samples
Sample | Viable count (CFU·ml−1) from water samples taken from:
| n |
---|
Water lines
| Air rotor water lines
|
---|
Geometric mean | Minimum | Maximum | Geometric mean | Minimum | Maximum |
---|
Water type |
Soft | 2,590 | 7 | 31,000 | 1,224 | 0 | 40,000 | 11 |
Hard | 3,290 | 32 | 64,000 | 5,063 | 0 | 95,000 | 33 |
Deionized | 1,740 | 68 | 40,000 | 2,699 | 55 | 30,000 | 9 |
Distilled | 5,970 | 4,400 | 8,100 | 2,013 | 1,350 | 3,000 | 2 |
Supply type |
Tank fed | 1,550 | 480 | 3,700 | 1,881 | 360 | 7,400 | 3 |
Bottle fed | 3,583 | 7 | 36,000 | 1,802 | 0 | 95,000 | 32 |
Main fed | 2,217 | 32 | 64,000 | 4,914 | 42 | 40,000 | 20 |
Total | 2,874 | 7 | 64,000 | 3,325 | 0 | 95,000 | 55 |
Surgeries 13, 14, 15, 16, and 43 had units that were reportedly sanitized (Fig. 1). Significantly greater numbers of bacteria were found in the water phase of units treated with disinfectant than in the untreated units (ANOVA, P = 0.048). A geometric mean of 1.6 × 104 CFU · ml−1 (range, 6.0 × 103 CFU · ml−1 to 3.6 × 104 CFU · ml−1) was recovered from treated units' water, compared with a geometric mean of 2.8 × 103 CFU · ml−1 (range, 7.0 CFU · ml−1 to 6.4 × 104 CFU · ml−1) from the untreated systems.
| FIG. 1 Total viable counts (TVC) of water samples from DUWS in different dental surgeries. |
Notable bacterial species isolated from the water lines of the DUWS were slow-growing Mycobacterium spp. from surgeries 21, 22, and 44, Fusobacterium spp. from surgery 19, and fluorescent Pseudomonas spp. from surgeries 16, 32, 35, 47, and 48.
(ii) Air rotor water line microbial contamination. Similar numbers of bacteria (geometric mean, 3.3 × 103 CFU · ml−1; range, not detectable to 9.5 × 104 CFU · ml−1) (Table 1) were found in the water from the air rotor water line and the water line. The numbers of bacteria in air rotor line water were significantly different for different DUWS types (two-way ANOVA, P = 0.04), with the numbers of microorganisms from main-fed units being significantly greater than from the bottle-supplied units (P < 0.05). Higher numbers of bacteria were recovered from hard water than from deionized, distilled, or soft water (Table 1), though these differences did not quite reach significance (two-way ANOVA, P = 0.055).
The following specific microorganisms were detected from air rotor water line samples: Candida spp. from surgery 21 and fluorescent Pseudomonas spp. from surgeries 7, 16, 33, 35, 44, 47, and 49.
(iii) Water line biofilm. A geometric mean of 9.2 × 102 CFU · cm−2 (range, not detectable to 6.4 × 104 CFU · cm−2) was recovered from the water line biofilm of the DUWS (Table 2). More bacteria were recovered from the distilled-water units than from the hard-, soft-, or deionized-water units; similarly, more were recovered from the main-fed units than from those supplied with either bottled or tank water (Table 2). However, the differences between the numbers of CFU recovered from biofilms in different DUWS types and from units supplied with different types of water were not significant (two-way ANOVA, P = 0.89 and 0.91, respectively). The numbers of bacteria in biofilms were significantly associated with the numbers in the corresponding planktonic phase (Kendall rank correlation; τ = 0.31, P = 0.04).
