doi:10.1111/iej.12248
Antibiofilm activity of irrigating solutions associated with cetrimide. Confocal laser scanning microscopy
nior1, M. S. Z. Graeff2, J. M. Guerreiro-Tanomaru1, C. A. Nascimento1, N. B. Faria-Ju 3 1 E. Watanabe & M. Tanomaru-Filho 1
Department of Restorative Dentistry, Araraquara Dental School, UNESP (Univ Estadual Paulista), Araraquara, SP; 2Integrated Research Center, Bauru Dental School, USP – University of S~ ao Paulo, Bauru, SP; and 3Department of Restorative Dentistry, Ribeir~ ao Preto Dental School, USP (University of S~ ao Paulo), Ribeir~ ao Preto, SP, Brazil
Abstract Guerreiro-Tanomaru JM, Nascimento CA, Faria nior NB, Graeff MSZ, Watanabe E, Tanomaru-Filho Ju M. Antibiofilm activity of irrigating solutions associated with cetrimide. Confocal laser scanning microscopy. International Endodontic Journal, 47, 1058–1063, 2014.
Aim To evaluate the antibiofilm activity of sodium hypochlorite (NaOCl) and chlorhexidine (CHX) solutions associated with cetrimide (CTR), and QMiX using confocal laser scanning microscopy. Methodology Enterococcus faecalis (ATCC- 29212) biofilms were induced on bovine dentine blocks for 14 days. The dentine blocks containing biofilm were immersed for 1 min in the following solutions: 2.5% NaOCl; 2.5% NaOCl + 0.2% CTR; 2% CHX; 2% CHX + 0.2% CTR; 0.2% CTR; QMiX. After contact with the solutions, the dentine blocks were stained with Live/Deadâ BacLight for analysis of the remaining biofilm using confocal laser scanning microscope. Images were evaluated using the BioImage_L software to determine the total biovolume (lm3), the green
Introduction The antimicrobial solutions used during root canal preparation are important for treatment success (Sjogren et al. 1997). Enterococcus faecalis is commonly
Correspondence: Mario Tanomaru-Filho, Rua Humait a, 1680, Caixa Postal 331, Centro, 14801-903 Araraquara, SP, Brazil (Tel.: +55-16-3301-6390; Fax: +55-16-33016392; e-mail: [email protected]).
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biovolume (live cells) (lm3) and the percentage of substrate coverage (%). The data were subjected to nonparametric statistical test using Kruskal–Wallis and Dunn’s tests at 5% significance level. Results After exposure to irrigants, the total biovolume observed for CHX, CHX+CTR, CTR, QMiX was similar to distilled water (P > 0.05). NaOCl and NaOCl+CTR had the lowest total and green biovolume. The CTR and QMiX had intermediate green biovolume, with greater antibacterial activity than CHX and CHX+CTR (P < 0.05). The NaOCl and NaOCl+CTR solutions were associated with microorganism removal and substrate cleaning ability. Conclusions NaOCl and NaOCl+CTR solutions were effective on microorganism viability and were able to eliminate biofilm. The addition of cetrimide did not influence antibacterial activity. Keywords: biofilm, chlorhexidine, confocal laser scanning microscopy, Enterococcus faecalis, sodium hypochlorite. Received 30 September 2013; accepted 10 January 2014
associated with root canal treatment failure (R^ ocßas et al. 2004, Siqueira & R^ oßcas 2004, 2008, Stuart et al. 2006, Gupta et al. 2013), due to its persistence within the root canal system. This microorganism has several resistance mechanisms and has the ability to form a biofilm (Stuart et al. 2006). When organized as a biofilm, microorganisms are surrounded by an extracellular polymeric matrix that confers resistance against antimicrobial agents (Donlan & Costerton 2002). The most commonly used irrigating solutions are sodium hypochlorite (NaOCl) and chlorhexidine
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Guerreiro-Tanomaru et al. Antibiofilm activity of irrigants solution with cetrimide
(CHX). NaOCl has a wide antibacterial activity and the ability to dissolve organic tissues, such as pulp tissue, collagen fibres and bacterial biofilm (Haapasalo et al. 2010). CHX has an antibacterial action, but is incapable of dissolving organic tissue (Mohammadi & Abbott 2009). A confocal laser scanning microscopy study demonstrated that 1% NaOCl acted on microbial viability and architecture of biofilm better than 2% CHX (Ordinola-Zapata et al. 2012). A 2% CHX solution did not eliminate biofilm from endodontic microbiota when applied for 15 or even for 30 min (Clegg et al. 2006, Del Carpio-Perochena et al. 2011). Addition of a surfactant to irrigating solutions may favour their antibiofilm activity. Surfactants can weaken the cohesive forces between the polymer matrix and bacterial cells, promoting biofilm disruption (Sim~ oes et al. 2005, Wang et al. 2012b). CHX-Plus, based on CHX with a surfactant, has demonstrated greater effectiveness than conventional CHX against a multispecies biofilm (Shen et al. 2009, 2011). Wang et al. (2012b) observed that after 1 min of contact with dentine infected by E. faecalis, CHXPlus exhibited antibacterial effects similar to 6% NaOCl and 2% NaOCl associated with cetrimide (CTR), a cationic surfactant. QMiX (Dentsply Tulsa Dental, Tulsa, OK, USA) is an irrigating solution that contains 2% CHX, 17% EDTA and CTR (Stojicic et al. 2012). This solution has shown antibacterial activity similar to 6% NaOCl when used for disinfection of dentine tubules contaminated with E. faecalis (Ma et al. 2011, Wang et al. 2012b). QMiX demonstrated stronger antibacterial action than CHX after 1 min of contact with E. faecalis biofilm (Stojicic et al. 2012). CTR is also present in other irrigants such as Tetraclean (Ogna Laboratori Farmaceutici, Muggio, Italy) (Giardino et al. 2009), Hypoclean (Palazzi et al. 2011) and SmearClear (da Silva et al. 2008). vCTR at 0.0312% resulted in negative cultures after 1 min of contact with E. faecalis biofilm induced for 24 h (Arias-Moliz et al. 2010). A 0.2% CTR solution eradicated microorganisms after 1 min (Baca et al. 2011). This surfactant has been shown to enhance antibiofilm activity when associated with lactic acid (Arias-Moliz et al. 2012), EDTA, citric acid (Ferrer-Luque et al. 2010) and BioPure MTAD (Dentsply Tulsa Dental, Tulsa) (Pappen et al. 2010). The aim of this study was to evaluate the antibiofilm activity of cetrimide associated with NaOCl or CHX, and QMiX using confocal laser scanning microscopy. The null hypothesis is that these solutions do not have antibiofilm activity.
