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Antimicrobial activity of different disinfectants against cariogenic microorganisms

Abstract

The aim of this study was to assess the in vitro antimicrobial effects of chlorhexidine digluconate (CHX), polyhexamethylene biguanide (PHBM), and octenidine dihydrochloride (OCT) on cariogenic microorganisms by using their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). CHX, PHBM, and OCT were diluted in distilled water to the final test concentrations. Using the in-tube dilution method, Streptococcus mutans, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Actinomyces viscosus were cultivated on blood agar and Mueller–Hinton broth (MHB) at 37°C for 48 h. They were read using a spectrophotometer to detect MIC. To determine MBC, samples in the range of the turbidity threshold after 24 h were transferred onto blood agar and evaluated for growth after 24 h. Different MICs and MBCs were observed in all disinfectants against each microorganism. The lowest MIC and MBC against S. mutans (60 mg/L) were obtained from PHBM. The lowest values against L. rhamnosus (15 mg/L, 30 mg/L), A. viscosus (30 mg/L), and L. acidophilus (15 mg/L, 30 mg/L) were determined by OCT. PHBM and OCT have the potential to be replaced with CHX because they were effective against cariogenic microorganisms.

Octenidine; Polyhexamethylene Biguanide; Chlorhexidine Gluconate

Introduction

Dental caries is a localized and transmissible pathological infectious process that results in the destruction of hard dental tissues.11. Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev. 1986;50(4):353-80. The elimination of cariogenic microorganisms is one of the crucial factors for the prevention of primary or residual caries lesions.22. Bjørndal L, Larsen T. Changes in the cultivable flora in deep carious lesions following a stepwise excavation procedure. Caries Res. 2000;34(6):502-8. doi:10.1159/000016631
https://doi.org/10.1159/000016631...
Recent research shows that Streptococcus mutans, Lactobacillus acidophilus, Actinomyces viscosus, and L. rhamnosus are the most common cariogenic microorganisms.33. Takahashi N, Nyvad B. Caries ecology revisited: microbial dynamics and the caries process. Caries Res. 2008;42(6):409-18. doi:10.1159/000159604
https://doi.org/10.1159/000159604...
,44. Oda Y, Hayashi F, Okada M. Longitudinal study of dental caries incidence associated with Streptococcus mutans and Streptococcus sobrinus in patients with intellectual disabilities. BMC Oral Health. 2015;15(1):102. doi:10.1186/s12903-015-0087-6
https://doi.org/10.1186/s12903-015-0087-...
,55. Cura F, Palmieri A, Girardi A, Martinelli M, Scapoli L, Carinci F. Lab-Test® 4: dental caries and bacteriological analysis. Dent Res J (Isfahan). 2012;9(Suppl 2):139-41. doi:10.4103/1735-3327.109723
https://doi.org/10.4103/1735-3327.109723...
S. mutans is the main etiological agent in dental caries.66. Anita P, Sivasamy S, Madan Kumar PD, Nanda Balan I, Ethiraj S. Streptococcus mutans and Lactobacillus acidophilus encourage the accumulation and adherence of plaque biofilm by metabolizing sucrose into sticky glucan. J Basic Clin Pharm. 2015;6(1):35-9. doi:10.4103/0976-0105.145777
https://doi.org/10.4103/0976-0105.145777...
It is associated with the initiation of caries and is also isolated both from enamel carious lesions and hidden dentin caries.77. Simón-Soro A, Mira A. Solving the etiology of dental caries. Trends Microbiol. 2015;23(2):76-82. doi:10.1016/j.tim.2014.10.010
https://doi.org/10.1016/j.tim.2014.10.01...
Lactobacillus is a group of bacteria that occurs at high levels in deep dentin caries.88. Byun R, Nadkarni MA, Chhour KL, Martin FE, Jacques NA, Hunter N. Quantitative analysis of diverse Lactobacillus species present in advanced dental caries. J Clin Microbiol. 2004;42(7):3128-36. doi:10.1128/JCM.42.7.3128-3136.2004
https://doi.org/10.1128/JCM.42.7.3128-31...
The analysis shows that among the genus Lactobacillus, L. acidophilus is most prevalent, and L. paracasei, L. rhamnosus, and L. fermentum are also present in deep caries lesions and promote caries progression.88. Byun R, Nadkarni MA, Chhour KL, Martin FE, Jacques NA, Hunter N. Quantitative analysis of diverse Lactobacillus species present in advanced dental caries. J Clin Microbiol. 2004;42(7):3128-36. doi:10.1128/JCM.42.7.3128-3136.2004
https://doi.org/10.1128/JCM.42.7.3128-31...
,99. Callaway A, Kostrzewa M, Willershausen B, Schmidt F, Thiede B, Küpper H et al. Identification of Lactobacilli from deep carious lesions by means of species-specific PCR and MALDI-TOF mass spectrometry. Clin Lab. 2013;59(11-12):1373-9. doi:10.7754/Clin.Lab.2013.121225
https://doi.org/10.7754/Clin.Lab.2013.12...
Furthermore, the fact that L. rhamnosus constitutes the dominant species in carious dentin both in deciduous and permanent teeth has been supported by numerous authors using biochemical or reliable genetically-based methods.22. Bjørndal L, Larsen T. Changes in the cultivable flora in deep carious lesions following a stepwise excavation procedure. Caries Res. 2000;34(6):502-8. doi:10.1159/000016631
https://doi.org/10.1159/000016631...
,1010. Marchant S, Brailsford SR, Twomey AC, Roberts GJ, Beighton D. The predominant microflora of nursing caries lesions. Caries Res. 2001;35(6):397-406. doi:10.1159/000047482
https://doi.org/10.1159/000047482...
,1111. Becker MR, Paster BJ, Leys EJ, Moeschberger ML, Kenyon SG, Galvin JL et al. Molecular analysis of bacterial species associated with childhood caries. J Clin Microbiol. 2002;40(3):1001-9. doi:10.1128/JCM.40.3.1001-1009.2002
https://doi.org/10.1128/JCM.40.3.1001-10...
,1212. Munson MA, Banerjee A, Watson TF, Wade WG. Molecular analysis of the microflora associated with dental caries. J Clin Microbiol. 2004;42(7):3023-9. doi:10.1128/JCM.42.7.3023-3029.2004
https://doi.org/10.1128/JCM.42.7.3023-30...
,1313. Wicht MJ, Haak R, Schütt-Gerowitt H, Kneist S, Noack MJ. Suppression of caries-related microorganisms in dentine lesions after short-term chlorhexidine or antibiotic treatment. Caries Res. 2004;38(5):436-41. doi:10.1159/000079624
https://doi.org/10.1159/000079624...
,1414. Edwardsson S. Bacteriological studies on deep areas of carious dentine. Odontol Rev Suppl. 1974;32:1-143. In addition to Lactobacillus species, anaerobic conditions have resulted in the isolation of Actinomyces species from dentin lesions.1515. Botha SJ, Boy SC, Botha FS, Senekal R. Lactobacillus species associated with active caries lesions. J Dent Assoc S Afr 1998;53(1):3-6.

