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 Table of Contents  
Year : 2017  |  Volume : 1  |  Issue : 2  |  Page : 124-128

Critical concentrations of surfactant combination regimens with MTAD™ on vancomycin-sensitive Enterococcus faecalis

1 Department of Conservative Dentistry and Endodontics, PMS College of Dental Science and Research, Thiruvananthapuram, Kerala, India
2 Department of Endodontics, ABSMIDS, Nitte University, Mangalore, Karnataka, India
3 Department of Microbiology, ABSMIDS, Nitte University, Mangalore, Karnataka, India
4 Department of Biochemistry, ABSMIDS, Nitte University, Mangalore, Karnataka, India

Date of Web Publication23-Nov-2017

Correspondence Address:
Manikandan Ravinanthanan
Department of Conservative Dentistry and Endodontics, PMS College of Dental Science and Research, Vattappara, Thiruvananthapuram, Kerala
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_78_17

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Background: Enterococcus faecalis is an opportunistic pathogen with greater virulence traits and has gained significance in endodontic literature. It is resistant to most irrigants and medicaments with significant posttreatment reversal cultures. The purpose of this study was to evaluate the critical concentrations of irrigants and surfactants and the possible outcome of the combination regimen on E. faecalis. Methods: Five percent sodium hypochlorite (NaOCl), chlorhexidine (CHX), iodine potassium iodide (IKI), and surfactants such as cetrimide (CTR) and sodium dodecyl sulfate (SDS) were prepared. BioPure™ MTAD™ served as control. Minimum inhibitory concentration was performed to determine the dilution fold and dilution factor by serial dilution, followed by minimum bactericidal concentration on agar plates. Results: Although CHX performed better than MTAD™, it proved to be bacteriostatic in action at lower concentrations. NaOCl and IKI were found to be inferior and were excluded. CTR was found to be better than MTAD™; while SDS showed insignificant results. The combination surfactant regimens (CHX + CTR; CHX + SDS) performed in a synergistic fashion and achieved higher eradication rates. Bactericidal action with narrow margins between critical concentrations was recorded and found to be superior to any test irrigant, including MTAD™. Conclusion: Critical concentrations of surfactant combination regimens were found to be highly effective at lower dilutions on E. faecalis. This combination can overcome the limitations, potentiate antimicrobial spectrum, and increase the substantive antimicrobial action of CHX application in clinical endodontics.

Keywords: CTR, combination regimen, SDS, surfactant

How to cite this article:
Ravinanthanan M, Hegde MN, Shetty V, Kumari S. Critical concentrations of surfactant combination regimens with MTAD™ on vancomycin-sensitive Enterococcus faecalis. Biomed Biotechnol Res J 2017;1:124-8

How to cite this URL:
Ravinanthanan M, Hegde MN, Shetty V, Kumari S. Critical concentrations of surfactant combination regimens with MTAD™ on vancomycin-sensitive Enterococcus faecalis. Biomed Biotechnol Res J [serial online] 2017 [cited 2022 Aug 11];1:124-8. Available from: https://www.bmbtrj.org/text.asp?2017/1/2/124/219110

  Introduction Top

The microorganisms colonizing the root canal system play an essential role in the pathogenesis of periradicular lesions. Endodontic treatment outcome depends primarily on how well the root canal system is shaped and cleaned. The current concept of root canal preparation is shaping to clean; to maximize the efficiency of endodontic irrigants at the apical terminus.[1] Disinfection of the root canal system in necrotic and infected pulps present greater challenges than vital pulps to achieve predictable results.[2]

Irrigants are used to flush out loose debris, lubricate dentinal walls, dissolve organic matter, and provide antimicrobial activity. Mechanical instrumentation with nonantimicrobial irrigants yielded positive cultures in 80% of initially infected root canals.[3] Thus, irrigants with antimicrobial properties are more effective in eliminating intracanal microorganisms. Therefore, chemomechanical preparation with an antimicrobial irrigant is recommended to ensure higher eradication rates.[3],[4]