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Materials and methods Biofilm growth on dentine blocks Forty-two autoclaved bovine root dentine blocks measuring 5 mm 9 5 mm 9 0.7 mm (width 9 length 9 thickness) were used as the substrate for biofilm growth. The dentine blocks were immersed in 17% EDTA (Odahcan-Herpo; Prod. Dent. Ltda., Rio de Janeiro, RJ, Brazil) for 3 min and then rinsed with sterile saline before autoclaving. Each dentine block was immersed in 1.8 mL Brain Heart Infusion (BHI; Difco, Detroit, MI, USA) and 200 lL of E. faecalis (ATCC29212) suspension, previously adjusted to optical density equivalent to 1 9 107 colony-forming units per millilitre (CFU mL 1). The dentine blocks were incubated for 14 days at 37 °C, under microaerophilic conditions on a rocking table. The BHI medium was changed every 48 h (Guerreiro-Tanomaru et al. 2013).
Direct contact of biofilm with irrigating solutions Antibiofilm activity was evaluated using root canal irrigants associated with CTR, divided into 7 groups (Table 1). The blocks were rinsed with saline before immersion in the irrigants for 1 min (n = 3). In the negative control group, the blocks were only rinsed with saline and received no further treatment. After the contact period, the dentine blocks were rinsed in the neutralizing solution (1% sodium thiosulfate/ Tween 80 + Lecithin) and rinsed once again in saline.
Analysis of the biofilm by confocal laser scanning microscopy For the analysis of antimicrobial activity using confocal laser scanning microscopy, the biofilm on the dentine blocks was stained with Live/Deadâ BacLight (Invitrogen, Eugene, OR, USA) and incubated for 15 min in Table 1 Irrigating solutions and associations evaluated Groups
Irrigating solutions
Group Group Group Group Group Group Group
2.5% NaOCla 2.5% NaOCl + 0.2% CTRb 2% CHXa 2% CHX + 0.2% CTR 0.2% CTR QMiXc Distilled water
I II III IV V VI VII
a
Pharmacy School of Araraquara, Araraquara, SP, Brazil. ~o Paulo, SP, Brazil. Sigma-Aldrich Brasil Ltda. Sa c Dentsply Tulsa Dental Specialties, Tulsa, OK, USA. b
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room temperature protected from light. The dentine blocks were analysed in a confocal laser scanning microscope (Leica TCS-SPE; Leica Microsystems GmbH, Mannheim, Germany) at 409 magnification. Six equidistant areas with representative fields for each sample were established, resulting in a total of 18 areas evaluated per group. The images were captured using Leica Application Suite Advanced Fluorescence software (LAS AF, Leica Microsystems GmbH) at 1.0-lm intervals and 512 9 512 pixel resolution. Each image represented an area of 275 9 275 lm2. The images were transferred to BioImage_L software. At the end of this process, an XLS file was generated (Excel; Microsoft Corp, Redmond, WA, USA), providing the total and the green biovolume (lm3) and the percentage of substrate coverage (%). The results were subjected to statistical analysis using the GraphPad Prism v. 5.0 software (GraphPad Software, San Diego, CA, USA). Nonparametric Kruskal–Wallis and Dunn’s tests were used with the significance level set at 5% (P < 0.05).