To reduce the potential of primary or residual caries and enhance the postoperative sensitivity, antibacterial agents may be chosen according to their ability to reduce or eliminate the possibility of existing bacteria. Thus, the use of antimicrobial solutions as an oral rinse or a cavity disinfectant for reducing cariogenic microorganisms according to the target of the application has been recommended. Chlorhexidine digluconate (CHX) is a commonly used antimicrobial agent because of its ability to significantly reduce the levels of cariogenic microorganisms.1616. Obata J, Takeshita T, Shibata Y, Yamanaka W, Unemori M, Akamine A et al. Identification of the microbiota in carious dentin lesions using 16S rRNA gene sequencing. PLoS One. 2014;9(8):e103712. doi:10.1371/journal.pone.0103712
https://doi.org/10.1371/journal.pone.010...

17. Rijkom HM, Truin GJ, Hof MA. A meta-analysis of clinical studies on the caries-inhibiting effect of chlorhexidine treatment. J Dent Res. 1996;75(2):790-5. doi:10.1177/00220345960750020901
https://doi.org/10.1177/0022034596075002...
-1818. Emilson CG. Potential efficacy of chlorhexidine against mutans streptococci and human dental caries. J Dent Res.1994;73(3):682-91. doi:10.1177/00220345940730031401
https://doi.org/10.1177/0022034594073003...

However, CHX had adverse effects, including staining of the teeth and tongue, mucosal soreness, and desquamation; temporary taste disturbances; hypersensitivity; and selective disturbance of the microbial balance within the oral ecosystem.1919. Walsh T, Oliveira-Neto JM, Moore D. Chlorhexidine treatment for the prevention of dental caries in children and adolescents. Cochrane Database Syst Rev. 2015;13(4):CD008457. doi:10.1002/14651858.CD008457.pub2
https://doi.org/10.1002/14651858.CD00845...
Previous studies have shown that CHX was cytotoxic to human fibroblasts via the inhibition of protein synthesis.2020. Goldschmidt P, Cogen R, Taubman S. Cytopathologic effects of chlorhexidine on human cells. J Periodontol. 1977;48(4):212-5. doi:10.1902/jop.1977.48.4.212
https://doi.org/10.1902/jop.1977.48.4.21...
In addition, the resistance of L. rhamnosus to CHX therapy was also reported.2121. Pucher JJ, Daniel JC. The effects of chlorhexidine digluconate on human fibroblasts in vitro. J Periodontol. 1993;63(6):526-32. doi:10.1902/jop.1992.63.6.526
https://doi.org/10.1902/jop.1992.63.6.52...
Therefore, antimicrobial agents, such as octenidine dihydrochloride (OCT) and polyhexamethylene biguanide (PHMB) have been investigated as alternatives to CHX because of their superior microbicidal activity and lower cytotoxicity than CHX.2222. Epstein JB, McBride BC, Stevenson-Moore P, Merilees H, Spinelli J. The efficacy of chlorhexidine gel in reduction of Streptococcus mutans and Lactobacillus species in patients treated with radiation therapy. Oral Surg Oral Med Oral Pathol. 1991;71(2):172-8. doi:10.1016/0030-4220(91)90461-K
https://doi.org/10.1016/0030-4220(91)904...
,2323. Müller G, Kramer A. Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity. J Antimicrob Chemother. 2008;61(6):1281-7. doi:10.1093/jac/dkn125
https://doi.org/10.1093/jac/dkn125...
,2424. Tirali RE, Bodur H, Sipahi B, Sungurtekin E. Evaluation of the antimicrobial activities of chlorhexidine gluconate, sodium hypochlorite and octenidine hydrochloride in vitro. Aust Endod J. 2013;39(1):15-8. doi:10.1111/j.1747-4477.2010.00266.x
https://doi.org/10.1111/j.1747-4477.2010...
,2525. Decker EM, Weiger R, Wiech I, Heide PE, Brecx M. Comparison of antiadhesive and antibacterial effects of antiseptics on Streptococcus sanguinis. Eur J Oral Sci. 2003;111(2):144-8. doi:10.1034/j.1600-0722.2003.00025.x
https://doi.org/10.1034/j.1600-0722.2003...

