Oral Biol Res 2022; 46(3): 106-110  https://doi.org/10.21851/obr.46.03.202209.106
Effect of cetylpyridinium chloride on dental unit waterline biofilms in-vitro
Si Young Lee1* and Hye Young Yoon2
1Professor, Department of Microbiology and Immunology, College of Dentistry, Gangneung-Wonju National University, Gangneung, Republic of Korea
2Ph.D. Student, Department of Microbiology and Immunology, College of Dentistry, Gangneung-Wonju National University, Gangneung, Republic of Korea
Correspondence to: Si Young Lee, Department of Microbiology and Immunology, College of Dentistry, Gangneung-Wonju National University, 7, Jukheon-gil, Gangneung 25457, Republic of Korea.
Tel: +82-33-640-2455, Fax: +82-33-642-6410, E-mail: siyoung@gwnu.ac.kr
Received: June 27, 2022; Revised: August 19, 2022; Accepted: August 22, 2022; Published online: September 30, 2022.
© Oral Biology Research. All rights reserved.

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Various disinfectants are used to remove biofilms formed in dental unit waterlines (DUWLs). This study aimed to assess the antibiofilm effects of cetylpyridinium chloride (CPC) on DUWL biofilms formed in a CDC Biofilm Reactor model and evaluate the feasibility of CPC as a novel DUWL disinfectant. For experiments, CPC was used at concentrations of 0.003125% to 0.00039%. Well-established DUWL disinfectants, such as 0.5% sodium hypochlorite (NaOCl), 0.12% chlorhexidine (CHX), and a mixture of 0.12% CHX and 12% ethanol, were used as standards. DUWL biofilms formed in the CDC Biofilm Reactor model were exposed to the disinfecting agents for 15 min. The number of living bacteria in the biofilms before and after exposure to chemicals were compared, and the biofilm reduction rate was calculated. The biofilm reduction rate of CPC at a concentration higher than 0.001562% was greater than that of all other conventional disinfecting agents. These findings suggest that CPC can be used for effective removal of DUWL biofilms.
Keywords: Biofilms; Cetylpyridinium chloride; Disinfectants

Biofilms formed in dental unit watcerlines (DUWLs) are considered as the cause of bacterial contamination of water discharged from the DUWLs [1,2]. Opportunistic pathogens such as Pseudomonas aeruginosa, Legionella pneumophila, and Mycobacterium spp. have been found in water from DUWLs. Thus, removal of biofilms in DUWLs is essential to provide safe dental care to patients [3-6]. There are various ways to remove biofilms in DUWLs. Among them, treating DUWLs with disinfectants was effective in removing biofilms [7-9]. Chemical agents used to disinfect DUWLs include chlorine dioxide, sodium hypochlorite (NaOCl), hydrogen peroxide, and chlorhexidine (CHX) gluconate [8-11]. In studies evaluating the effects of different disinfectants on DUWL biofilms, disinfectants containing NaOCl or hydrogen peroxide showed high anti-biofilm effects [8,11]. However, side effects such as corrosion of dental equipment, blockage of water tubes, and irritation of oral mucosa were also observed, limiting the use of these disinfectants [8,9,11]. Therefore, it is necessary to develop products that have few side effects on dental units and oral cavity of patients, while ensuring a high rate of disinfection.

Cetylpyridinium chloride (CPC) is a widely used antibacterial compound. This compound is positively charged and is thus able to bind to the negatively charged surface of bacteria. This allows the compound to interact with the bacterial cell membrane, damage the cells, and exert antibacterial effects [12-14]. CPC is known as a safe preservative used in oral hygiene products (up to 0.1% in mouthwashes), has antibacterial effects against various microorganisms including oral bacteria, and has anti-biofilm effects [14-16]. Additionally, in a study evaluating the susceptibility of bacterial species isolated from DUWLs to various chemical agents, the isolates were found to be highly susceptible to CPC, suggesting that it may be used as a disinfectant for DUWLs [17].

The purpose of this study was to confirm the effects of CPC on DUWLs biofilm and compare to the effects of other conventional chemical agents used to disinfect DUWLs.

Materials and Methods

Biofilm formation on DUWLs

To form DUWL biofilms in the laboratory, the CDC biofilm reactor model suggested by Yoon and Lee [18] was used. One liter of water discharged from DUWLs was collected and filtered through a 0.2-µm filter paper (Millipore, Billerica, MA, USA). The filter paper was suspended in 20 mL of phosphate buffered saline (PBS, pH 7.4), and the suspension was incubated in R2A liquid medium (Becton Dickinson and Company, Sparks, MD, USA) for 10 days and stored in a freezer at –70°C. This bacterial stock was used in every experiment. After inoculating 2.5 mL of the bacterial stock into 50 mL of R2A liquid medium, batch culture was performed for 5 days at 26°C.