Results Figure 1 shows the biofilm images obtained by confocal laser scanning microscopy after 1 min of contact with the irrigating solutions and associations with cetrimide. The mean, median, maximum and minimum values for total and green biovolume (lm3) are showed in Table 2. The percentages of substrate coverage, showing the biofilm remaining on dentine blocks after treatments, are presented in Fig. 2. After 1 min of direct contact, NaOCl and NaOCl+CTR were associated with the greatest reduction in total and green biovolume (P < 0.05). The CTR and QMiX solutions reduced the green biovolume compared with control group (distilled water) (P < 0.05). The CHX and CHX+CTR solutions had results similar to those of distilled water (P > 0.05). NaOCl and NaOCl+CTR demonstrated the ability to remove microorganisms and cleared biofilm from the dentine blocks after 1 min of direct contact.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Figure 1 Three-dimensional reconstruction of the biofilm after 1 min of contact with the solutions and combinations: (a) distilled water, (b) CHX, (c) CHX+CTR, (d) CTR, (e) QMiX, (f) NaOCl and (g) NaOCl+CTR.
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© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Guerreiro-Tanomaru et al. Antibiofilm activity of irrigants solution with cetrimide
Table 2 Mean, median, minimum and maximum values of total and green biovolume (lm3) after 1 min of contact of Enterococcus faecalis biofilm with the irrigating solutions and associations Total biovolume (lm3) Distilled waterA Mean Median Minimum Maximum
4.03 2.40 3.20 1.95
9 9 9 9
105 105 104 106
NaOClB 5.70 2.66 3.80 1.50
9 9 9 9
102 102 101 103
NaOCl+CTRB 1.32 9 104 3.58 9 102 0 9.13 9 104
CHXA 4.38 1.21 3.01 2.9
9 9 9 9
CHX+CTRA
105 105 104 106
4.34 2.62 5.68 1.30
9 9 9 9
105 105 104 106
CTRA 2.22 1.63 2.11 6.64
9 9 9 9
105 105 103 105
QMiXA 1.05 9.96 2.55 2.17
9 9 9 9
105 104 104 105
Green biovolume (lm3) Distilled watera Mean Median Minimum Maximum
3.06 1.54 2.87 1.89
9 9 9 9
105 105 104 106
NaOClc 4.93 9 102 2.21 9 102 0 1.37 9 103
NaOCl+CTRc 4.06 9 103 3.07 9 102 0 3.43 9 104
CHXa 2.04 3.01 7.98 1.29
9 9 9 9
105 104 102 106
CHX+CTRa 3.27 2.11 3.40 1.15
9 9 9 9
105 105 103 106
CTRb 1.92 5.59 1.71 1.07
9 9 9 9
104 103 102 105
QMiXb 1.72 1.24 1.35 5.82
9 9 9 9
104 104 103 104
Values followed by different superscript uppercase letters present significant difference for the total biovolume (lm3) after 1 min of E. faecalis biofilm contact with the irrigating solutions and combinations (P < 0.05). Values followed by superscript lowercase letters present statistically significant difference for the green biovolume (lm3) (P < 0.05).
Figure 2 Box plots of the substrate coverage percentage after 1 min of contact with the irrigating solutions and combinations. Different letters indicate significant difference between the groups (P < 0.05).
Discussion In the present study, NaOCl and NaOCl+CTR solutions were able to reduce the total number of cells in the biofilm and were associated with the lowest number of remaining viable cells on the dentine blocks. The CTR and QMiX solutions had more effective antibacterial activity against E. faecalis than CHX and
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
CHX+CTR. The stronger antibiofilm activity of QMiX when compared with CHX was also reported by Stojicic et al. (2012) and Wang et al. (2012b). Wang et al. (2012b) evaluated the effectiveness of dentine disinfection by different solutions in the presence and absence of detergents. It was concluded that the addition of detergents in the disinfecting solutions increased their antibacterial effects against E. faecalis in the dentinal tubules. However, E. faecalis was incubated only for 1 day before being exposed to the solutions. On the other hand, the present study tested the solutions against a biofilm incubated for 14 days. This may explain the differences between the present results that revealed the addition of cetrimide did not influence the antibiofilm action of the irrigation solution. In the present study, QMiX did not reduce cell viability in biofilms as effectively as NaOCl, corroborating with the results of Ordinola-Zapata et al. (2013). NaOCl and NaOCl+CTR were more effective in cleaning dentine. Indeed, it is well known that sodium hypochlorite solution promotes biofilm disruption and dissolution (Clegg et al. 2006, Chavez de Paz et al. 2010, Del Carpio-Perochena et al. 2011, Ordinola-Zapata et al. 2012, 2013, Stojicic et al. 2012). QMiX solution reduced the percentage of bacterial cells on dentine, resulting in intermediate values. The presence of a greater amount of surfactant and EDTA may explain the greater ability of this solution to remove biofilm cells from the substrate compared with 2% CHX + 0.2% CTR used in the present study.