OCT is an antiseptic agent recently used for the management of skin burns, wounds, and as a mouth rinse. OCT belongs to the bipyridines, carrying two cationic active centers per molecule, and demonstrates the broad-spectrum antimicrobial effects on both gram-positive and gram-negative bacteria, fungi, and several viral species.2626. Hübner NO, Kramer A. Review on the efficacy, safety and clinical applications of polihexanide, a modern wound antiseptic. Skin Pharmacol Physiol. 2010;23(suppl 1):17-27. doi:10.1159/000318264
https://doi.org/10.1159/000318264...
The bactericidal and fungicidal effects are primarily achieved by interfering with the cell wall and membranes of such microbes. Previous studies have shown that the efficacy of OCT against dental plaque-associated bacteria (e.g., S. mutans and A. viscosus) was comparable to that of CHX.2424. Tirali RE, Bodur H, Sipahi B, Sungurtekin E. Evaluation of the antimicrobial activities of chlorhexidine gluconate, sodium hypochlorite and octenidine hydrochloride in vitro. Aust Endod J. 2013;39(1):15-8. doi:10.1111/j.1747-4477.2010.00266.x
https://doi.org/10.1111/j.1747-4477.2010...
,2727. Sedlock DM, Bailey DM. Microbicidal activity of octenidine hydrochloride, a new alkanediylbis[pyridine] germicidal agent. Antimicrob Agents Chemother. 1985;28(6):786-90. doi:10.1128/AAC.28.6.786
https://doi.org/10.1128/AAC.28.6.786...

PHMB is a type of modern antiseptic that combines a broad antimicrobial spectrum with low toxicity, high tissue compatibility, no reported adsorption, and is used as a solution, gel, ointment, or foam. As such, it is one of the most promising antiseptic substances.2525. Decker EM, Weiger R, Wiech I, Heide PE, Brecx M. Comparison of antiadhesive and antibacterial effects of antiseptics on Streptococcus sanguinis. Eur J Oral Sci. 2003;111(2):144-8. doi:10.1034/j.1600-0722.2003.00025.x
https://doi.org/10.1034/j.1600-0722.2003...

Although some studies have been published concerning the antimicrobial properties of OCT on S. mutans and A. viscosus as well as that of PHMB on S. mutans,2828. Slee AM, O’Connor JR, Bailey DM. Relationship between structure and antiplaque and antimicrobial activities for a series of bispyridines. Antimicrob Agents Chemother. 1983;23(4):531-5. doi:10.1128/AAC.23.4.531
https://doi.org/10.1128/AAC.23.4.531...
,2929. Slee AM, O’Connor JR. In vitro antiplaque activity of octenidine dihydrochloride (WIN 41464-2) against preformed plaques of selected oral plaque-forming microorganisms. Antimicrob Agents Chemother. 1983;23(3):379-84. doi:10.1128/AAC.23.3.379
https://doi.org/10.1128/AAC.23.3.379...
there is a lack of studies regarding their antibacterial activity on other essential cariogenic microorganisms (e.g., L. rhamnosus and L. acidophilus). Therefore, the aim of the present study was to assess the in vitro antimicrobial effects of CHX, OCT, and PHMB on cariogenic microorganisms using both their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).

Methodology

The experiments were performed at the Microbiology Laboratory in the Department of Biology, Faculty of Science, Ege University. Culture media brain-heart infusion (BHI) (Difco, Detroit, MI, USA) broth and agar were made. The tubes and the plates were sterilized in an autoclave, tested for sterility, and kept under refrigeration until use.

CHX (Drogsan, Ankara, Turkey), PHMG (Mertsel Ilac, Izmir, Turkey), and OCT (Schülke & Mayr GmbH, Norderstedt, Germany) were obtained as solutions and diluted in water of standardized hardness (WSH, according to DIN EN 1040) to the test concentrations. The initial concentrations of OCT and PHMB were 0.2% (2,000 mg/L), and 2% (20,000 mg/L) for CHX. The tube dilution method was used for determining MIC and MBC of the antimicrobial agents against the selected microorganisms.

In this study, S. mutans (ATCC 25175), L. acidophilus (ATCC 4356), A. viscosus (ATCC 15987), and L. rhamnosus (ATCC 7469) were used; all were previously frozen. All bacterial strains were obtained from the American-type culture collection.

The microorganisms were reactivated in sterile BHI broth and incubated at 37°C in a carbon dioxide incubator (SANYO Electric Biomedical Co., Ltd., Osaka, Japan) at 5% CO2. After 48 h of culture growth, the Gram staining was used and the cultures were replated to verify their purity. The microorganisms were transferred to the broth media and their absorbance indexes were verified hourly using a spectrophotometer at a wavelength of 540 nm. Bacterial suspensions equal to the No. 0.5 McFarland standard were prepared.

Ten sterile test tubes were collected and 1 mL of sterile tryptic soy broth (TSB) was added to each tube. Then, 1 mL of each solution with specified dilutions was prepared using the serial dilution (1:2) method and added to each of the tubes. A bacterial suspension of 1.5 × 108 CFU equal to the No. 0.5 McFarland standard was prepared from the standard strains.