To form biofilms, a polyurethane disc (Nitta Moore Corp., Gumi, Korea) with a diameter of 5 mm was used. A polyurethane disc was attached to the disc holder in the CDC biofilm reactor (BioSurface Technologies Corp., Bozeman, MT, USA) using double-sided tape. Afterwards, the CDC biofilm reactor was sterilized, and 300 mL of R2A liquid medium and 50 mL of batch-cultured bacterial medium were added. The CDC biofilm reactor was incubated at 26°C for 4 days. During the incubation period, the reactor was vortexed at 50 rpm using an agitator, and R2A liquid medium was supplied at 12.5 mL/h using a peristaltic pump (JenieWell, Seoul, Korea) [19]. Vortex and medium supply were maintained from 9 a.m. to 6 p.m., to replicate normal dental treatment hours. Steady culture condition was applied outside of those hours.

Chemical agents and treatment

CPC was diluted two-fold from 0.003125% to 0.00039% for use to check the effective concentration. CPC’s efficiency of disinfection was compared to that of 0.5% NaOCl (Junsei Chemical Co., Ltd., Tokyo, Japan), 0.12% CHX (Sigma-Aldrich Chemical Co., St. Louis, MO, USA), and a mixture of 0.12% CHX and 12% ethanol (Honeywell, Ulsan, Korea), which have previously been used as DUWL disinfectants. All chemical agents were prepared using sterile distilled water, and the chemical agents used in the experiments are shown in Table 1.

Concentrations of disinfecting agents used in this study

Chemical agent Concentration (%)
CPC 0.003125
NaOCl 0.5
CHX 0.12
CHX, EtOH 0.12, 12

CPC, cetylpyridinium chloride; NaOCl, sodium hypochlorite; CHX, chlorhexidine; EtOH, ethanol.

Polyurethane discs with biofilms were placed in a 24-well plate (SPL Life Science, Pocheon, Korea) and treated with 2 mL of chemical agents. The plate was incubated at 26°C for 15 min. The experiment was performed in triplicates.

Assessment of biofilm accumulation

After chemical treatment, the polyurethane discs were separated from the 24-well plate and washed twice with PBS. The discs were placed in 1 mL of PBS containing 0.09-mm glass beads and vortexed. The PBS suspension was diluted 10-fold, and the diluted solution was plated on R2A solid medium (Becton Dickinson and Company) using a spiral plater (IUL, S.A., Barcelona, Spain). The smeared R2A solid medium was cultured at 26°C for 7 days, and bacterial colonies were counted using a colony counter (IUL). Colony forming unit per mL was calculated.

Biofilm accumulation-reduction effect of each chemical was expressed as biofilm reduction rate, which was obtained by comparing the biofilm accumulation amounts of the disinfectant-treated and sterile distilled water-treated groups. The formula for calculating biofilm reduction rate is as follows.

Biofilm reduction rate (%)=NwNdNw×100

where, Nw is the number living bacteria in biofilm after sterile distilled water treatment, and Nd is the number living bacteria in biofilm after disinfectant treatment.

Statistical analysis

Kruskal-Wallis test and Mann-Whitney U-test were conducted to compare biofilm reduction rate of each chemical. A p-value <0.05 was considered statistically significant, and all statistical analysis were conducted using the SPSS statistical program for Windows, Version 25.0 (IBM SPSS Inc., Armonk, NY, USA).


Effects of CPC on DUWL biofilms

When the concentration of CPC was greater than 0.001562%, biofilm reduction rate was greater than 90%. Additionally, biofilm reduction rate of CPC was greater than 99% at a concentration of 0.003125% or greater (data not shown). The biofilm reduction rate of CPC at concentrations higher than 0.000781% was greater than that of 0.5% NaOCl (67.7%) and 0.12% CHX (60.4%). The biofilm reduction rate of CPC at concentrations higher than 0.001562% was greater than that of 0.12% CHX+12% EtOH (86.6%). At a concentration of 0.003125% or higher, the biofilm reduction rate of CPC was significantly greater than that of other chemicals (p<0.05) (Fig. 1).

Fig. 1. Percentage of decrease in biofilm CFU/mL after exposure to CPC, NaOCl, CHX, and CHX+EtOH. The error bars indicate standard deviations from the mean for triplicate polyurethane discs. Asterisks indicate a significant difference (p<0.05) between % reduction of chemical agents. CFU, colony forming unit; CPC, cetylpyridinium chloride; NaOCl, sodium hypochlorite; CHX, chlorhexidine; EtOH, ethanol.

An ideal disinfectant for DUWLs must be able to remove not only bacterial planktons floating in water, but also biofilms formed on the surface of DUWLs, and should also have only limited side effects on the dental units and patients [20,21]. To develop an ideal DUWL disinfectant, we used CPC and assessed its effects on DUWLs biofilms.

