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.

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.

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 (%)=Nw−NdNw×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.

None.

The authors declare that they have no competing interests.