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Bovine dentine was used as substrate for biofilm growth due to its similarity to human dentine (Del Carpio-Perochena et al. 2011, Ordinola-Zapata et al. 2012, Guerreiro-Tanomaru et al. 2013). However, dentine itself may decrease the antimicrobial activity of root canal irrigants such as CHX and QMiX (Haapasalo et al. 2010, Morgental et al. 2013). Morgental et al. 2013 evaluated the antibacterial effect of QMiX and conventional irrigation solutions in the presence or absence of dentine powder. The results showed that dentine delayed the antibacterial activity of NaOCl and QMiX but did not completely prevent their action, which may justify the weak antibiofilm action of these solutions in the present study. Evaluation of antimicrobial activity may be performed using biofilms in different stages of growth (Arias-Moliz et al. 2010, Chavez de Paz et al. 2010, Shen et al. 2011). In the present study, biofilm was induced for 14 days on bovine dentine, promoting the formation of mature biofilm as previously demonstrated (Guerreiro-Tanomaru et al. 2013). This mature biofilm generally has greater resistance to antimicrobial substances (Liu et al. 2010, Guerreiro-Tanomaru et al. 2013). Using a similar dentine biofilm model, Faria-J unior et al. (2013) evaluated antibiofilm activity of endodontic sealers against E. faecalis and found that Sealapex and MTA-Fillapex promoted a reduction in the number of bacteria in biofilms. Confocal laser scanning microscopy allows threedimensional image analysis. The fluorescent marker Live/Deadâ BacLight™ used in the present study allows evaluation of microorganism cell membrane integrity. The kit is composed of SYTO-9 and propidium iodide. SYTO-9 is a green fluorescent dye that stains the cell nucleic acids, regardless of whether the microorganisms are live or dead. Propidium iodide, on the other hand, only stain cells with damaged cytoplasmic membrane, marking the dead microorganisms (Shen et al. 2009, Pappen et al. 2010, OrdinolaZapata et al. 2012). Specific software as BioImage_L (Chavez de Paz 2009) allows evaluation of different parameters: total biovolume represents the volume for all cells in biofilm, and green biovolume, on the other hand, indicates the live cells only. Also, it is possible to evaluate the substrate coverage percentage and the amount of residual biofilm after treatment. The ratio of live and dead cells over the total biovolume has been used when biofilm activity of irrigating solutions is analysed (Shen et al. 2009, Pappen et al. 2010, Ordinola-Zapata et al. 2012, Wang et al. 2012a,b). In
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the present study, the absolute volume (lm3) was verified for both the total and the green biovolume (Ordinola-Zapata et al. 2012, Guerreiro-Tanomaru et al. 2013).
Conclusions The addition of cetrimide did not influence the antibiofilm action of sodium hypochlorite and chlorhexidine solutions. Sodium hypochlorite solutions and their association with cetrimide were able to act on microbial viability and promoted elimination of biofilm.
Acknowledgements The authors thank Dr Luiz Chavez de Paz (University of Connecticut Health Center) for providing the BioImage_L software for analysis of the biofilms. The authors deny any conflict of interests related to this study.
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© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
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Faria-J unior NB, Tanomaru-Filho M, Berbert FL, GuerreiroTanomaru JM (2013) Antibiofilm activity, pH and solubility of endodontic sealers. International Endodontic Journal 46, 755–62. Ferrer-Luque CM, Arias-Moliz MT, Gonzalez-Rodriguez MP, Baca P (2010) Antimicrobial activity of maleic acid and combinations of cetrimide with chelating agents against Enterococcus faecalis biofilm. Journal of Endodontics 36, 1673–5. Giardino L, Savoldi E, Ambu E, Rimondini R, Palezona A, Debbia EA (2009) Antimicrobial effect of MTAD, Tetraclean, Cloreximid, and sodium hypochlorite on three common endodontic pathogens. Indian Journal of Dental Research 20, 391. Guerreiro-Tanomaru JM, de Faria-Junior NB, Duarte MA, Ordinola-Zapata R, Graeff MS, Tanomaru-Filho M (2013) Comparative analysis of Enterococcus faecalis biofilm formation on different substrates. Journal of Endodontics 39, 346–50. Gupta A, Duhan J, Tewari S et al. (2013) Comparative evaluation of antimicrobial efficacy of Syzygium aromaticum, Ocimum sanctum and Cinnamomum zeylanicum plant extracts against Enterococcus faecalis: a preliminary study. International Endodontic Journal 46, 775–83. Haapasalo M, Shen Y, Qian W, Gao Y (2010) Irrigation in endodontics. Dental Clinics of North America 54, 291–312. Liu H, Wei X, Ling J, Wang W, Huang X (2010) Biofilm formation capability of Enterococcus faecalis cells in starvation phase and its susceptibility to sodium hypochlorite. Journal of Endodontics 36, 630–5. Ma J, Wang Z, Shen Y, Haapasalo M (2011) A new noninvasive model to study the effectiveness of dentin disinfection by using confocal laser scanning microscopy. Journal of Endodontics 37, 1380–5. Mohammadi Z, Abbott PV (2009) The properties and applications of chlorhexidine in endodontics. International Endodontic Journal 42, 288–302. Morgental RD, Singh A, Sappal H, Kopper PM, Vier-Pelisser FV, Peters OA (2013) Dentin inhibits the antibacterial effect of new and conventional endodontic irrigants. Journal of Endodontics 39, 406–10. Ordinola-Zapata R, Bramante CM, Cavenago B et al. (2012) Antimicrobial effect of endodontic solutions used as final irrigants on a dentine biofilm model. International Endodontic Journal 45, 162–8. Ordinola-Zapata R, Bramante CM, Brandao Garcia R et al. (2013) The antimicrobial effect of new and conventional endodontic irrigants on intra-orally infected dentin. Acta Odontologica Scadinavica 71, 424–31. Palazzi F, Morra M, Mohammadi Z, Grandini S, Giardino L (2011) Comparison of the surface tension of 5.25% sodium hypochlorite solution with three new sodium