The intracanal environment consists of a plethora of microorganisms ranging from Gram-positive and Gram-negative ones.[5] The complexities in the root canal system such as accessory and lateral canals which harbor these microorganisms effectively shield them from the action of antimicrobial agents. The facultative microbial floras are not readily eliminated than their obligate counterparts, thus contributing to higher resistance to intracanal irrigants and medicaments.[6]

Sodium hypochlorite (NaOCl) is the most popular irrigating solution; it has the advantage of being effective at concentrations between 1% and 6%, has potent antimicrobial activity, and dissolves necrotic tissue. Disadvantage being it does not remove smear layer from dentinal walls, highly destructive on contact with periapical and gingival tissues. Higher concentrations were found to affect the microhardness significantly and erode dentinal microstructure.[7],[8]

Ethylene diamine tetraacetic acid (EDTA) has been advocated as it removes smear layer by affecting the inorganic component of dentin, facilitates removal of infected tissue and bacteria in root canal. However, it has low or no antibacterial activity and contributes to dentinal erosion.[9] Thus, a combination of NaOCl and EDTA is recommended to facilitate root canal disinfection.[10]

Chlorhexidine digluconate (0.12%–2%) has been used an adjunct irrigant as it does not affect the smear layer;[11] iodine potassium iodide (1%–2%) although a potent irrigant finds limited application in clinical practice.[12] Torabinejad reported development of a new irrigant for root canal disinfection and smear layer removal. BioPure™ MTAD™ (Dentsply, Tulsa), which consists of tetracycline isomer (doxycycline), an acid (citric acid), and a detergent (tween 80), has been claimed to have superior antibacterial properties.[13]

Newer irrigants such as Cetrixidin (0.2% CHX and 0.2% cetrimide), Endo CHX (2% CHX with surfactants), and Chlor-XTRA™ (6% NaOCl with surface modifiers) are commercially available. The surfactants/modifiers added have been claimed to enable intimate contact of the irrigant to the microbes to enhance their antibacterial effect.[14] Thus, the purpose of our study was to determine the minimum effective concentrations of surfactant combination irrigant regimen on Enterococcus faecalis. We hypothesize that there will be a significant difference between primary and combination surfactant regimens. We postulate this based on our previous study results obtained.[15],[16]

  Methods Top

Place of study and bacterial strain used

The present study was performed in Central Research laboratory, A. B Shetty Memorial Institute of Dental Sciences, Nitte University. E. faecalis ATCC 29212 (HiMedia) was used in the study. Bacteria were subcultured from the stock culture. The suspension culture of the test microorganism was prepared in Brain Heart Infusion broth.

Standardization of microorganisms

Brain Heart Infusion broth was inoculated with the test microorganism (E. faecalis) and incubated for 6–7 h to get mean optical density of 0.5 McFarland constant; equivalent to 1.5 × 108 CFU/ml. Then, 1 ml of each suspension culture was transferred to the required number of sterile screw cap tubes (HIMEDIA). All procedures were performed using sterilized instruments and materials.

Irrigants and surfactants used

The primary irrigant group comprised of 5% NaOCl (Prevest Denpro), 5% CHX (Sigma), and 5% IKI (HiMedia). Surfactants such as cetrimide (CTR), HiMedia, and sodium dodecyl sulfate (SDS), Merck, were prepared at 5% to standardize the concentrations for test protocol. BioPure™ MTAD™ (Tulsa, Dentsply) was used as per manufacturer's instructions which served as control. All the prepared irrigants were stored in sterile bottles.

Determination of minimum inhibitory concentration

Minimum inhibitory concentration (MIC) of primary, surfactants, combination regimens, and MTAD™ on E. faecalis was determined by broth dilution method.[17] All the irrigants were diluted in 1 ml of Brain Heart Infusion broth followed by 2-fold serial dilution of the irrigants subsequently. To the serially diluted irrigants, 20 μl of the bacterial suspension was inoculated and tubes were kept for overnight incubation at 37°C in an incubator. Test tubes were evaluated for visible growth of the bacteria (turbidity). MIC of the irrigants was determined by evaluating the minimum concentration of the irrigants inhibiting visible growth (number of negative tubes) of the bacteria in broth.