A 1 mL volume of the dilute suspension was added to each set of eight tubes that contained the TSB medium and solution. After the bacterial suspension was added to the test tubes, they were placed in a carbon dioxide incubator (to provide 5% CO2) and were incubated at 37°C for 24–48 h. After this period had elapsed, the tubes were examined for the presence of turbidity, which indicated microbial growth. The last tube, or the last dilution of solutions at which turbidity was not observed, was considered MIC of the respective solutions for certain microorganisms. MIC of each of the three solutions was then compared for the ability to inhibit microbial growth. After a 24 h incubation, the tubes without turbidity (transparent), which indicated the inhibition of bacterial growth by the respective solutions, were transferred to a solid medium (TSA agar) and evaluated for microbial growth to determine the MBC of the solutions. The last tube, which was negative regarding the presence of cultures on a solid medium, indicated MBC of the solutions. This procedure was performed for all bacterial strains. The experiment was performed in triplicate.

Results

All disinfectants tested had bactericidal and bacteriostatic effects on the cariogenic microorganisms tested in this study. However, different MICs and MBCs were observed in all disinfectants against each microorganism (Figure).

Figure
The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of various antimicrobial agents.

The lowest MIC values against S. mutans were obtained from CHX (70 mg/L) and PHBM (60 mg/L), while MIC of OCT (120 mg/L) was two times greater than that of CHX and PHBM. The lowest MBC was obtained from PHBM (60 mg/L), while MBC of CHX (150 mg/L) and OCT (120 mg/L) was nearly twice that of PHBM.

The lowest MIC against L. rhamnosus was achieved by OCT and PHBM (15 mg/L), while MIC of CHX (150 mg/L) was ten times greater than OCT and PHBM. The lowest MBC was determined by OCT and PHBM (30 mg/L), while MBC of CHX (150 mg/L) was five times greater than OCT and PHBM.

The lowest MIC against L. acidophilus was found to be OCT (15 mg/L), while MIC of PHMB (30 mg/L) was two times and MIC of CHX (70 mg/L) was nearly four times greater than OCT. The lowest MBC was obtained from OCT and PHMB (30 mg/L), while MBC of CHX (150 mg/L) was five times greater than OCT and PHMB.

The lowest MIC and MBC against A. viscosus (30 mg/L) was determined by OCT, while MIC and MBC of CHX (70 mg/L) were two times and MIC and MBC of PHBM (120 mg/L) were four times greater than OCT.

Discussion

MICs and MBCs of the antimicrobial agents investigated in the present study were determined using the microdilution test method. MICs are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after an overnight incubation and are considered the gold standard for determining the susceptibility of organisms to antimicrobials. On the other hand, MBCs are the lowest concentrations of an antimicrobial that will prevent the growth of an organism following a subculture on antimicrobial-free media.3030. Kocak MM, Ozcan S, Kocak S, Topuz O, Erten H. Comparison of the efficacy of three different mouthrinse solutions in decreasing the level of streptococcus mutans in saliva. Eur J Dent. 2009;3(1):57-61. MBCs are used for determining the potential resistance of an antimicrobial and making rational decisions in determining how successful an antimicrobial treatment is likely to be.3030. Kocak MM, Ozcan S, Kocak S, Topuz O, Erten H. Comparison of the efficacy of three different mouthrinse solutions in decreasing the level of streptococcus mutans in saliva. Eur J Dent. 2009;3(1):57-61. However, MBC is more relevant than MIC for assessing the antibacterial activity of a solution and is also higher than MIC.3131. Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother. 2001;48(suppl 1):5-16. doi:10.1093/jac/48.suppl_1.5
https://doi.org/10.1093/jac/48.suppl_1.5...
In this study, the initial concentration of the tested solutions were different and are the commonly used concentrations for these solutions.

Three commonly used antimicrobial agents were tested. CHX is used at a concentration of 0.12% and 0.2% as oral rinses, and 2% as an endodontic irrigation solution and cavity disinfectant.3232. Peterson LR, Shanholtzer CJ. Tests for bactericidal effects of antimicrobial agents: technical performance and clinical relevance. Clin Microbiol Rev. 1992;5(4):420-32. doi:10.1128/CMR.5.4.420
https://doi.org/10.1128/CMR.5.4.420...
Additionally, for CHX to be used as an anticarcinogenic agent, a 1% concentration in a gel formulation is available.3333. Najafi MH, Taheri M, Mokhtari MR, Forouzanfar A, Farazi F, Mirzaee M et al. Comparative study of 0.2% and 0.12% digluconate chlorhexidine mouth rinses on the level of dental staining and gingival indices. Dent Res J (Isfahan). 2012;9(3):305-8.,3434. Slot DE, Rosema NA, Hennequin-Hoenderdos NL, Versteeg PA, Van Der Velden U, Van Der Weijden GA. The effect of 1% chlorhexidine gel and 0.12% dentifrice gel on plaque accumulation: a 3-day non-brushing model. Int J Dent Hyg 2010;8(4):294-300. doi:10.1111/j.1601-5037.2010.00487.x
https://doi.org/10.1111/j.1601-5037.2010...
OCT is commonly used at a concentration of 0.1%–0.2% and PHMB is used at 0.2% as an antiseptic.2525. Decker EM, Weiger R, Wiech I, Heide PE, Brecx M. Comparison of antiadhesive and antibacterial effects of antiseptics on Streptococcus sanguinis. Eur J Oral Sci. 2003;111(2):144-8. doi:10.1034/j.1600-0722.2003.00025.x
https://doi.org/10.1034/j.1600-0722.2003...
,3535. Maltz, M, Zickert I, Krasse B. Effect of intensive treatment with chlorhexidine on number of Streptococcus mutans in saliva. Scand J Dent Res. 1981;89(6):445-9.,3636. Hübner NO, Siebert J, Kramer A. Octenidine dihydrochloride, a modern antiseptic for skin, mucous membranes and wounds. Skin Pharmacol Physiol 2010;23(5):244-58. doi:10.1159/000314699
https://doi.org/10.1159/000314699...