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hypochlorite-based endodontic irrigants. International Endodontic Journal 45, 129–35. Pappen FG, Shen Y, Qian W, Leonardo MR, Giardino L, Haapasalo M (2010) In vitro antibacterial action of Tetraclean, MTAD and five experimental irrigation solutions. International Endodontic Journal 43, 528–35. R^ oßcas IN, Siqueira JF Jr, Santos KR (2004) Association of Enterococcus faecalis with different forms of periradicular diseases. Journal of Endodontics 30, 315–20. Shen Y, Qian W, Chung C, Olsen I, Haapasalo M (2009) Evaluation of the effect of two chlorhexidine preparations on biofilm bacteria in vitro: a three-dimensional quantitative analysis. Journal of Endodontics 35, 981–5. Shen Y, Stojicic S, Haapasalo M (2011) Antimicrobial efficacy of chlorhexidine against bacteria in biofilms at different stages of development. Journal of Endodontics 37, 657–61. da Silva LA, Sanguino AC, Rocha CT, Leonardo MR, Silva RA (2008) Scanning electron microscopic preliminary study of the efficacy of SmearClear and EDTA for smear layer removal after root canal instrumentation in permanent teeth. Journal of Endodontics 34, 1541–4. Sim~ oes M, Pereira MO, Vieira MJ (2005) Effect of mechanical stress on biofilms challenged by different chemicals. Water Research 39, 5142–52. Siqueira JF Jr, R^ ocßas IN (2004) Polymerase chain reactionbased analysis of microorganisms associated with failed endodontic treatment. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics 97, 85–94. Siqueira JF Jr, R^ oßcas IN (2008) Clinical implications and microbiology of bacterial persistence after treatment procedures. Journal of Endodontics 34, e3. Sjogren U, Figdor D, Persson S, Sundqvist G (1997) Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis. International Endodontic Journal 30, 297–306. Stojicic S, Shen Y, Qian W, Johnson B, Haapasalo M (2012) Antibacterial and smear layer removal ability of a novel irrigant, QMiX. International Endodontic Journal 45, 363– 71. Stuart CH, Schwartz SA, Beeson TJ, Owatz CB (2006) Enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. Journal of Endodontics 32, 93–8. Wang Z, Shen Y, Haapasalo M (2012a) Effectiveness of endodontic disinfecting solutions against young and old Enterococcus faecalis biofilms in dentin canals. Journal of Endodontics 38, 1376–9. Wang Z, Shen Y, Ma J, Haapasalo M (2012b) The effect of detergents on the antibacterial activity of disinfecting solutions in dentin. Journal of Endodontics 38, 948–53.
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Antibiofilm activity of irrigating solutions associated with cetrimide. Confocal laser scanning microscopy
nior1, M. S. Z. Graeff2, J. M. Guerreiro-Tanomaru1, C. A. Nascimento1, N. B. Faria-Ju 3 1 E. Watanabe & M. Tanomaru-Filho 1
Department of Restorative Dentistry, Araraquara Dental School, UNESP (Univ Estadual Paulista), Araraquara, SP; 2Integrated Research Center, Bauru Dental School, USP – University of S~ ao Paulo, Bauru, SP; and 3Department of Restorative Dentistry, Ribeir~ ao Preto Dental School, USP (University of S~ ao Paulo), Ribeir~ ao Preto, SP, Brazil
Abstract Guerreiro-Tanomaru JM, Nascimento CA, Faria nior NB, Graeff MSZ, Watanabe E, Tanomaru-Filho Ju M. Antibiofilm activity of irrigating solutions associated with cetrimide. Confocal laser scanning microscopy. International Endodontic Journal, 47, 1058–1063, 2014.
Aim To evaluate the antibiofilm activity of sodium hypochlorite (NaOCl) and chlorhexidine (CHX) solutions associated with cetrimide (CTR), and QMiX using confocal laser scanning microscopy. Methodology Enterococcus faecalis (ATCC- 29212) biofilms were induced on bovine dentine blocks for 14 days. The dentine blocks containing biofilm were immersed for 1 min in the following solutions: 2.5% NaOCl; 2.5% NaOCl + 0.2% CTR; 2% CHX; 2% CHX + 0.2% CTR; 0.2% CTR; QMiX. After contact with the solutions, the dentine blocks were stained with Live/Deadâ BacLight for analysis of the remaining biofilm using confocal laser scanning microscope. Images were evaluated using the BioImage_L software to determine the total biovolume (lm3), the green
Introduction The antimicrobial solutions used during root canal preparation are important for treatment success (Sjogren et al. 1997). Enterococcus faecalis is commonly
Correspondence: Mario Tanomaru-Filho, Rua Humait a, 1680, Caixa Postal 331, Centro, 14801-903 Araraquara, SP, Brazil (Tel.: +55-16-3301-6390; Fax: +55-16-33016392; e-mail: [email protected]).