CPC is a quaternary ammonium compound used for various purposes. In particular, CPC is commonly used in dental products such as mouthwash, toothpaste, and varnish. The effects of CPC on oral bacteria and dental plaque have been demonstrated in previous studies [12,15,22]. Additionally, CPC has antibacterial effects on food-borne Salmonella spp. and is used to disinfect various food products including poultry meat [13,23,24]. CPC has the lowest level of risk (level 1) for safety as classified by the United States Food and Drug Administration (FDA) [25], suggesting its high safety.

The CPC concentration of 0.003125% used in our study, which showed a 99% DUWLs biofilm reduction rate, is significantly lower than the CPC concentrations of 0.05%, 0.1%, and 0.5% that are used in dental or food disinfection. Moreover, 0.003125% of CPC showed higher biofilm reduction effects than conventional disinfecting chemicals at the concentrations used to disinfect DUWLs.

In a previous study by Yoon and Lee [17], susceptibility of bacterial species isolated from DUWLsto various chemicals including CPC was evaluated. In single biofilm state, all bacterial species showed greatest susceptibility to CPC (minimum biofilm inhibitory concentration and minimum biofilm eradication concentration). Similarly, in this study, CPC showed the highest reduction rate against various types of DUWLs biofilms formed in the CDC biofilm reactor. These findings suggest that CPC may be used as a DUWL disinfectant. However, further studies must assess the effects of CPC on clinical DUWL biofilms for the use of CPC as a DUWL disinfectant.



Conflicts of Interest

The authors declare that they have no competing interests.

