Previous studies have demonstrated that water discharged from dental unit waterlines (DUWLs) is contaminated with many bacteria [1-3]. Additionally, opportunistic bacteria such as Legionella, which can be fatal to immunocompromised patients, have also been observed [4-6]. The excretion of bacteria from DUWLsis caused by the detachment of bacteria from biofilms formed in DUWLs [2,7,8]. Therefore, it is necessary to effectively prevent the formation of biofilms in DUWLs to prevent contamination.

Biofilms mature through the adhesion of bacteria to the conditioning film formed on the surface, co-aggregation of early- and late-adherent bacteria, and binding of extracellular polymeric substances produced by the bacteria [8]. Bacterial adhesion is the first and most important step for biofilm formation [9]. In other words, the absence of bacterial adhesion prevents the formation of biofilms. Therefore, studies have been seeking methods to prevent bacterial adhesion in order to prevent biofilm formation [10,11].

A simple method to prevent bacterial adhesion is to construct DUWL tubes using materials that inhibit adhesion. However, there is a lack of studies on various materials that are be used to make DUWL tubes. The purpose of this study was to compare biofilm formation in DUWL tubes made of different materials.

A total of 6 types of DUWL tube materials with an inner diameter of 2 mm were used in this study: polyvinyl chloride (PVC; Hanmi hose Corp., Anyang, Korea), polytetra fluorethylene (PTFE; Tommyheco Corp., Seoul, Korea), silicone (Tommyheco Corp.), polyvinylidene fluoride (PVDF; HAKKO Corp., Tokyo, Japan), polyethylene (PE; AS One Corp., Osaka, Japan), and polyurethane (PU; Nitta Moore Corp., Gumi, Korea). To induce biofilm formation on the inner surface, each tube material was cut in half lengthwise and 1-cm wide.

To form DUWL biofilms in the laboratory, the CDC biofilm reactor model (BioSurface Technologies Corp., Bozeman, MT, USA) established by Yoon and Lee was used [12]. One liter of water collected from DUWLs was filtered through a 0.2-µm filter paper (Millipore Billerica, MA, USA), and this filter paper was then suspended in 20 mL of phosphate buffered saline (PBS, pH 7.4). The suspension was incubated in R2A liquid medium (Becton, Dickinson and Company, Sparks, NV, USA) for 10 days, and the culture medium was stored in a freezer at –70°C. R2A is a generally used medium for detection of low-temperature bacteria in water. This bacterial culture medium stock was used in every experiment. For each experiment, 2.5 mL of the bacterial culture medium stock was inoculated into 50 mL of R2A liquid medium and batch cultured at 26°C for 5 days.

Each piece of the water tube was attached to the disc holder in the CDC biofilm reactor, and the CDC biofilm reactor was sterilized. A total of 300 mL of R2A liquid medium and 50 mL of bacterial culture medium (batch cultured for 5 days) were added to the CDC biofilm reactor. 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). Vortex as well as medium supply were maintained from 9 am to 6 pm to replicate normal dental treatment hours. A steady culture condition was maintained outside of those hours. Biofilms were formed for 4 days inside the CDC biofilm reactor model.

The tube pieces of each material were removed from the CDC biofilm reactor model, and only their outer surface was washed using 70% ethanol. Loosely attached bacteria were washed away twice using PBS (pH 7.4). The tube pieces were then placed in 1 mL of PBS containing 0.09-mm glass beads and vortexed to separate the biofilms from the tubes. The solution, from which the biofilms were separated, was diluted 10-fold and smeared on R2A solid medium 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 S.A.). Colony forming units per 1 mL (CFU/mL) were calculated.

Kruskal–Wallis test and Mann–Whitney U-test were conducted to compare biofilm accumulation between the tube materials. A p-value<0.05 was considered statistically significant, and all statistical analyses were conducted using SPSS statistical program (IBM Corp., Armonk, NY, USA).

Biofilm accumulation on the PE tube (4.3×10⁵ CFU/mL) was significantly higher than that in tubes of other materials (p<0.05). Biofilm accumulation in PU and silicone tubes was the least at 8.0×10⁴ CFU/mL and 9.1×10⁴ CFU/mL, respectively (p<0.05). Biofilm accumulation in PVDF and PTFE tubes was greater than that in PVC, PU, and silicone tubes (p<0.05) (Fig. 1).

Fig. 1. The biofilm accumulation on 6 materials using the CDC biofilm reactor model (BioSurface Technologies Corporation, Bozeman, MT, USA). Each value represents the mean value of 4 replicate samples in duplicate on R2A. The error bars indicate standard deviations from the mean. Asterisks indicate a significant difference (p<0.05) between biofilm accumulation of materials. CFU, colony forming unit; PE, polyethylene; PVC, polyvinyl chloride; PU, polyurethane; Si, silicone; PVDF, polyvinylidene fluoride; PTFE, polytetra fluorethylene.

In this study, we compared biofilm formation in various materials used to make DWULs. PVC, silicone, PE, and PU are tube materials that are currently used to make DUWLs. PVDF and PTFE are materials that contain fluorine, which is antibacterial. They were tested in this study to assess their anti-adhesion effects compared to that of conventional materials.

Yabune et al. [10] used dental units with PU and PVDF for up to 185 days and assessed bacterial contamination in the tubes. They found that PVDF effectively reduced the number of bacteria in DUWL water and inhibited the formation of biofilms. In another study, Sacchetti et al. [11] compared the inhibition of DUWL bacterial adhesion between PE and PTFE, and observed that PTFE inhibited bacterial growth and colonization better than PE. Similarly, in our study, we also found that PTFE exhibited better inhibition of biofilm formation than PE. However, contrary to the findings of Yabune et al. [10], we showed that PU and silicone were more effective in inhibiting biofilm formation than PVDF. But, there is no statistical significance. Additionally, PU and silicone were the most effective in inhibiting biofilm formation. These differences may be attributed to the different bacterial species constituting the biofilms formed in the tubes. The bacterial species found in DUWLs are different in each country, and such differences may have led to different levels of biofilm formation in tubes of same materials [13-15].

Previous studies have compared various materials for inhibition of bacterial adhesion in catheters and pipes for distribution of drinking water. First, in a study that compared bacterial adhesion on various catheter materials, PU showed the highest resistance to adhesion of Staphylococcus spp. They also presented that Teflon (PTFE) had the highest resistance to adhesion of Escherichia coli and Pseudomonas aeruginosa [16]. In another study that assessed bacterial adhesion in drinking water distribution pipes of different materials, the inhibitory effects of the materials on bacterial adhesion differed depending on the bacterial species [17].

Bacterial adhesion on the surface is a complex process affected by the physicochemical properties of both the bacteria and material [16,17]. Among the properties, hydrophobicity and surface charge were shown to be the most important factors [17]. As the hydrophobicity and surface charge differ for each bacterial and material, bacterium adhesion may also differ depending on the material [16,17].

Previous studies showed that bacterial contamination can be reduced by using tube materials that can inhibit bacterial adhesion and biofilm formation for different bacterial species [10,11]. PU, which led to the least amount of biofilms formed in this study, may be an ideal material for DUWLs. Further studies are needed to assess the inhibitory effects of different tube materials on the adhesion of individual bacterial species that constitute DUWLs biofilms. In addition, further research is also needed to evaluate and determine the most suitable material for DUWLs.

None.

The authors declare that they have no competing interests.