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Applied and Environmental Microbiology, October 2008, p. 5958-5964, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.00610-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Assessment of Inhibitory Effects of Fluoride-Coated Tubes on Biofilm Formation by Using the In Vitro Dental Unit Waterline Biofilm Model

Toshiaki Yabune, Satoshi Imazato,^* and Shigeyuki Ebisu

Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan

Received 13 March 2008/ Accepted 27 July 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study aimed to establish an in vitro model to simulate biofilms formed in dental unit waterlines (DUWLs) and to investigate the ability of polyvinylidene fluoride (PVDF)-coated tubes to inhibit biofilm formation using this model. The water and biofilm samples were obtained from DUWLs which had been clinically used for 2.5 years, and the predominant bacteria were identified. A conventional polyurethane tube was incubated for 24 to 96 h in the mixed flora of isolated bacteria, and the optimal incubation conditions to simulate a clinically formed biofilm were determined by observation with a scanning electron microscope. Biofilm formation on a PVDF-coated tube was observed using this in vitro model, and the adherence of different bacterial species to conventional and PVDF-coated tubes was assessed. Sphingomonas paucimobilis, Acinetobacter haemolytics, and Methylobacterium mesophilicum were predominantly isolated from contaminated DUWLs. Incubation of the polyurethane tube with the mixed flora containing these three species for 96 h resulted in the formation of a mature biofilm similar to the one clinically observed. The PVDF-coated tube was significantly less adhesive to all three bacterial species than the polyurethane tube (P < 0.05 by the Mann-Whitney U test), and the attachment of small amounts of rods was observed even after incubation with the mixed flora for 96 h. In conclusion, an in vitro biofilm model was obtained by using a mixed flora of bacteria isolated from DUWLs, and the PVDF-coated tube was found to be effective in preventing biofilm formation using this model.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Attention has been paid to the prevention of bacterial contamination of dental unit waterlines (DUWLs) due to the increase in occurrence of nosocomial infections. Many studies demonstrated that the water discharged from DUWLs contains a number of bacteria (4, 6, 16, 17, 21, 23, 24, 26, 27, 29, 32-37), and pathogenic bacteria such as Legionella, which are virulent to an immunocompromised host, have been reported to be present occasionally (2, 18, 22). The bacterial outflow from DUWLs has been suggested to be caused by sloughing of bacteria from biofilms which had formed on the surfaces of the water supply tubes (16, 23, 32-34). Therefore, to prevent the contamination of DUWLs, it is essential to develop effective treatments that can inhibit the formation of a bacterial biofilm in the tube.

Several studies (1, 2, 16-18, 22, 33, 36, 37) identified the different bacteria in the water discharged from DUWLs. However, little information is available on the process of biofilm formation. The affinity of the biofilm-forming bacteria to the tube surface or the mutual interactions of multiple species to create a community remain to be determined. To further elucidate the processes involved in the formation of the DUWL biofilm, the establishment of an in vitro model would be helpful. Moreover, an in vitro model can provide a powerful tool for developing fundamental measures to prevent DUWL contamination. Such a model will enable the screening of the effectiveness of various preventive methods in a short period, while the present clinical usage tests are time-consuming and have been shown to give nonreproducible results (8, 10, 14, 15, 20, 38).

In this study, aiming at establishment of such an in vitro model, the bacterial species involved in the formation of biofilm in DUWLs were identified, and attempts to produce an artificial biofilm were conducted using the bacteria isolated. In addition, using the established in vitro model, the inhibitory effects of a polyvinylidene fluoride (PVDF)-coated tube, which was previously demonstrated to be effective in reducing bacterial outflow from DUWLs in a clinical study (38), were evaluated.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Identification of the bacteria responsible for the formation of biofilms in DUWLs.
To identify the predominant bacterial species forming biofilms in DUWLs, water and biofilm samples were obtained clinically from two dental units. These units were used for 2.5 years in the Conservative Dentistry Clinic of Osaka University Dental Hospital. Five milliliters of water was collected from a high-speed handpiece line before the commencement of the daily clinic work. For the biofilm samples, a 10-mm portion of the high-speed handpiece line tubing was taken from the same units, and the biofilm inside the tube was scraped with a dental excavator and dispersed in sterile distilled water (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan). Each sample was serially 10-fold diluted with sterile distilled water, and a 100-µl aliquot was inoculated onto R2A agar plates (Becton Dickinson & Co., NJ) suitable for the growth of heterotrophic bacteria (15). After aerobic incubation for 7 days at 25°C, the morphologies and colors of the colonies formed on the agar plates were examined under a dissecting microscope. The predominant colonies were then isolated, and the bacterial species were identified by Gram staining, catalase activity tests, oxidase tests, and sugar fermentation tests and using an identification kit for gram-negative rods (ID test NF-18; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan).