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biovolume (live cells) (lm3) and the percentage of substrate coverage (%). The data were subjected to nonparametric statistical test using Kruskal–Wallis and Dunn’s tests at 5% significance level. Results After exposure to irrigants, the total biovolume observed for CHX, CHX+CTR, CTR, QMiX was similar to distilled water (P > 0.05). NaOCl and NaOCl+CTR had the lowest total and green biovolume. The CTR and QMiX had intermediate green biovolume, with greater antibacterial activity than CHX and CHX+CTR (P < 0.05). The NaOCl and NaOCl+CTR solutions were associated with microorganism removal and substrate cleaning ability. Conclusions NaOCl and NaOCl+CTR solutions were effective on microorganism viability and were able to eliminate biofilm. The addition of cetrimide did not influence antibacterial activity. Keywords: biofilm, chlorhexidine, confocal laser scanning microscopy, Enterococcus faecalis, sodium hypochlorite. Received 30 September 2013; accepted 10 January 2014
associated with root canal treatment failure (R^ ocßas et al. 2004, Siqueira & R^ oßcas 2004, 2008, Stuart et al. 2006, Gupta et al. 2013), due to its persistence within the root canal system. This microorganism has several resistance mechanisms and has the ability to form a biofilm (Stuart et al. 2006). When organized as a biofilm, microorganisms are surrounded by an extracellular polymeric matrix that confers resistance against antimicrobial agents (Donlan & Costerton 2002). The most commonly used irrigating solutions are sodium hypochlorite (NaOCl) and chlorhexidine
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Guerreiro-Tanomaru et al. Antibiofilm activity of irrigants solution with cetrimide
(CHX). NaOCl has a wide antibacterial activity and the ability to dissolve organic tissues, such as pulp tissue, collagen fibres and bacterial biofilm (Haapasalo et al. 2010). CHX has an antibacterial action, but is incapable of dissolving organic tissue (Mohammadi & Abbott 2009). A confocal laser scanning microscopy study demonstrated that 1% NaOCl acted on microbial viability and architecture of biofilm better than 2% CHX (Ordinola-Zapata et al. 2012). A 2% CHX solution did not eliminate biofilm from endodontic microbiota when applied for 15 or even for 30 min (Clegg et al. 2006, Del Carpio-Perochena et al. 2011). Addition of a surfactant to irrigating solutions may favour their antibiofilm activity. Surfactants can weaken the cohesive forces between the polymer matrix and bacterial cells, promoting biofilm disruption (Sim~ oes et al. 2005, Wang et al. 2012b). CHX-Plus, based on CHX with a surfactant, has demonstrated greater effectiveness than conventional CHX against a multispecies biofilm (Shen et al. 2009, 2011). Wang et al. (2012b) observed that after 1 min of contact with dentine infected by E. faecalis, CHXPlus exhibited antibacterial effects similar to 6% NaOCl and 2% NaOCl associated with cetrimide (CTR), a cationic surfactant. QMiX (Dentsply Tulsa Dental, Tulsa, OK, USA) is an irrigating solution that contains 2% CHX, 17% EDTA and CTR (Stojicic et al. 2012). This solution has shown antibacterial activity similar to 6% NaOCl when used for disinfection of dentine tubules contaminated with E. faecalis (Ma et al. 2011, Wang et al. 2012b). QMiX demonstrated stronger antibacterial action than CHX after 1 min of contact with E. faecalis biofilm (Stojicic et al. 2012). CTR is also present in other irrigants such as Tetraclean (Ogna Laboratori Farmaceutici, Muggio, Italy) (Giardino et al. 2009), Hypoclean (Palazzi et al. 2011) and SmearClear (da Silva et al. 2008). vCTR at 0.0312% resulted in negative cultures after 1 min of contact with E. faecalis biofilm induced for 24 h (Arias-Moliz et al. 2010). A 0.2% CTR solution eradicated microorganisms after 1 min (Baca et al. 2011). This surfactant has been shown to enhance antibiofilm activity when associated with lactic acid (Arias-Moliz et al. 2012), EDTA, citric acid (Ferrer-Luque et al. 2010) and BioPure MTAD (Dentsply Tulsa Dental, Tulsa) (Pappen et al. 2010). The aim of this study was to evaluate the antibiofilm activity of cetrimide associated with NaOCl or CHX, and QMiX using confocal laser scanning microscopy. The null hypothesis is that these solutions do not have antibiofilm activity.
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Materials and methods Biofilm growth on dentine blocks Forty-two autoclaved bovine root dentine blocks measuring 5 mm 9 5 mm 9 0.7 mm (width 9 length 9 thickness) were used as the substrate for biofilm growth. The dentine blocks were immersed in 17% EDTA (Odahcan-Herpo; Prod. Dent. Ltda., Rio de Janeiro, RJ, Brazil) for 3 min and then rinsed with sterile saline before autoclaving. Each dentine block was immersed in 1.8 mL Brain Heart Infusion (BHI; Difco, Detroit, MI, USA) and 200 lL of E. faecalis (ATCC29212) suspension, previously adjusted to optical density equivalent to 1 9 107 colony-forming units per millilitre (CFU mL 1). The dentine blocks were incubated for 14 days at 37 °C, under microaerophilic conditions on a rocking table. The BHI medium was changed every 48 h (Guerreiro-Tanomaru et al. 2013).
