5 The aim of this study was to determine the relative antimicrobial effectiveness of some commerciallyavailable medicaments and their bases against several bacterial species commonly found in root canals using a standardized in vitro well diffusion assay system to measure inhibition of bacterial growth. MATERIALS AND METHODS The root canal medicaments evaluated were a commercially-available corticosteroid-antibiotic compound, Ledermix paste (Lederle Pharmaceuticals, Wolfsratshausen, Germany); two commercially-available Ca(OH) 2 pastes, Pulpdent paste (Pulpdent Corporation of America, Watertown, MA, USA) and Ultracal ª 2009 Australian Dental Association 141

B Athanassiadis et al. paste (Ultradent Products, South Jordan, UT, USA); plus a 50:50 mixture of Ledermix and Pulpdent pastes. Polyethylene glycol (also known as PEG) (Huntsman, Victoria, Australia) was also examined in the study as this is part of the base of Ledermix paste. PEG 400 is a clear viscous liquid, whilst PEG 3350 appears as small flakes. Ledermix paste is comprised of a mixture of PEG 400, PEG 3350 and water approximately 72%, various inactives (zinc oxide, calcium chloride, triethanolamine and sodium sulphite) approximately 24%, and the two active agents demeclocycline HCl 3% and triamcinolone 1%. The antimicrobial action of the vehicle (PEG 400 3350) was tested with water and in combination with the inactives (zinc oxide, calcium chloride, triethanolamine and sodium sulphite), which serve as preservatives, buffers and fillers. 6 The bases used in the Pulpdent and Ultracal pastes consist mainly of water and methylcellulose, hence a mixture of methylcellulose (Methocel A4M premium, Swift and Company, Rosehill, NSW, Australia) and water was tested. The calcium hydroxide used to replicate the commercial pastes was from Mallinckrodt Baker Inc (Phillipsburg, New Jersey, USA). The organisms tested were Enterococcus faecalis ATCC (American Type Culture Collection) 29212, Peptostreptococcus micros (Parvimonas micra) ATCC 33270, Prevotella intermedia wild strain, and Candida albicans ATCC14053. The anaerobic organisms (Peptococcus and Prevotella) were cultured on Brain Heart Infusion agar (BHI) (BBL 211065 Becton Dickinson, Sparks, MD, USA) with added yeast extract, haematin, Vitamin K and 5% whole horse blood. Enterococcus and Candida strains were cultured onto 5% horse blood agar (Cat No 04059, Biomerieux, Murrarrie, Australia). All subcultures were incubated at 35 C for 48 hours prior to the testing of the medicaments and their base components. Purity of strains was checked by subculture of inocula onto horse blood agar for E. faecalis and C. albicans, and Brain Heart Infusion agar for Peptostreptococcus and Prevotella. Purity checks were performed at the time of inoculation of test samples. Agar well diffusion assays were performed in 90 mm diameter Petri dishes containing Mueller Hinton Agar (BBL 211438 Becton Dickinson, Sparks, MD, USA) to a depth of 4 mm for Enterococcus and Candida. Brain Heart Infusion Agar (BBL 211065 Becton Dickinson, Sparks, MD, USA) was used for Peptostreptococcus and Prevotella. A direct colony suspension of each test isolate was prepared in 0.85% sterile saline, and the turbidity was adjusted to a 0.5 McFarland standard for E. faecalis and a 1.0 McFarland standard for Peptostreptococcus, Prevotella and Candida. The agar plates were flooded with the test suspension and a well (4 mm deep x 6 mm in diameter) was cut into the centre of the agar. A sterile pipette was used to place 50 ll of the test medicament or component into each well. The plates were then incubated at 35 C for 48 hours for Enterococcus and Candida Peptococcus, andprevotella tests were incubated under anaerobic conditions achieved using a gas generating kit (Biomerieux, Mitsubishi Gas Chemical Company Inc, Japan). After incubation for 48 hours, the diameters of the growth inhibition zones were measured in millimetres to the nearest 0.1 mm using electronic callipers. Each experiment was repeated five times, and the means and standard deviations calculated. The ph of each medicament and component was measured in triplicate (to the nearest 0.01 ph unit) using a ph meter with temperature compensation (Aqua-pH, TPS, Brisbane, Australia). The meter was calibrated to known standard solutions of ph 4.00 and 6.88. The