It was demonstrated that all antiseptic solutions had both bacteriostatic and bactericidal effects against the test microorganisms, as CHX was used at 150 mg/L and OCT and PHMB were used at 120 mg/L in the present study. All of the bacterial groups that were chosen as the test microorganisms in this study were the most common cariogenic bacteria isolated from caries lesions.33. Takahashi N, Nyvad B. Caries ecology revisited: microbial dynamics and the caries process. Caries Res. 2008;42(6):409-18. doi:10.1159/000159604
https://doi.org/10.1159/000159604...
,44. Oda Y, Hayashi F, Okada M. Longitudinal study of dental caries incidence associated with Streptococcus mutans and Streptococcus sobrinus in patients with intellectual disabilities. BMC Oral Health. 2015;15(1):102. doi:10.1186/s12903-015-0087-6
https://doi.org/10.1186/s12903-015-0087-...
,55. Cura F, Palmieri A, Girardi A, Martinelli M, Scapoli L, Carinci F. Lab-Test® 4: dental caries and bacteriological analysis. Dent Res J (Isfahan). 2012;9(Suppl 2):139-41. doi:10.4103/1735-3327.109723
https://doi.org/10.4103/1735-3327.109723...
,77. Simón-Soro A, Mira A. Solving the etiology of dental caries. Trends Microbiol. 2015;23(2):76-82. doi:10.1016/j.tim.2014.10.010
https://doi.org/10.1016/j.tim.2014.10.01...
,88. Byun R, Nadkarni MA, Chhour KL, Martin FE, Jacques NA, Hunter N. Quantitative analysis of diverse Lactobacillus species present in advanced dental caries. J Clin Microbiol. 2004;42(7):3128-36. doi:10.1128/JCM.42.7.3128-3136.2004
https://doi.org/10.1128/JCM.42.7.3128-31...
,99. Callaway A, Kostrzewa M, Willershausen B, Schmidt F, Thiede B, Küpper H et al. Identification of Lactobacilli from deep carious lesions by means of species-specific PCR and MALDI-TOF mass spectrometry. Clin Lab. 2013;59(11-12):1373-9. doi:10.7754/Clin.Lab.2013.121225
https://doi.org/10.7754/Clin.Lab.2013.12...
,1515. Botha SJ, Boy SC, Botha FS, Senekal R. Lactobacillus species associated with active caries lesions. J Dent Assoc S Afr 1998;53(1):3-6. The antimicrobial effects of these agents against oral microorganisms, such as S. mutans, S. sanguinis, C. a1lbicans, and F. nucleatum were previously confirmed by Kocak et al.3030. Kocak MM, Ozcan S, Kocak S, Topuz O, Erten H. Comparison of the efficacy of three different mouthrinse solutions in decreasing the level of streptococcus mutans in saliva. Eur J Dent. 2009;3(1):57-61. and Rohrer et al.3737. Rohrer N, Widmer AF, Waltimo T, Kulik EM, Weiger R, Filipuzzi-Jenny E et al. Antimicrobial efficacy of 3 oral antiseptics containing octenidine, polyhexamethylene biguanide, or Citroxx: can chlorhexidine be replaced? Infect Control Hosp Epidemiol. 2010;31(7):733-9. doi:10.1086/653822
https://doi.org/10.1086/653822...
However, the antibacterial effects of these agents on the other crucial microorganisms in caries progression (e.g., L. rhamnosus and L. acidophilus) were not assessed. The most important findings in the present study included the effectiveness of these antimicrobials against common cariogenic bacteria and MIC of these agents.

PHMB and CHX were both effective against all microorganisms tested; however, against Lactobacillus species and S. mutans, PHMB was effective at the same or slightly lower concentrations than CHX. This finding is in accordance with Rohrer et al.3737. Rohrer N, Widmer AF, Waltimo T, Kulik EM, Weiger R, Filipuzzi-Jenny E et al. Antimicrobial efficacy of 3 oral antiseptics containing octenidine, polyhexamethylene biguanide, or Citroxx: can chlorhexidine be replaced? Infect Control Hosp Epidemiol. 2010;31(7):733-9. doi:10.1086/653822
https://doi.org/10.1086/653822...
who stated that the antimicrobial activity of PHMB was comparable to that of CHX against S. mutans and S. sanguinis. PHMB is a biguanide with effective antibacterial properties. The antibacterial effect is mediated by an increase in the permeability of the bacterial cell membrane, which leads to an osmotic imbalance and an outpouring of cytoplasm. Polyhexanides have a broad antibacterial spectrum, mainly against Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Enterobacter cloacae, and Streptococcus lactis. In addition to its effective antibacterial activity against the cariogenic microorganisms tested, the cytotoxicity of PHMB is low, and thus, the tissue compatibility is high. The local tolerability of PHMB is superior compared to other disinfectants, such as iodine, hydrogen peroxide, or CHX.3838. Dogan AA, Adiloglu AK, Onal S, Cetin ES, Polat E, Uskun E et al. Short-term relative antibacterial effect of octenidine dihydrochloride on the oral microflora in orthodontically treated patients. Int J Infect Dis. 2008;12(6):19-25. doi:10.1016/j.ijid.2008.03.013
https://doi.org/10.1016/j.ijid.2008.03.0...