Direct contact of biofilm with irrigating solutions Antibiofilm activity was evaluated using root canal irrigants associated with CTR, divided into 7 groups (Table 1). The blocks were rinsed with saline before immersion in the irrigants for 1 min (n = 3). In the negative control group, the blocks were only rinsed with saline and received no further treatment. After the contact period, the dentine blocks were rinsed in the neutralizing solution (1% sodium thiosulfate/ Tween 80 + Lecithin) and rinsed once again in saline.
Analysis of the biofilm by confocal laser scanning microscopy For the analysis of antimicrobial activity using confocal laser scanning microscopy, the biofilm on the dentine blocks was stained with Live/Deadâ BacLight (Invitrogen, Eugene, OR, USA) and incubated for 15 min in Table 1 Irrigating solutions and associations evaluated Groups
Irrigating solutions
Group Group Group Group Group Group Group
2.5% NaOCla 2.5% NaOCl + 0.2% CTRb 2% CHXa 2% CHX + 0.2% CTR 0.2% CTR QMiXc Distilled water
I II III IV V VI VII
a
Pharmacy School of Araraquara, Araraquara, SP, Brazil. ~o Paulo, SP, Brazil. Sigma-Aldrich Brasil Ltda. Sa c Dentsply Tulsa Dental Specialties, Tulsa, OK, USA. b
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room temperature protected from light. The dentine blocks were analysed in a confocal laser scanning microscope (Leica TCS-SPE; Leica Microsystems GmbH, Mannheim, Germany) at 409 magnification. Six equidistant areas with representative fields for each sample were established, resulting in a total of 18 areas evaluated per group. The images were captured using Leica Application Suite Advanced Fluorescence software (LAS AF, Leica Microsystems GmbH) at 1.0-lm intervals and 512 9 512 pixel resolution. Each image represented an area of 275 9 275 lm2. The images were transferred to BioImage_L software. At the end of this process, an XLS file was generated (Excel; Microsoft Corp, Redmond, WA, USA), providing the total and the green biovolume (lm3) and the percentage of substrate coverage (%). The results were subjected to statistical analysis using the GraphPad Prism v. 5.0 software (GraphPad Software, San Diego, CA, USA). Nonparametric Kruskal–Wallis and Dunn’s tests were used with the significance level set at 5% (P < 0.05).
Results Figure 1 shows the biofilm images obtained by confocal laser scanning microscopy after 1 min of contact with the irrigating solutions and associations with cetrimide. The mean, median, maximum and minimum values for total and green biovolume (lm3) are showed in Table 2. The percentages of substrate coverage, showing the biofilm remaining on dentine blocks after treatments, are presented in Fig. 2. After 1 min of direct contact, NaOCl and NaOCl+CTR were associated with the greatest reduction in total and green biovolume (P < 0.05). The CTR and QMiX solutions reduced the green biovolume compared with control group (distilled water) (P < 0.05). The CHX and CHX+CTR solutions had results similar to those of distilled water (P > 0.05). NaOCl and NaOCl+CTR demonstrated the ability to remove microorganisms and cleared biofilm from the dentine blocks after 1 min of direct contact.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
Figure 1 Three-dimensional reconstruction of the biofilm after 1 min of contact with the solutions and combinations: (a) distilled water, (b) CHX, (c) CHX+CTR, (d) CTR, (e) QMiX, (f) NaOCl and (g) NaOCl+CTR.
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© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Guerreiro-Tanomaru et al. Antibiofilm activity of irrigants solution with cetrimide
Table 2 Mean, median, minimum and maximum values of total and green biovolume (lm3) after 1 min of contact of Enterococcus faecalis biofilm with the irrigating solutions and associations Total biovolume (lm3) Distilled waterA Mean Median Minimum Maximum
4.03 2.40 3.20 1.95
9 9 9 9
105 105 104 106
NaOClB 5.70 2.66 3.80 1.50
9 9 9 9
102 102 101 103
NaOCl+CTRB 1.32 9 104 3.58 9 102 0 9.13 9 104
CHXA 4.38 1.21 3.01 2.9
9 9 9 9
CHX+CTRA
105 105 104 106
4.34 2.62 5.68 1.30
9 9 9 9
105 105 104 106
CTRA 2.22 1.63 2.11 6.64
9 9 9 9
105 105 103 105
QMiXA 1.05 9.96 2.55 2.17
9 9 9 9
105 104 104 105
Green biovolume (lm3) Distilled watera Mean Median Minimum Maximum
3.06 1.54 2.87 1.89
9 9 9 9
105 105 104 106
NaOClc 4.93 9 102 2.21 9 102 0 1.37 9 103
NaOCl+CTRc 4.06 9 103 3.07 9 102 0 3.43 9 104
CHXa 2.04 3.01 7.98 1.29
9 9 9 9
105 104 102 106
CHX+CTRa 3.27 2.11 3.40 1.15
9 9 9 9
105 105 103 106
CTRb 1.92 5.59 1.71 1.07
9 9 9 9
104 103 102 105
QMiXb 1.72 1.24 1.35 5.82
9 9 9 9
104 104 103 104
Values followed by different superscript uppercase letters present significant difference for the total biovolume (lm3) after 1 min of E. faecalis biofilm contact with the irrigating solutions and combinations (P < 0.05). Values followed by superscript lowercase letters present statistically significant difference for the green biovolume (lm3) (P < 0.05).