Determination of minimum bactericidal concentration

To determine the minimum bactericidal concentration (MBC) of irrigant, 20 μl of the solution from the MIC concentration obtained was transferred to the test tubes containing fresh Brain Heart Infusion broth and incubated overnight in an incubator. Then, each tube was streaked on Muller–Hinton Agar plates. After overnight incubation, plates were examined for the colonies of E. faecalis. The concentration that effectively inhibited growth was taken as MBC.

  Results Top

The MIC and MBC of primary, surfactants, and combination regimens by fold dilution and dilution factor are summarized in [Table 1] and [Table 2], respectively. The number of negative tubes obtained, denoted as fold dilution (in numbers), is depicted in [Figure 1], [Figure 2], [Figure 3].
Table 1: Minimum inhibitory concentration values obtained with primary, surfactant, and combination irrigant regimens with MTAD™

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Table 2: Minimum bactericidal concentration values obtained with primary, surfactant, and combination irrigant regimens with MTAD™

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Figure 1: Bar graph depicting fold dilution of primary irrigants with MTAD™

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Figure 2: Bar graph depicting fold dilution of surfactants with MTAD™

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Figure 3: Bar graph depicting fold dilution of combination regimens with chlorhexidine and MTAD™

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Minimum inhibitory concentration

In the primary irrigant group, the dilution fold (in numbers) obtained was as follows; 5% CHX (22), MTAD™ (14), 5% IKI, (6) and 5% NaOCl (4). The surfactants 5% CTR and 5% SDS yielded 16- and 10-fold dilution, respectively. Surfactant combination regimens with 5% CHX showed synergistic effect, while those of NaOCl and IKI were inferior to MTAD™ and were thus excluded. A combination of 5% CHX + 5% CTR by serial dilution was effective at dilution fold of 30, while a combination of 5% CHX + 5% SDS yielded significant results even at 33.

Minimum bactericidal concentration

In the primary irrigant group, the dilution fold (in numbers) obtained was as follows; 5% CHX (13), MTAD™ (12), 5% IKI (5), and 5% NaOCl (3). The surfactants 5% CTR and 5% SDS yielded 14- and 9- fold dilution, respectively. Surfactant combination regimens with 5% CHX showed synergistic effect. A combination of 5% CHX + 5% CTR by serial dilution was effective at dilution fold of 25, while a combination of 5% CHX + 5% SDS was effective even at dilution folds of 31.

  Discussion Top

E. faecalis is a Gram-positive facultative anaerobe commonly detected in asymptomatic persistent endodontic infections. Its prevalence in such infections ranges from 24% to 77%. This finding can be explained by its various survival and virulence factors; including its ability to compete with other microorganisms, invading dentinal tubules, and resisting nutritional deprivation.[18]E. faecalis (ATCC 29212) is a commonly used quality control strain for in vitro studies[19] and thus was selected.

Torabinejad et al. found that MTAD™ was more effective in killing E. faecalis than NaOCl when the solutions were diluted; however, EDTA did not exhibit any antibacterial activity.[20] Newberry et al. reported that MIC/MLC tests showed that MTAD™ inhibited most strains of E. faecalis growth.[21] BioPure™ MTAD™ requires 1.3% NaOCl before its application as per manufacturer's instruction and hence restricted as a final rinse only. Although this combination was found to be superior,[22] certain studies find it insignificant as compared with NaOCl/EDTA regimen.[23] Currently, E. faecalis has been found to be resistant even to higher concentrations of NaOCl,[24] and clinical strains have emerged resistant to tetracycline as well.[25] Tong et al. stated that nisin improves the postantibiotic sub-MIC effects of MTAD™ against E. faecalis as well as sensitizes it to alkaline environments, and thus nisin might be used to further develop the application of MTAD™ in root canal irrigation.[26]

Estrela et al. assessed the efficacy of NaOCl and CHX on E. faecalis and concluded that both irrigants failed to eliminate E. faecalis when evaluated by either PCR or culture techniques.[27] Newer irrigants are on the rise with modifications with NaOCl (Chlor-XTRA™) or CHX (CHX Plus) and have claimed to be effective than their counterparts; however, no concrete data exist to support this claim. Thus, this study evaluated minimum effective concentrations (MIC and MBC) of primary irrigants and with surfactant combination regimens.