In vitro biofilm model.
We attempted to produce an in vitro biofilm simulating the one formed in DUWLs in the clinics by incubating with the mixed flora of the predominant biofilm-forming bacteria identified. Portions of R2A broth containing Sphingomonas paucimobilis at 5.4 x 10⁶ CFU/ml, Acinetobacter haemolytics at 3.0 x 10⁵ CFU/ml, and Methylobacterium mesophilicum at 3.0 x 10⁵ CFU/ml were prepared and mixed together to give a 10-ml suspension. The mixing ratio of the three species was 18:1:1, determined on the basis of the findings obtained in previous experiments to identify the bacterial species present in clinical samples of water and biofilm. A polyurethane tube (length, 10 mm) sterilized with ethylene oxide gas was immersed in the suspension containing the mixed flora of the three bacterial species and incubated at 25°C for 24, 48, or 96 h. After each incubation period, the tube was sectioned longitudinally and immersed in 2% paraformaldehyde-2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for fixation. The specimens were dehydrated in an ascending series of ethanol solutions (50, 70, 80, 90, 95, and 100%), immersed in t-butyl alcohol, and freeze-dried. The inner surface of the tube was coated with platinum using a plasma multicoater (PMC-5000; Meiwa Co., Osaka, Japan) and observed using a scanning electron microscope (SEM) (JSM-5310LV; JEOL, Tokyo, Japan). The cross section of the tube was also obtained after 96 hours of incubation and observed under SEM.

Inhibitory effects of the PVDF-coated tube on bacterial adherence and biofilm formation.
The adherences of the single species of the three dominant biofilm-forming bacteria identified to the conventional polyurethane and PVDF-coated tubes were compared. Ten milliliters of R2A broth containing each bacterial species (3.0 x 10⁷ CFU/ml for S. paucimobilis and A. haemolytics and 3.0 x 10⁵ CFU/ml for M. mesophilicum) were prepared, and a polyurethane or PVDF-coated tube (length, 10 mm) (Soft Fluoro Hose E-PD-2; Hakko Co., Tokyo, Japan) sterilized with ethylene oxide gas was immersed in each broth. After incubation at 25°C for 24 h, the tube was sectioned longitudinally, fixed, dehydrated, and freeze-dried. The inner surface was coated with platinum and observed by SEM. The number of adhered bacteria (cells/mm²) was calculated from the bacterial count on five randomly selected SEM images at a magnification of x2,000. Three specimens were assessed for each tube, and the results were compared using the Mann-Whitney U test with the significance level at a P value <0.05.

Following the incubation of the tube in a mixed flora of S. paucimobilis, A. haemolytics, and M. mesophilicum (18:1:1) for 24 to 96 h, biofilm formation on the surface of the PVDF-coated tube was assessed by SEM observation.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Identification of the bacteria forming the biofilm in DUWLs.
For each of the two different dental units, three predominant colonies were isolated from water and biofilm samples; there were yellow and white colonies with rough margins and a rounded smaller red colony with smooth margins (Fig. 1a, c, and e). The yellow colonies were the most prevalent among the three colonies, and the colonies formed on the agar plates were in a ratio of 18:1:1 for yellow, white, and red colonies for both water and biofilm samples. There were few colonies other than these three types.

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FIG. 1. The three predominant bacterial colonies formed and SEM images of bacteria corresponding to each colony. (a and b) The yellow colony-forming rods were identified as Sphingomonas paucimobilis. (c and d) The white colony-forming rods were Acinetobacter haemolytics. (e and f) The small, red, colony-forming filamentous rods were Methylobacterium mesophilicum.

Figures 1b, d, and f show the SEM images of the bacteria corresponding to the yellow, white, and red colonies. The yellow and white colonies were characterized by rods, while the red colony contained filamentous rods. These three bacteria were gram negative and were classified as heterotrophic bacteria living in water. The bacteria forming the yellow, white, and red colonies were identified as Sphingomonas paucimobilis (Fig. 1b), Acinetobacter haemolytics (Fig. 1d), and Methylobacterium mesophilicum (Fig. 1f), respectively.