In the present study, OCT was found to be more effective than CHX at lower concentrations against each microorganism. Similarly, Kocak et al.3030. Kocak MM, Ozcan S, Kocak S, Topuz O, Erten H. Comparison of the efficacy of three different mouthrinse solutions in decreasing the level of streptococcus mutans in saliva. Eur J Dent. 2009;3(1):57-61. and Dogan et al.3838. Dogan AA, Adiloglu AK, Onal S, Cetin ES, Polat E, Uskun E et al. Short-term relative antibacterial effect of octenidine dihydrochloride on the oral microflora in orthodontically treated patients. Int J Infect Dis. 2008;12(6):19-25. doi:10.1016/j.ijid.2008.03.013
https://doi.org/10.1016/j.ijid.2008.03.0...
found in their previous studies that OCT was more favorable than CHX regarding its antibacterial activity against S. mutans and Lactobacillus species, both in vitro and in vivo. OCT was originally developed as a potential broad-spectrum topical antimicrobial agent,3939. Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother. 2001;48(Suppl 1):5-16. doi:10.1093/jac/48.suppl_1.5
https://doi.org/10.1093/jac/48.suppl_1.5...
and its use as an oral rinse has been reported to inhibit the development of dental plaque in both primates2727. Sedlock DM, Bailey DM. Microbicidal activity of octenidine hydrochloride, a new alkanediylbis[pyridine] germicidal agent. Antimicrob Agents Chemother. 1985;28(6):786-90. doi:10.1128/AAC.28.6.786
https://doi.org/10.1128/AAC.28.6.786...
and in humans.4040. Emilson CG, Bowen WH, Robrish SA, Kemp CW. Effect of the antibacterial agents octenidine and chlorhexidine on the plaque flora in primates. Scand J Dent Res 1981;89(5):384-92.,4141. Pithon MM, Sant’Anna LI, Baião FC, Santos RL, Coqueiro RS, Maia LC. Assessment of the effectiveness of mouthwashes in reducing cariogenic biofilm in orthodontic patients: a systematic review. J Dent. 2015;43(3):297-308. doi:10.1016/j.jdent.2014.12.010
https://doi.org/10.1016/j.jdent.2014.12....
One recent study showed that a 0.1% OCT mouth rinse provided a statistically significant reduction of 39% of plaque, 50% of gingivitis, and 60% of gingival bleeding sites.4242. Patters MR, Anerud K, Trummel CL, Kornman KS, Nalbandian J, Robertson PB. Inhibition of plaque formation in humans by octenidine mouth rinse. J Periodontal Res. 1983;18(2):212-9. doi:10.1111/j.1600-0765.1983.tb00354.
https://doi.org/10.1111/j.1600-0765.1983...
Moreover, in an in vivo study, it was observed that 0.1% OCT had a significant effect on S. mutans in both 1 and 10 min time periods, and the antimicrobial efficiency was preserved even after 60 min.2929. Slee AM, O’Connor JR. In vitro antiplaque activity of octenidine dihydrochloride (WIN 41464-2) against preformed plaques of selected oral plaque-forming microorganisms. Antimicrob Agents Chemother. 1983;23(3):379-84. doi:10.1128/AAC.23.3.379
https://doi.org/10.1128/AAC.23.3.379...

CHX, OCT, and PHMB were found to be effective against the most common cariogenic microorganisms in this study. However, their potential use in clinical procedures and their effect on biofilm formation, bacterial adhesion, and bond strength of adhesive systems should be investigated with future in vitro and in vivo studies.

Conclusion

The findings of the present study suggest that OCT and PHMB are significantly effective against cariogenic microorganisms at 120 mg/L concentrations. OCT and PHMB can be considered potential alternatives to CHX for reducing cariogenic microorganisms during antibacterial procedures.