materials to be tested were placed in direct contact with the sensor, and left in place for at least 10 seconds or until a stable ph reading was obtained (maximum of 30 seconds). The same procedures described above were used to measure the ph and zones of inhibition in three preparations made to evaluate the effect of alkalinity on the antimicrobial properties of demeclocycline hydrochloride (Sigma 6140-5G, St Louis, USA). The three preparations consisted of: (a) PEG 400 3350, water and 3.2% demeclocycline HCl; (b) PEG 400 3350, water, 3.2% demeclocycline HCl and 35% Ca(OH) 2 ; and (c) PEG 400 3350, water, 3.2% demeclocycline HCl plus the inactive components of Ledermix paste base (thereby replicating Ledermix paste but without the corticosteroid). The results were tabulated and data sets pooled from replicates examined for their distribution using the Kolmogorov-Smirnov test (GraphPad Instat version 3.0 software, Palo Alto, California, USA). As some groups showed non-normal distributions, all data were examined using the Kruskal-Wallis (non-parametric) test, followed by Dunn s multiple comparison test, with significance set at the 5% level. RESULTS The mean diameters of inhibition zones and the ph values for each preparation are presented in Table 1. The range of inhibition values observed between the preparations varied over a large range, and not all preparations exhibited antimicrobial properties. Overall, the largest zones of growth inhibition in the well diffusion assay were seen with Ledermix paste and with the 50:50 mix of Ledermix and Pulpdent pastes. The PEG 400 3350 and water combination showed a small inhibitory effect on P. intermedia, however the addition of zinc oxide, calcium chloride, triethanolamine and sodium sulphite (inactives) to PEG 400 3350 resulted in inhibition of C. albicans as well. 142 ª 2009 Australian Dental Association

Antimicrobial action of medicaments Table 1. Diameter of zones of inhibition for each organism for the various preparations examined, measured (in mm) after 48 hours Preparation E. faecalis Peptostrep. micros Prevotella intermedia Candida albicans ph PEG + water only 0.00 (0.00) 0.00 (0.00) 12.56 (0.39) 0.00 (0.00) 8.83 (0.05) PEG + inactive components (i.e. ZnO, CaCl, etc) 0.00 (0.00) 0.00 (0.00) 17.36 (3.36) 13.54 (0.60) 8.50 (0.01) Methylcellulose 2% + water 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 9.64 (0.15) Ledermix paste 9.58 (0.42) 0.00 (0.00) 37.84 (2.38) 12.4 (1.52) 7.63 (0.05) Pulpdent paste 10.38 (0.59) 0.00 (0.00) 0.00 (0.00) 20.78 (1.13)* 12.64 (0.03) Ultracal paste 10.90 (1.19) 0.00 (0.00) 0.00 (0.00) 20.66 (1.92)* 12.53 (0.09) 50:50 mix of Ledermix + Pulpdent pastes 10.8 (0.89) 0.00 (0.00) 35.76 (6.25) 19.45 (0.55)* 12.00 (0.06) PEG + water + demeclocycline HCl 3.2% 21.56 (0.46)* 22.10 (1.41)* 49.90 (1.50)* 12.76 (1.61) 3.58 (0.03)* PEG + water + demeclocycline HCl 3.2% + inactives 12.20 (0.57) 11.94 (0.95)* 60.8 (1.30)* 12.66 (0.51) 7.52 (0.02)* PEG + water + demeclocycline HCl 3.2% 0.00 (0.00) 0.00 (0.00) 21.90 (1.19) 20.11 (2.48)* 11.47 (0.05) (no inactives) + Ca(OH) 2 35% Methylcellulose 2% + water + Ca(OH) 2 35% 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 12.43 (0.01) Data shown are the means (and standard deviation) from five separate experiments. *Indicates a significant change (P < 0.05) compared with the baseline. PEG = PEG 400 3350. All commercial products showed similar in vitro effectiveness against E. faecalis, but none were active against Peptostreptococcus micros. This latter species was therefore the most resistant organism in vitro to all medicaments tested using this assay system. Ultracal and Pulpdent pastes showed the least effectiveness of the commercial products tested, with no inhibition of Peptostreptococcus micros or Prevotella intermedia. Methylcellulose was not effective in vitro against any of the organisms tested when used alone. Table 2 lists the mean diameters of growth inhibition for P. intermedia against varying dilutions of PEG 400 3350 combinations. There was no in vitro antimicrobial activity for PEG 400 3350 and its various dilutions against E. faecalis, P. micros and C. albicans, however a graduated decrease was noted in its activity to P. intermedia as the dilution factor increased. The demeclocycline PEG water combination (with or without