In vitro biofilm model.
Figure 2a to c show the SEM images of the inner surface of the polyurethane tube incubated for 24, 48, and 96 h in R2A broth containing the mixed flora of S. paucimobilis, A. haemolytics, and M. mesophilicum. Many rods adhered to the tube after 24 hour of incubation, and the amount of attached rods increased with cell aggregation after 48 h. When the tube was incubated for 96 h, the filamentous rods adhered on the underlying layer of rods, and the artificially formed biofilm exhibited characteristics similar to those observed in contaminated DUWLs after 6 months of clinical usage (Fig. 2d). From the SEM images of the cross-sectioned tube, formation of 2- to 5-µm-thick biofilm with adherence of the filamentous rods on the bottom layer of rods was confirmed (Fig. 2e).

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FIG. 2. SEM images of the inner surface of the polyurethane tube incubated in a mixed flora of the three bacteria identified, showing the longitudinal section after incubation for 24 (a), 48 (b), or 96 h (c) and the cross section after 96 hour of incubation (e). A biofilm formed after incubation for 96 h (c) was similar to the one observed in contaminated polyurethane tubes after 6 months of clinical use (d).

Inhibitory effects of the PVDF-coated tube on bacterial adherence and biofilm formation.
Figure 3 shows the adherences of each species of bacteria to the polyurethane and PVDF-coated tubes after 24 hour of incubation. S. paucimobilis and A. haemolytics adhered in large amounts to the polyurethane tube (Fig. 3a and c). M. mesophilicum adhered less to the polyurethane tube than the other two species, but the bacterial cells were clearly aggregating with each other (Fig. 3e). Only a few bacteria adhered on the surface of the PVDF-coated tube, with little aggregation for all of the species tested (Fig. 3b, d, and f). Significant differences in the number of attached cells were obtained between the two types of tubes for all bacteria (P < 0.05), demonstrating that there was less bacterial attachment on the PVDF-coated tube than on the polyurethane tube (Table 1).

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FIG. 3. Adherence of each species to a polyurethane (a, c, and e) or a PVDF-coated tube (b, d, and f) after 24 hour of incubation. (a and b) S. paucimobilis; (c and d) A. haemolytics; (e and f) M. mesophilicum.

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TABLE 1. Numbers of bacterial cells attached to the inner surfaces of the tubes

Figure 4 shows the SEM images of the inner surface of the PVDF-coated tube incubated in the suspension of mixed flora for 24 to 96 h. A small number of rods adhered after 24 h, and sparse attachment of rods was observed even after 48 h. The number of attached rods increased by 96 h, but little aggregation of the cells was observed. Unlike with the polyurethane tube, adherence of filamentous rods was not seen with the PVDF-coated tube even after 96 h.

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FIG. 4. SEM images of the inner surface of the PVDF-coated tube incubated for 24 (a), 48 (b), or 96 h (c) in the mixed flora of the three bacteria identified. No mature biofilm formation was observed even after 96 h of incubation.

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All of the studies conducted so far reported no contamination of DUWLs by oral bacteria but did report contamination by bacteria present in the water supply. These include Sphingomonas paucimobilis, Methylobacterium mesophilicum, Xanthomonas maltophilia, Micrococcus luteus, Pseudomonas spp., Flavobacterium spp., Legionella spp., Bacillus spp., and Acinetobacter spp.; most of them are not strong pathogens (1, 2, 18, 22, 36, 37). In the present study, the heterotrophic bacteria Sphingomonas paucimobilis, Acinetobacter haemolytics, and Methylobacterium mesophilicum were identified to be the predominant bacteria in water and biofilm samples obtained from DUWLs used clinically for 2.5 years. These findings coincided with those of a previous study which reported the isolation of S. paucimobilis and M. mesophilicum from DUWLs used for approximately 4 years in Japanese clinics (1).