References

  • 1
    Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev. 1986;50(4):353-80.
  • 2
    Bjørndal L, Larsen T. Changes in the cultivable flora in deep carious lesions following a stepwise excavation procedure. Caries Res. 2000;34(6):502-8. doi:10.1159/000016631
    » https://doi.org/10.1159/000016631
  • 3
    Takahashi N, Nyvad B. Caries ecology revisited: microbial dynamics and the caries process. Caries Res. 2008;42(6):409-18. doi:10.1159/000159604
    » https://doi.org/10.1159/000159604
  • 4
    Oda Y, Hayashi F, Okada M. Longitudinal study of dental caries incidence associated with Streptococcus mutans and Streptococcus sobrinus in patients with intellectual disabilities. BMC Oral Health. 2015;15(1):102. doi:10.1186/s12903-015-0087-6
    » https://doi.org/10.1186/s12903-015-0087-6
  • 5
    Cura F, Palmieri A, Girardi A, Martinelli M, Scapoli L, Carinci F. Lab-Test® 4: dental caries and bacteriological analysis. Dent Res J (Isfahan). 2012;9(Suppl 2):139-41. doi:10.4103/1735-3327.109723
    » https://doi.org/10.4103/1735-3327.109723
  • 6
    Anita P, Sivasamy S, Madan Kumar PD, Nanda Balan I, Ethiraj S. Streptococcus mutans and Lactobacillus acidophilus encourage the accumulation and adherence of plaque biofilm by metabolizing sucrose into sticky glucan. J Basic Clin Pharm. 2015;6(1):35-9. doi:10.4103/0976-0105.145777
    » https://doi.org/10.4103/0976-0105.145777
  • 7
    Simón-Soro A, Mira A. Solving the etiology of dental caries. Trends Microbiol. 2015;23(2):76-82. doi:10.1016/j.tim.2014.10.010
    » https://doi.org/10.1016/j.tim.2014.10.010
  • 8
    Byun R, Nadkarni MA, Chhour KL, Martin FE, Jacques NA, Hunter N. Quantitative analysis of diverse Lactobacillus species present in advanced dental caries. J Clin Microbiol. 2004;42(7):3128-36. doi:10.1128/JCM.42.7.3128-3136.2004
    » https://doi.org/10.1128/JCM.42.7.3128-3136.2004
  • 9
    Callaway A, Kostrzewa M, Willershausen B, Schmidt F, Thiede B, Küpper H et al. Identification of Lactobacilli from deep carious lesions by means of species-specific PCR and MALDI-TOF mass spectrometry. Clin Lab. 2013;59(11-12):1373-9. doi:10.7754/Clin.Lab.2013.121225
    » https://doi.org/10.7754/Clin.Lab.2013.121225
  • 10
    Marchant S, Brailsford SR, Twomey AC, Roberts GJ, Beighton D. The predominant microflora of nursing caries lesions. Caries Res. 2001;35(6):397-406. doi:10.1159/000047482
    » https://doi.org/10.1159/000047482
  • 11
    Becker MR, Paster BJ, Leys EJ, Moeschberger ML, Kenyon SG, Galvin JL et al. Molecular analysis of bacterial species associated with childhood caries. J Clin Microbiol. 2002;40(3):1001-9. doi:10.1128/JCM.40.3.1001-1009.2002
    » https://doi.org/10.1128/JCM.40.3.1001-1009.2002
  • 12
    Munson MA, Banerjee A, Watson TF, Wade WG. Molecular analysis of the microflora associated with dental caries. J Clin Microbiol. 2004;42(7):3023-9. doi:10.1128/JCM.42.7.3023-3029.2004
    » https://doi.org/10.1128/JCM.42.7.3023-3029.2004
  • 13
    Wicht MJ, Haak R, Schütt-Gerowitt H, Kneist S, Noack MJ. Suppression of caries-related microorganisms in dentine lesions after short-term chlorhexidine or antibiotic treatment. Caries Res. 2004;38(5):436-41. doi:10.1159/000079624
    » https://doi.org/10.1159/000079624
  • 14
    Edwardsson S. Bacteriological studies on deep areas of carious dentine. Odontol Rev Suppl. 1974;32:1-143.
  • 15
    Botha SJ, Boy SC, Botha FS, Senekal R. Lactobacillus species associated with active caries lesions. J Dent Assoc S Afr 1998;53(1):3-6.
  • 16
    Obata J, Takeshita T, Shibata Y, Yamanaka W, Unemori M, Akamine A et al. Identification of the microbiota in carious dentin lesions using 16S rRNA gene sequencing. PLoS One. 2014;9(8):e103712. doi:10.1371/journal.pone.0103712
    » https://doi.org/10.1371/journal.pone.0103712
  • 17
    Rijkom HM, Truin GJ, Hof MA. A meta-analysis of clinical studies on the caries-inhibiting effect of chlorhexidine treatment. J Dent Res. 1996;75(2):790-5. doi:10.1177/00220345960750020901
    » https://doi.org/10.1177/00220345960750020901
  • 18
    Emilson CG. Potential efficacy of chlorhexidine against mutans streptococci and human dental caries. J Dent Res.1994;73(3):682-91. doi:10.1177/00220345940730031401
    » https://doi.org/10.1177/00220345940730031401
  • 19
    Walsh T, Oliveira-Neto JM, Moore D. Chlorhexidine treatment for the prevention of dental caries in children and adolescents. Cochrane Database Syst Rev. 2015;13(4):CD008457. doi:10.1002/14651858.CD008457.pub2
    » https://doi.org/10.1002/14651858.CD008457.pub2
  • 20
    Goldschmidt P, Cogen R, Taubman S. Cytopathologic effects of chlorhexidine on human cells. J Periodontol. 1977;48(4):212-5. doi:10.1902/jop.1977.48.4.212
    » https://doi.org/10.1902/jop.1977.48.4.212
  • 21
    Pucher JJ, Daniel JC. The effects of chlorhexidine digluconate on human fibroblasts in vitro. J Periodontol. 1993;63(6):526-32. doi:10.1902/jop.1992.63.6.526
    » https://doi.org/10.1902/jop.1992.63.6.526
  • 22
    Epstein JB, McBride BC, Stevenson-Moore P, Merilees H, Spinelli J. The efficacy of chlorhexidine gel in reduction of Streptococcus mutans and Lactobacillus species in patients treated with radiation therapy. Oral Surg Oral Med Oral Pathol. 1991;71(2):172-8. doi:10.1016/0030-4220(91)90461-K
    » https://doi.org/10.1016/0030-4220(91)90461-K