TABLE 2
Comparison of viable counts from the DUWS biofilmsamples
Sample | Viable count (CFU·cm−2) from biofilm samples taken from:
| n |
---|
Water lines
| Air lines
|
---|
Geometric mean | Minimum | Maximum | Geometric mean | Minimum | Maximum |
---|
Water source |
Soft | 892 | 2 | 42,000 | 33 | 0 | 30,000 | 10 |
Hard | 1,103 | 0 | 64,000 | 85 | 0 | 3,200 | 27 |
Deionized | 411 | 5 | 500 | 10 | 0 | 1,600 | 8 |
Distilled | 2,200 | 1,100 | 4,400 | 259 | 12 | 5,600 | 2 |
Supply type |
Tank fed | 1,268 | 40 | 8,800 | 33 | 0 | 2,000 | 3 |
Bottle fed | 717 | 2 | 42,000 | 26 | 0 | 30,000 | 29 |
Main fed | 1,383 | 0 | 64,000 | 199 | 0 | 30,000 | 15 |
Total | 917 | 0 | 64,000 | 51 | 0 | 30,000 | 47 |
The numbers of bacteria recovered from the biofilms in the “sanitized” units (surgeries 13, 14, 15, 16, and 43) were significantly lower than the numbers recovered from the untreated surgeries (ANOVA, P = 0.04). Mycobacterium spp. were recovered from the biofilms of surgeries 4 and 23.
(iv) Air line biofilm. Significantly lower numbers of bacteria were found in the DUWS air lines than in the water lines. A geometric mean of 5.1 × 101 CFU · cm−2 (range, not detectable to 3.0 × 104 CFU · cm−2) were recovered from the air lines, compared with a geometric mean of 9.2 × 102 CFU · cm−2 from the water line (paired t test, P 0.0001) (Table 2).
However, a number of medically important bacteria were detected in air line biofilm samples: L. pneumophila in surgery 24, Candida spp. in surgery 30, and Lactobacillus spp. and Streptococcus spp. in surgery 19.
(v) Percentage of biofilm coverage in water and air lines. The average coverage of water line surfaces was 43% (range, 0.01 to 94%) (Table 3). Significantly less coverage was observed on the DUWS units supplied with soft water than on those supplied with hard, distilled, or deionized water (Kruskal-Wallis test, P = 0.035). The highest coverage was found on those units supplied with deionized water, followed by those using hard, distilled, and soft water (Table 3). There was no significant difference among units with different types of water supply, although it was observed that a higher percentage of coverage was recorded in the units supplied by water mains than in bottle- or tank-fed systems.
TABLE 3
Comparison of percentage of coverage from the DUWS biofilmsamples
Sample | % Coverage from biofilm samples taken from:
| n |
---|
Water lines
| Air lines
|
---|
Avg | Minimum | Maximum | Avg | Minimum | Maximum |
---|
Water source |
Soft | 21 | 0.1 | 62 | 0.0 | 0.1 | 0.1 | 10 |
Hard | 48 | 0.01 | 94 | 8.8 | 0.1 | 74 | 27 |
Deionized | 54 | 0.5 | 94 | 1.9 | 0.1 | 6.4 | 8 |
Distilled | 44 | 5 | 83 | 0.1 | 0.1 | 0.1 | 2 |
Supply type |
Tank fed | 33 | 25 | 50 | 3.5 | 0.1 | 8 | 3 |
Bottle fed | 41 | 10 | 94 | 2.9 | 0.1 | 64 | 29 |
Main fed | 50 | 0.1 | 92 | 10 | 0.1 | 74 | 15 |
Total | 43 | 0.01 | 94 | 5.2 | 0.1 | 74 | 47 |
The results of the percent coverage analysis of the air line demonstrated that the average coverage was 5.2% (range, 0.1 to 74%) (Table 3). A higher degree of biofouling was observed on the tubing surfaces of those DUWS that were supplied with hard water than in those supplied by deionized, distilled, or soft water (Table 3). This difference was statistically significant (Kruskal-Wallis test, P = 0.04). In terms of the type of water supply, more extensive biofouling coverage was observed on those units supplied by mains water than on those supplied by tanks or bottles, although this difference was not significant (Kruskal-Wallis test, P = 0.35).
(vi) Assessment of viability. Due to fluorescein binding to the DUWS tubing surfaces, viable cells could not be discriminated against the background; therefore, biofilm counts could not be assessed in situ using the BacLight technique. Resuspended biofilm samples were analyzed and a mean of 68% of the total visible bacterial population was determined to be viable. A mean of 5% (three determinations) of this “viable by BacLight” fraction produced viable colonies on agar plates.
(vii) Detection of viruses. None of the samples taken from the DUWS were positive for HBsAg.