S. paucimobilis is known to be an opportunistic pathogen causing meningitis (7), septicemia (28), and bacteremia and peritonitis (19, 30) in immunocompromised hosts. M. mesophilicum is a pink-pigmented rod and was reported to cause bacteremia in a child with lymphoma (5). In the event of an opportunistic infection, the size of the bacterial population has a significant impact, as does the virulence of the bacteria. However, detailed information on the populations of bacteria isolated from contaminated DUWLs in the present study has not been clarified. Using R2A medium suitable for the growth of heterotrophic bacteria, we found that S. paucimobilis, A. haemolytics, and M. mesophilicum existed in a ratio of 18:1:1 in both water and biofilm samples, and S. paucimobilis showed the largest population among these three species. Increased risks of pyrogenic and septicemic complications have been reported when the level of contamination exceeds 10,000 bacteria/ml in dialysis fluids (11). Considering the total number of bacteria in DUWLs equipped with conventional polyurethane tubes (1, 8, 15, 22, 24, 34, 36, 38) and the population determined in this study, the amount of S. paucimobilis discharged can be calculated to be greater than 10,000 CFU/ml. Although no information is available on the definitive concentration of S. paucimobilis that causes symptomatic infection, more attention should be paid to reduce its outflow from DUWLs to eliminate the risk of opportunistic infections.

Many approaches to develop in vitro oral biofilm models, using specially developed equipment such as the modified Robbins device (12) or chemostat (13), are available. These devices are useful for in vitro investigations with the simulation of dental caries or periodontal diseases. However, less attention has been given to the biofilm-forming ability of heterotrophic bacteria, and in vitro biofilm models simulating bacterial contamination of DUWLs have not yet been established. In the present study, we attempted to produce a biofilm model of DUWLs by incubating the tube in a mixed suspension of three predominant bacteria, i.e., S. paucimobilis, A. haemolytics, and M. mesophilicum, prepared in the same ratio as that found in clinical specimens. We found that when the tube was incubated for 96 h in this mixed flora, a biofilm similar to that observed after 6 months of clinical use was produced. This setup provided a biofilm model by simple static culture without the need of a flowing bacterial suspension. In addition, by culturing in the presence of several types of bacteria, the processes of biofilm formation in DUWLs were also clarified. Until 48 h of incubation, the attachment of the rods was the major event, and a mature biofilm-like structure with overlaid filamentous rods was produced by incubating for a further 48 h. In the adherence assay using the single species, S. paucimobilis showed a stronger affinity to the surface of the polyurethane tube than A. haemolytics. These results suggested that in the course of biofilm formation in DUWLs, the rods of mainly S. paucimobilis initially adhered to the tube surface and proliferated with aggregation to form a dense bottom layer. Then, the filamentous rods of M. mesophilicum attached to this rod layer as a maturation process. Therefore, it is considered that the inhibition of initial attachment of the rods is crucial to prevent biofilm formation in DUWLs.

Using clinical tests, we have previously reported that contamination of DUWLs can be effectively reduced by using a PVDF-coated tube instead of a conventional polyurethane tube (38). A surface coated with PVDF has a low free energy (38), and PVDF inhibits the attachment of organic materials in a nonspecific manner similarly to other fluoridated surfaces (3, 31). In the present study, the use of the PVDF-coated tube inhibited the attachment of each bacterium identified from water and biofilm samples in DUWLs, resulting in the interruption of the biofilm-forming processes even when the incubation conditions were optimal to produce a mature biofilm on a conventional tube. On the surface of the PVDF-coated tube, the adherence of the rods up to 48 h was limited, and the attachment of sparse rods but no filaments was observed even after 96 h of incubation. Therefore, the PVDF-coated tube was able to disturb the initial step of the biofilm formation process, i.e., attachment of rods, and hamper the subsequent maturation process. These in vitro results confirmed the effectiveness of the PVDF-coated tube as shown in a previous clinical study (38). In addition, it has been reported that chloride, which was added to drinking water to inhibit bacterial growth, adsorbed to the polyurethane tube, and thereby the concentration of chlorine in the water was decreased (9). Less chloride adsorbs to fluoride-coated surface (9), and a PVDF-coated tube is further advantageous for prevention of bacterial growth in DUWLs.

The established in vitro model is useful to assess the longevity of the effectiveness of various methods for prevention of DUWL contamination in a short period with low cost. It may also be possible to investigate the detailed mechanisms of biofilm formation in DUWLs, including the analysis of the genes involved, by using this biofilm model.

 
This work was supported in part by Grant-in-Aid for Scientific Research no. 19791396 from the Japan Society for the Promotion of Science and by the 21st Century COE entitled "Origination of Frontier BioDentistry" at Osaka University Graduate School of Dentistry, supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan.

 
* Corresponding author. Mailing address: 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81 (0)6 6879 2928. Fax: 81 (0)6 6879 2929. E-mail: imazato{at}dent.osaka-u.ac.jp

Published ahead of print on 1 August 2008.

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Applied and Environmental Microbiology, October 2008, p. 5958-5964, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.00610-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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