  • 23
    Müller G, Kramer A. Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity. J Antimicrob Chemother. 2008;61(6):1281-7. doi:10.1093/jac/dkn125
    » https://doi.org/10.1093/jac/dkn125
  • 24
    Tirali RE, Bodur H, Sipahi B, Sungurtekin E. Evaluation of the antimicrobial activities of chlorhexidine gluconate, sodium hypochlorite and octenidine hydrochloride in vitro. Aust Endod J. 2013;39(1):15-8. doi:10.1111/j.1747-4477.2010.00266.x
    » https://doi.org/10.1111/j.1747-4477.2010.00266.x
  • 25
    Decker EM, Weiger R, Wiech I, Heide PE, Brecx M. Comparison of antiadhesive and antibacterial effects of antiseptics on Streptococcus sanguinis. Eur J Oral Sci. 2003;111(2):144-8. doi:10.1034/j.1600-0722.2003.00025.x
    » https://doi.org/10.1034/j.1600-0722.2003.00025.x
  • 26
    Hübner NO, Kramer A. Review on the efficacy, safety and clinical applications of polihexanide, a modern wound antiseptic. Skin Pharmacol Physiol. 2010;23(suppl 1):17-27. doi:10.1159/000318264
    » https://doi.org/10.1159/000318264
  • 27
    Sedlock DM, Bailey DM. Microbicidal activity of octenidine hydrochloride, a new alkanediylbis[pyridine] germicidal agent. Antimicrob Agents Chemother. 1985;28(6):786-90. doi:10.1128/AAC.28.6.786
    » https://doi.org/10.1128/AAC.28.6.786
  • 28
    Slee AM, O’Connor JR, Bailey DM. Relationship between structure and antiplaque and antimicrobial activities for a series of bispyridines. Antimicrob Agents Chemother. 1983;23(4):531-5. doi:10.1128/AAC.23.4.531
    » https://doi.org/10.1128/AAC.23.4.531
  • 29
    Slee AM, O’Connor JR. In vitro antiplaque activity of octenidine dihydrochloride (WIN 41464-2) against preformed plaques of selected oral plaque-forming microorganisms. Antimicrob Agents Chemother. 1983;23(3):379-84. doi:10.1128/AAC.23.3.379
    » https://doi.org/10.1128/AAC.23.3.379
  • 30
    Kocak MM, Ozcan S, Kocak S, Topuz O, Erten H. Comparison of the efficacy of three different mouthrinse solutions in decreasing the level of streptococcus mutans in saliva. Eur J Dent. 2009;3(1):57-61.
  • 31
    Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother. 2001;48(suppl 1):5-16. doi:10.1093/jac/48.suppl_1.5
    » https://doi.org/10.1093/jac/48.suppl_1.5
  • 32
    Peterson LR, Shanholtzer CJ. Tests for bactericidal effects of antimicrobial agents: technical performance and clinical relevance. Clin Microbiol Rev. 1992;5(4):420-32. doi:10.1128/CMR.5.4.420
    » https://doi.org/10.1128/CMR.5.4.420
  • 33
    Najafi MH, Taheri M, Mokhtari MR, Forouzanfar A, Farazi F, Mirzaee M et al. Comparative study of 0.2% and 0.12% digluconate chlorhexidine mouth rinses on the level of dental staining and gingival indices. Dent Res J (Isfahan). 2012;9(3):305-8.
  • 34
    Slot DE, Rosema NA, Hennequin-Hoenderdos NL, Versteeg PA, Van Der Velden U, Van Der Weijden GA. The effect of 1% chlorhexidine gel and 0.12% dentifrice gel on plaque accumulation: a 3-day non-brushing model. Int J Dent Hyg 2010;8(4):294-300. doi:10.1111/j.1601-5037.2010.00487.x
    » https://doi.org/10.1111/j.1601-5037.2010.00487.x
  • 35
    Maltz, M, Zickert I, Krasse B. Effect of intensive treatment with chlorhexidine on number of Streptococcus mutans in saliva. Scand J Dent Res. 1981;89(6):445-9.
  • 36
    Hübner NO, Siebert J, Kramer A. Octenidine dihydrochloride, a modern antiseptic for skin, mucous membranes and wounds. Skin Pharmacol Physiol 2010;23(5):244-58. doi:10.1159/000314699
    » https://doi.org/10.1159/000314699
  • 37
    Rohrer N, Widmer AF, Waltimo T, Kulik EM, Weiger R, Filipuzzi-Jenny E et al. Antimicrobial efficacy of 3 oral antiseptics containing octenidine, polyhexamethylene biguanide, or Citroxx: can chlorhexidine be replaced? Infect Control Hosp Epidemiol. 2010;31(7):733-9. doi:10.1086/653822
    » https://doi.org/10.1086/653822
  • 38
    Dogan AA, Adiloglu AK, Onal S, Cetin ES, Polat E, Uskun E et al. Short-term relative antibacterial effect of octenidine dihydrochloride on the oral microflora in orthodontically treated patients. Int J Infect Dis. 2008;12(6):19-25. doi:10.1016/j.ijid.2008.03.013
    » https://doi.org/10.1016/j.ijid.2008.03.013
  • 39
    Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother. 2001;48(Suppl 1):5-16. doi:10.1093/jac/48.suppl_1.5
    » https://doi.org/10.1093/jac/48.suppl_1.5
  • 40
    Emilson CG, Bowen WH, Robrish SA, Kemp CW. Effect of the antibacterial agents octenidine and chlorhexidine on the plaque flora in primates. Scand J Dent Res 1981;89(5):384-92.
  • 41
    Pithon MM, Sant’Anna LI, Baião FC, Santos RL, Coqueiro RS, Maia LC. Assessment of the effectiveness of mouthwashes in reducing cariogenic biofilm in orthodontic patients: a systematic review. J Dent. 2015;43(3):297-308. doi:10.1016/j.jdent.2014.12.010
    » https://doi.org/10.1016/j.jdent.2014.12.010
  • 42
    Patters MR, Anerud K, Trummel CL, Kornman KS, Nalbandian J, Robertson PB. Inhibition of plaque formation in humans by octenidine mouth rinse. J Periodontal Res. 1983;18(2):212-9. doi:10.1111/j.1600-0765.1983.tb00354.
    » https://doi.org/10.1111/j.1600-0765.1983.tb00354.

Publication Dates

  • Publication in this collection
    2016

History

  • Received
    15 Jan 2016
  • Reviewed
    15 July 2016
  • Accepted
    15 Sept 2016
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