MIC and MBC are quantitative approaches to determine the antibacterial sensitivity of test organism. MIC determines the concentration of the antimicrobial agent inhibiting the visible growth and MBC determines the lowest concentration that kills the bacteria in cultured plates. MIC and MBC use serial dilutions of a solution to determine the minimum concentration that would still exert antimicrobial properties.[28],[29]

Five percent of primary irrigants, surfactants and combination regimens was chosen to standardize the dilution factor and fold dilution of test irrigants. Tube dilution[29] was performed for MIC determination as it can be visualized for the presence of turbidity which can be cross veri fied by individual examiners. The lowest MIC concentration obtained was cultured to obtain and confirm MBC values. The results obtained were recorded as fold dilution and dilution factor for analyzing the results. An irrigant is said to be bactericidal if the MIC and MBC values recorded were same. If the MBC is greater than the MIC obtained, then it is said to be bacteriostatic. The data obtained enable us to predict the nature of the mechanism of action at the lowest levels achieved.[29]

Among the primary irrigants, although CHX elicited greater MIC values than MTAD™, the MBC values obtained were similar to MTAD™, thus confirming it to be only bacteriostatic at lower concentrations. The results obtained with NaOCl and IKI proved to be inferior and thus were excluded. In the surfactant group, CTR showed better MIC and MBC values than MTAD™, while results with SDS by fold dilution proved to be inadequate. Both CTR and SDS displayed narrow margin between the MIC and MBC values similar to MTAD™, thus suggesting near-bactericidal action.[30]

The combination regimens comprising 5% CHX + 5% CTR and 5% CHX + 5% SDS did not show any chemical interaction and were stable. Serial 2-fold dilutions revealed antibacterial efficacy even at extremely very low concentrations, thus suggesting synergetic effect of this combination regimens. The possible explanation could be due to the surfactants' lytic effect and solubilizing action on organic component of the cell wall,[30],[31] thereby allowing adherence and deeper penetration of CHX into the cellular organelles causing cell death.[32],[33] Thus, these combination regimens achieved bactericidal action as depicted by their narrow margins between the obtained MIC and MBC values.

The results of our study are in accordance with Gomes et al. and Vianna et al. who reported that antibacterial activity of NaOCl was effective at higher concentrations only.[34],[35],[36] Jungbluth stated that Chlor-XTRA™ (6% NaOCl + surface modifiers) has no unique features other than its price; its reduced surface tension with surfactants did not result in greater soft-tissue dissolution by NaOCl.[37] Radcliffe et al. and Retamozo et al. also found that higher concentrations of NaOCl and longer exposure times are required to eliminate E. faecalis.[38],[39] IKI with surfactant combinations yielded a lesser antimicrobial activity as compared with IKI alone. This could be due to interaction of IKI with the organic surfactants, resulting in decreased antibacterial activity.[40]

In addition, in favor of our study, Giardino et al. also stated that cetrexedin (0.2% CTR + 0.2% CHX) has the lowest surface tension value, thus increasing the intimate contact of irrigant solution with the dentinal walls, permitting deeper penetration of the irrigant.[41] Baca et al. stated that 2% CHX + 0.2% CTR would be an effective alternative as the final irrigation regimen, given its antimicrobial action over time.[42]

Our study results were not in accordance with Estrela et al., who reported that 1% NaOCl was effective on all microorganisms; however, 2% CHX was ineffective on certain species individually and in mixed culture.[43] Portenier et al., who compared the antimicrobial activity of MTAD™ to that of CHX, found MTAD™ and CHX to be equally effective in killing E. faecalis.[44]

  Conclusion Top

Within the limitations of our study, we conclude that combination surfactant irrigant regimens at lower concentrations can effectively eradicate E. faecalis. The clinical significance of its bactericidal action may overcome the limitations of conventional endodontic irrigants. Resistance to this regimen is unlikely and the synergistic effect will potentiate the substantive antimicrobial action of CHX. Further studies on various microorganisms and other parameters need to be evaluated before the final conclusion can be drawn regarding its clinical application.

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Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2]

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