inactives) resulted in the greatest zones of inhibition overall for E. faecalis, P. micros, and P. intermedia (P < 0.01 compared with other groups). The addition of the inactives to the demeclocycline PEG water combination resulted in a significant decrease in zone sizes for E. faecalis and P. micros, while the addition of calcium hydroxide to the same Table 2. Mean dose responses for PEG 400 3350 PEG 400 3350 Dilutions with water (%) Prevotella intermedia 0 14.5 5 14.3 10 13.7 20 12.1 40 0 60 0 Data for this dilution study are the diameters of the zone of inhibition (in mm) after 48 hours. Data are the mean from two separate experiments. demeclocycline PEG water mix also resulted in a significant reduction in the zones of inhibition seen, except in the case of C. albicans. With regard to ph, both Pulpdent and Ultracal pastes had a similar ph of 12.6, while the PEG 400 3350 water and PEG 400 3350 inactives gave similar ph readings. The addition of demeclocycline to PEG 400 3350 water resulted in a significant drop of ph, whilst the addition of 35% calcium hydroxide to methylcellulose and water resulted in a ph which was similar to that seen with the Pulpdent and Ultracal. The Dunn s multiple comparison test revealed that there was a significant difference between the bases and the active preparations (P < 0.05) for the demeclocycline in PEG (with without the inactives added) across all the micro-organisms tested (except C. albicans). The preparations found to have significant inhibitory activity against individual micro-organisms were as follows: For E. faecalis, demeclocycline in PEG (with without inactives) and PEG demeclocycline HCl Ca(OH) 2. For P. micros and P. intermedia, PEG demeclocycline HCl (with without inactives) and PEG demeclocycline HCl Ca(OH) 2. Preparations that were significantly active against C. albicans were those with higher ph (Pulpdent, Ultracal, Ledermix Pulpdent 50:50, PEG demeclocycline HCl Ca(OH) 2, methylcellulose Ca(OH) 2 water). DISCUSSION The bacterial species Peptostreptococcus micros and Prevotella intermedia were chosen to represent organisms typically isolated from untreated infected root canal systems, 7,8 whilst E. faecalis and C. albicans were selected to represent organisms commonly isolated from the root canals of teeth that have been previously root-filled. 4 Chu et al. 9 showed that both E. faecalis and C. albicans (which are known to be more resistant ª 2009 Australian Dental Association 143

B Athanassiadis et al. to endodontic therapy) are linked to unfavourable treatment outcomes. The agar diffusion test method employed in this study is useful for evaluating the in vitro antimicrobial activity of medicaments before other more advanced tests are performed. The results obtained from this in vitro test must be interpreted with caution as this assay may not demonstrate the full clinical potential of the material being tested. The Clinical and Laboratory Standards Institute (CLSI) Performance Standards for Antimicrobial Susceptibility Testing (M100-S17) 10 state that cephalosporins, aminoglycosides, clindamycin and trimethoprim-sulfamethoxazole may appear active in vitro against Enterococcus spp., but they are not effective clinically, and isolates should not be reported as susceptible. Although the agents examined were not in these classes, it highlights the precautions required in assessing agar diffusion results. The Therapeutic Guidelines used for prescribing in Australia 11 specifies the resistance of common bacterial pathogens in Australia, and states that E. faecalis, Peptostreptococcus spp., Fusobacteria spp., Propionibacterium spp., and some other species are resistant to doxycycline tetracycline minocycline. Moreover, resistance of Enterococcus spp. and Peptostreptococcus spp. to clindamycin is fairly common. Both tetracycline and clindamycin are bacteristatic antibiotics, 12 14 and this characteristic may explain the inactivity of these medicaments and their bases seen in the agar diffusion model for E. faecalis and P. micros. The greater resistance of these two Gram-positive bacterial species to the various medicaments and bases evaluated in this study may explain why in some cases persisting infection may remain despite the use of medicaments. Due to the issues of resistance outlined above as well as a limited spectrum