  1. Williams HN, Baer ML, Kelley JI. Contribution of biofilm bacteria to the contamination of the dental unit water supply. J Am Dent Assoc 1995;126:1255-1260. doi: 10.14219/jada.archive.1995.0360.
    Pubmed CrossRef
  2. Walker JT, Marsh PD. Microbial biofilm formation in DUWS and their control using disinfectants. J Dent 2007;35:721-730. doi: 10.1016/j.jdent.2007.07.005.
    Pubmed CrossRef
  3. Fotos PG, Westfall HN, Snyder IS, Miller RW, Mutchler BM. Prevalence of Legionella-specific IgG and IgM antibody in a dental clinic population. J Dent Res 1985;64:1382-1385. doi: 10.1177/00220345850640121101.
    Pubmed CrossRef
  4. Walker JT, Bradshaw DJ, Bennett AM, Fulford MR, Martin MV, Marsh PD. Microbial biofilm formation and contamination of dental-unit water systems in general dental practice. Appl Environ Microbiol 2000;66:3363-3367. doi: 10.1128/AEM.66.8.3363-3367.2000.
    Pubmed KoreaMed CrossRef
  5. Costa D, Mercier A, Gravouil K, Lesobre J, Delafont V, Bousseau A, Verdon J, Imbert C. Pyrosequencing analysis of bacterial diversity in dental unit waterlines. Water Res 2015;81:223-231. doi: 10.1016/j.watres.2015.05.065.
    Pubmed CrossRef
  6. Szymańska J, Sitkowska J. Opportunistic bacteria in dental unit waterlines: assessment and characteristics. Future Microbiol 2013;8:681-689. doi: 10.2217/fmb.13.33.
    Pubmed CrossRef
  7. Kettering JD, Muñoz-Viveros CA, Stephens JA, Naylor WP, Zhang W. Reducing bacterial counts in dental unit waterlines: distilled water vs. antimicrobial agents. J Calif Dent Assoc 2002;30:735-741.
  8. Schel AJ, Marsh PD, Bradshaw DJ, Finney M, Fulford MR, Frandsen E, Østergaard E, ten Cate JM, Moorer WR, Mavridou A, Kamma JJ, Mandilara G, Stösser L, Kneist S, Araujo R, Contreras N, Goroncy-Bermes P, O’Mullane D, Burke F, O’Reilly P, Hourigan G, O’Sullivan M, Holman R, Walker JT. Comparison of the efficacies of disinfectants to control microbial contamination in dental unit water systems in general dental practices across the European Union. Appl Environ Microbiol 2006;72:1380-1387. doi: 10.1128/AEM.72.2.1380-1387.2006.
    Pubmed KoreaMed CrossRef
  9. O’Donnell MJ, Boyle MA, Russell RJ, Coleman DC. Management of dental unit waterline biofilms in the 21st century. Future Microbiol 2011;6:1209-1226. doi: 10.2217/fmb.11.104.
    Pubmed CrossRef
  10. Liaqat I, Sabri AN. Effect of biocides on biofilm bacteria from dental unit water lines. Curr Microbiol 2008;56:619-624. doi: 10.1007/s00284-008-9136-6.
    Pubmed CrossRef
  11. Lin SM, Svoboda KK, Giletto A, Seibert J, Puttaiah R. Effects of hydrogen peroxide on dental unit biofilms and treatment water contamination. Eur J Dent 2011;5:47-59. doi: 10.1055/s-0039-1698858.
    Pubmed KoreaMed CrossRef
  12. Sreenivasan PK, Haraszthy VI, Zambon JJ. Antimicrobial efficacy of 0·05% cetylpyridinium chloride mouthrinses. Lett Appl Microbiol 2013;56:14-20. doi: 10.1111/lam.12008.
    Pubmed CrossRef
  13. Lim K, Mustapha A. Inhibition of Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus on sliced roast beef by cetylpyridinium chloride and acidified sodium chlorite. Food Microbiol 2007;24:89-94. doi: 10.1016/j.fm.2006.04.005.
    Pubmed CrossRef
  14. Ioannou CJ, Hanlon GW, Denyer SP. Action of disinfectant quaternary ammonium compounds against Staphylococcus aureus. Antimicrob Agents Chemother 2007;51:296-306. doi: 10.1128/AAC.00375-06.
    Pubmed KoreaMed CrossRef
  15. Pandit S, Cai JN, Jung JE, Lee YS, Jeon JG. Effect of brief cetylpyridinium chloride treatments during early and mature cariogenic biofilm formation. Oral Dis 2015;21:565-571. doi: 10.1111/odi.12312.
    Pubmed CrossRef
  16. Ramalingam K, Amaechi BT, Ralph RH, Lee VA. Antimicrobial activity of nanoemulsion on cariogenic planktonic and biofilm organisms. Arch Oral Biol 2012;57:15-22. doi: 10.1016/j.archoralbio.2011.07.001.
    Pubmed KoreaMed CrossRef
  17. Yoon HY, Lee SY. Susceptibility of bacteria isolated from dental unit waterlines to disinfecting chemical agents. J Gen Appl Microbiol 2019;64:269-275. doi: 10.2323/jgam.2018.02.001.
    Pubmed CrossRef
  18. Yoon HY, Lee SY. Establishing a laboratory model of dental unit waterlines bacterial biofilms using a CDC biofilm reactor. Biofouling 2017;33:917-926. doi: 10.1080/08927014.2017.1391950.
    Pubmed CrossRef
  19. Walker JT, Bradshaw DJ, Fulford MR, Marsh PD. Microbiological evaluation of a range of disinfectant products to control mixed-species biofilm contamination in a laboratory model of a dental unit water system. Appl Environ Microbiol 2003;69:3327-3332. doi: 10.1128/AEM.69.6.3327-3332.2003.
    Pubmed KoreaMed CrossRef
  20. Gawande PV, LoVetri K, Yakandawala N, Romeo T, Zhanel GG, Cvitkovitch DG, Madhyastha S. Antibiofilm activity of sodium bicarbonate, sodium metaperiodate and SDS combination against dental unit waterline-associated bacteria and yeast. J Appl Microbiol 2008;105:986-992. doi: 10.1111/j.1365-2672.2008.03823.x.
    Pubmed CrossRef
  21. Percival RS, Devine DA, Nattress B, Kite P, Marsh PD. Control of microbial contamination in dental unit water systems using tetra-sodium EDTA. J Appl Microbiol 2009;107:1081-1088. doi: 10.1111/j.1365-2672.2009.04299.x.
    Pubmed CrossRef
  22. Van Leeuwen MP, Rosema NA, Versteeg PA, Slot DE, Van Winkelhoff AJ, Van der Weijden GA. Long-term efficacy of a 0.07% cetylpyridinium chloride mouth rinse in relation to plaque and gingivitis: a 6-month randomized, vehicle-controlled clinical trial. Int J Dent Hyg 2015;13:93-103. doi: 10.1111/idh.12082.
    Pubmed CrossRef
  23. Kim JW, Slavik MF. Cetylpyridinium chloride (CPC) treatment on poultry skin to reduce attached Salmonella. J Food Prot 1996;59:322-326. doi: 10.4315/0362-028x-59.3.322.
    Pubmed CrossRef
  24. Saucedo-Alderete RO, Eifert JD, Boyer RR, Williams RC, Welbaum GE. Cetylpyridinium chloride direct spray treatments reduce Salmonella on cantaloupe rough surfaces. J Food Saf 2018;38:e12471. doi: 10.1111/jfs.12471.
    Pubmed KoreaMed CrossRef
  25. Food and Drug Administration. Oral health care drug products for overthe-counter human use; antigingivitis/antiplaque drug products; establishment of a monograph; proposed rules. Fed Reg 2003;68:32231-32287.

This Article