Figure 2 Box plots of the substrate coverage percentage after 1 min of contact with the irrigating solutions and combinations. Different letters indicate significant difference between the groups (P < 0.05).
Discussion In the present study, NaOCl and NaOCl+CTR solutions were able to reduce the total number of cells in the biofilm and were associated with the lowest number of remaining viable cells on the dentine blocks. The CTR and QMiX solutions had more effective antibacterial activity against E. faecalis than CHX and
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
CHX+CTR. The stronger antibiofilm activity of QMiX when compared with CHX was also reported by Stojicic et al. (2012) and Wang et al. (2012b). Wang et al. (2012b) evaluated the effectiveness of dentine disinfection by different solutions in the presence and absence of detergents. It was concluded that the addition of detergents in the disinfecting solutions increased their antibacterial effects against E. faecalis in the dentinal tubules. However, E. faecalis was incubated only for 1 day before being exposed to the solutions. On the other hand, the present study tested the solutions against a biofilm incubated for 14 days. This may explain the differences between the present results that revealed the addition of cetrimide did not influence the antibiofilm action of the irrigation solution. In the present study, QMiX did not reduce cell viability in biofilms as effectively as NaOCl, corroborating with the results of Ordinola-Zapata et al. (2013). NaOCl and NaOCl+CTR were more effective in cleaning dentine. Indeed, it is well known that sodium hypochlorite solution promotes biofilm disruption and dissolution (Clegg et al. 2006, Chavez de Paz et al. 2010, Del Carpio-Perochena et al. 2011, Ordinola-Zapata et al. 2012, 2013, Stojicic et al. 2012). QMiX solution reduced the percentage of bacterial cells on dentine, resulting in intermediate values. The presence of a greater amount of surfactant and EDTA may explain the greater ability of this solution to remove biofilm cells from the substrate compared with 2% CHX + 0.2% CTR used in the present study.
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Bovine dentine was used as substrate for biofilm growth due to its similarity to human dentine (Del Carpio-Perochena et al. 2011, Ordinola-Zapata et al. 2012, Guerreiro-Tanomaru et al. 2013). However, dentine itself may decrease the antimicrobial activity of root canal irrigants such as CHX and QMiX (Haapasalo et al. 2010, Morgental et al. 2013). Morgental et al. 2013 evaluated the antibacterial effect of QMiX and conventional irrigation solutions in the presence or absence of dentine powder. The results showed that dentine delayed the antibacterial activity of NaOCl and QMiX but did not completely prevent their action, which may justify the weak antibiofilm action of these solutions in the present study. Evaluation of antimicrobial activity may be performed using biofilms in different stages of growth (Arias-Moliz et al. 2010, Chavez de Paz et al. 2010, Shen et al. 2011). In the present study, biofilm was induced for 14 days on bovine dentine, promoting the formation of mature biofilm as previously demonstrated (Guerreiro-Tanomaru et al. 2013). This mature biofilm generally has greater resistance to antimicrobial substances (Liu et al. 2010, Guerreiro-Tanomaru et al. 2013). Using a similar dentine biofilm model, Faria-J unior et al. (2013) evaluated antibiofilm activity of endodontic sealers against E. faecalis and found that Sealapex and MTA-Fillapex promoted a reduction in the number of bacteria in biofilms. Confocal laser scanning microscopy allows threedimensional image analysis. The fluorescent marker Live/Deadâ BacLight™ used in the present study allows evaluation of microorganism cell membrane integrity. The kit is composed of SYTO-9 and propidium iodide. SYTO-9 is a green fluorescent dye that stains the cell nucleic acids, regardless of whether the microorganisms are live or dead. Propidium iodide, on the other hand, only stain cells with damaged cytoplasmic membrane, marking the dead microorganisms (Shen et al. 2009, Pappen et al. 2010, OrdinolaZapata et al. 2012). Specific software as BioImage_L (Chavez de Paz 2009) allows evaluation of different parameters: total biovolume represents the volume for all cells in biofilm, and green biovolume, on the other hand, indicates the live cells only. Also, it is possible to evaluate the substrate coverage percentage and the amount of residual biofilm after treatment. The ratio of live and dead cells over the total biovolume has been used when biofilm activity of irrigating solutions is analysed (Shen et al. 2009, Pappen et al. 2010, Ordinola-Zapata et al. 2012, Wang et al. 2012a,b). In
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the present study, the absolute volume (lm3) was verified for both the total and the green biovolume (Ordinola-Zapata et al. 2012, Guerreiro-Tanomaru et al. 2013).
Conclusions The addition of cetrimide did not influence the antibiofilm action of sodium hypochlorite and chlorhexidine solutions. Sodium hypochlorite solutions and their association with cetrimide were able to act on microbial viability and promoted elimination of biofilm.
Acknowledgements The authors thank Dr Luiz Chavez de Paz (University of Connecticut Health Center) for providing the BioImage_L software for analysis of the biofilms. The authors deny any conflict of interests related to this study.
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