of activity and lack of antifungal properties, antibiotics may not be the ideal agent for use in intracanal medicaments. This same thinking has obvious application to the issue of agents which may be used as irrigants, where once again the choice of material used needs to account for the likelihood of resistant organisms being present. With regard to the assay system used, the size of the inhibition zone seen will depend upon the solubility and diffusibility of the test substance in agar. In this regard, the vehicles on which these pastes are based may dramatically influence their chemical characteristics (such as solubility and diffusion). Materials in waterbased vehicles (i.e., distilled water or saline solution) will have a higher rate of ionic dissociation in agar than those in viscous vehicles (e.g., PEG) and oil-based vehicles (e.g., camphorated paramonochlorophenol). As reported by Estrela et al. 15 the vehicle plays a supportive role by giving pastes their chemical characteristics (i.e., dissociation and diffusion) as well as the appropriate consistency for placement within, and removal from, the root canal system. While agar diffusion tests, although aqueous in nature, do not mirror exactly the antimicrobial effect of the medicaments within dentine tubules, they do allow direct comparisons of the ability of various agents to diffuse through agar and inhibit the test organisms. Poorly diffusing materials will give very small zones of inhibition, even if they are potent antimicrobial agents. 16 In addition, agar diffusion tests only show inhibition of growth (i.e., bacteristatic), which may not be the same as bacterial death (i.e., bactericidal). 2 An important finding in the present study was the inhibitory effect of PEG 400 3350 against Prevotella intermedia, a species which was inhibited only by PEG and by preparations based on PEG. The lack of inhibition of this organism has been reported with calcium hydroxide preparations based on water and glycerine (such as Hy-Cal, Root-cal and Hypo-cal) 17 rather than those with PEG bases. However, the effect of PEG per se on endodontic pathogens has not hitherto been recognized. The inhibitory effect of PEG against Gram-negative species is clearly an advantage when used as a base for the formulation of endodontic medicaments. Chifre et al. showed that PEG 400 only can exert antimicrobial effects by lowering the water content of Gram-negative bacterial cells, causing plasmolysis, clumping and morphological changes. 18 In fact, PEG 400 meets many of the physiochemical requirements for an ideal topical disinfectant it is neutral, highly soluble in water, non-irritating to the skin, does not decompose, and its toxicity from acute oral administration is extremely low. 18 Currently, PEG 400 is used to form the base for pastes such as Ledermix and Calen (a calcium hydroxide paste) (SS White, Rio de Janeiro, Brazil). Its biocompatibility is demonstrated by its wide use in cosmetic and medical preparations. There are no reports in the literature of studies that have examined the antimicrobial properties of the components that make up the base for Ledermix paste and the various calcium hydroxide pastes. The results of the current study indicate that PEG 400 3350 only provides some in vitro antimicrobial activity against P. intermedia, whilst methylcellulose has no in vitro antimicrobial properties. In the study by Chifre et al., 18 PEG 400 showed significant antibacterial activity against Gram-negative bacteria, including Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli while Gram-positive bacteria such as Staphylococcus aureus were far more resistant. This effect has also been demonstrated in the current study. With regard to mixtures of preparations, it was shown that for two cariogenic micro-organisms, Lactobacillus casei and Streptococcus mutans, the 50:50 mixture of Ledermix and calcium hydroxide pastes was very marginally more effective than either paste alone. 19 This finding was not corroborated by Seow 20 144 ª 2009 Australian Dental Association

Antimicrobial action of medicaments who reported that the 50:50 combination showed reduced activity against Streptococcus sanguis and Staphylococcus aureus. The results presented in Table 1 show that, when compared to Pulpdent paste, the addition of Ledermix paste to Pulpdent increased the in vitro activity against P. intermedia only. This increased activity is in fact most likely to be due to the carry over effect of the demeclocycline HCl and PEG 400 3350 within the Ledermix paste. When compared to Ledermix paste, the combination showed improved in vitro activity against C. albicans. Demeclocycline hydrochloride, as with other tetracyclines, is a yellow, odourless, crystalline powder that darkens in strong sunlight and in moisture. The potency of all tetracyclines is reduced in solutions having a ph below 2, and they are rapidly destroyed in solutions of alkali hydroxides. A 1% demeclocycline HCl solution in water has a ph of 2 to 3. Other properties of demeclocycline HCl include an extreme affinity for binding to calcium ions, the greatest of all the tetracyclines. 12 The demeclocycline PEG water mixture with a low ph (3.58) gave the largest inhibition zones against P. micros and E. faecalis, and most of this can be attributed to the low ph of the mix. The effect of ph on demeclocycline was investigated in this study, and the results presented in Table 1 show a significant reduction in inhibition zone diameters when 35% calcium hydroxide was added to the demeclocycline HCl PEG mix. In this case the inactives consisting of zinc oxide, calcium chloride, triethanolamine and sodium sulphite were not added in order to assess the direct effect of calcium hydroxide on demeclocycline only. Thus, the addition of calcium hydroxide to the demeclocycline HCl PEG mix reduced the effect of demeclocycline HCl, raising the ph from 3.58 to 11.47. The combined individual zone sizes of Ledermix and Pulpdent paste do not equate to the zone size of the 50:50 combination, hence this 50:50 combination of Ledermix Pulpdent pastes is not additive or synergistic. The reported ph values for calcium hydroxide products in the literature correspond to those noted in the present study. The ph of PEG 400 3350 in water was very similar to the ph of PEG 400 3350 combined with all the inactive components found in the Ledermix paste base. The methylcellulose base was alkaline, and hence the addition of calcium hydroxide gave it an even higher ph. The ph of the 50:50 mix of Ledermix and Pulpdent pastes was found to be lowered from the 12.64 of Pulpdent paste alone to 12.00. This was similar to the results reported by Taylor et al. 19 who found that mixing these two pastes marginally lowered the ph from 12.8 to 12.5 and Anderson et al. 21 who found a small decrease in ph from 12.18 to 12.06. The final point of interest from this study relates to the decrease in the zone of growth inhibition as the PEG 400 3350 (with no inactives or demeclocycline) was diluted with water. The greatest zone of inhibition occurred within the 0 10% range of water dilution. In the current study, E. faecalis, P. micros and C. albicans were not affected by PEG 400 3350 (with no inactives or demeclocycline). CONCLUSIONS This agar well diffusion study, which measured changes in the size of inhibition zones, has shown the importance of assessing the in vitro activity of basal components as well as active ingredients in endodontic medicaments. PEG 400 3350 alone can exert a limited level of antimicrobial action, whilst methylcellulose has no detectable in vitro activity. The addition of a tetracycline compound to the PEG 400 3350 (as in Ledermix paste) gave improved antimicrobial properties. The 50:50 mixture of Ledermix and Pulpdent pastes showed no additive or synergistic effects compared to Ledermix paste alone, and this was confirmed by the combination of demeclocycline HCl and calcium hydroxide in PEG 400 3350. Hence, in the in vitro agar well diffusion model, Ledermix paste and the 50:50 combination with Pulpdent paste were the most effective commercial medicaments, when compared to Pulpdent or Ultracal paste. Clinically, in cases where periapical inflammation is present, the use of a 50:50 combination of Ledermix and Pulpdent may be of value because of the antimicrobial actions of the calcium hydroxide, and the anti-inflammatory actions of the corticosteroid. REFERENCES 1. Kakehashi S, Stanley HR, Fitzgerald RJ, Bethesda BS. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 1965; 20:340 349. 2. Haapasalo M, Endal U, Zandi H, Coil JM. 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