DISCUSSION Water supplied by 95% of general dental practice DUWS units failed current European Union potable-water guidelines on microbial load (i.e., loads were >100 CFU · ml−1) (2), and 83% failed American Dental Association recommendations for DUWS water quality (<200 CFU · ml−1) (1). The bacterial numbers reported here were comparable to those found in a number of other studies (6, 28) and lower than some (19, 35). These values probably underestimate the true microbial load to which a patient is exposed, since we also demonstrated that only 3% of the microscopically visible bacteria produced colonies on agar plates. Other bacteria may be either in a temporarily nonculturable state or may represent the large fraction of the microflora from many natural habitats which remain “as yet uncultured” (as discussed in the review by Barer and Harwood [7]). The most common pathogens detected were fluorescent Pseudomonas spp. (16% of samples were positive). L. pneumophila was isolated on only one occasion, which was far less frequent than reported in previous studies in a dental hospital and in a mixture of institutional and private practices (25 and 6% isolation frequencies, respectively) (4, 8). Larger water distribution systems are known frequently to harbor this organism (C. L. Bartlett, J. B. Kurtz, J. G. Hutchison, G. C. Turner, and A. E. Wright, Letter, Lancet ii:1315, 1983). We detected Mycobacterium spp. in ca. 5% of surgeries, a lower detection rate than that reported by a previous study (30). These isolates were not identified, so their pathogenic potential is unknown, although several non-Mycobacterium tuberculosis, non-Mycobacterium avium species of mycobacteria are associated with a variety of infections in humans (18). Presumptive oral streptococci were identified in 7% of DUWS water samples, suggesting the failure of antiretraction devices in these systems and thus raising the possibility of cross-infection between successive patients. No HBsAg was detected in any sample. Some dentists perceive that certain types of DUWS may be less prone to microbial contamination than others. We found no significant differences between different DUWS systems, regardless of whether these systems were main, bottle, or header tank fed or whether the water supplied to them was hard, soft, deionized, or distilled. Thus, no DUWS can be considered superior in microbiological terms to any other or can be called microbiologically “clean.” Water from air rotor lines and from 3-in-1 handpieces was contaminated to a similar degree, emphasizing that air rotors should not be used as an aid in any dental surgical procedures. The significant correlation between the numbers of bacteria recovered from biofilms and from water samples from the same units suggests that the biofilms may seed the water with bacteria and vice versa. Strategies developed for the control of DUWS contamination must eliminate both the biofilms and the waterborne bacteria in these systems (23, 31, 36). In addition, although significantly lower levels of biofilm contamination were found in the five units reported to have been recently decontaminated, more bacteria were recovered from the water phase in these systems. Decontamination with detergents or with inorganic acids (including hypochlorous acid) could increase the risk of release of organisms from biofilms and thus increase the numbers of bacteria in the water phase (J. S. Colborne, P. J. Dennis, J. V. Lee, and M. R. Bailey, Letter, Lancet i:684, 1987). Inadequate decontamination regimens may thus increase the hazards associated with DUWS water. In conclusion, improved, evidence-based practical methods for controlling the microbial contamination of DUWS are urgently needed. This is particularly important in view of the increasing numbers of medically compromised and immunocompromised patients receiving regular dental treatment. |
ACKNOWLEDGMENTS This investigation was supported by the Primary Dental Care R & D Programme and National Research Register Research Grant PDC97-213 from the NHS Executive, North West, Warrington, United Kingdom. We thank the staff and patients of the participating practices for their cooperation and support during the study. |
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REFERENCES 1. Anonymous. ADA statement on dental unit waterlines. J Am Dent Assoc. 1996;127:185–186. 2. Anonymous. Council directive 98/83/EC of 3 November 1998 on the quality of water intended for human consumption. Off J Eur Community. 1998;L330:32–54. 3. Anonymous. The microbiology of water 1994. Part 1—drinking water. London, United Kingdom: Her Majesty's Stationery Office; 1994. 4. Atlas, R M; Williams, J F; Huntington, M K. Legionella contamination of dental-unit waters. Appl Environ Microbiol. 1995;61:1208–1213. [PubMed]5. Barbeau, J; Gauthier, C; Payment, P. Biofilms, infectious agents, and dental unit waterlines: a review. Can J Microbiol. 1998;44:1019–1028. [PubMed]6. Barbeau, J; Tanguay, R; Faucher, E; Avezard, C; Trudel, L; Côté, L; Prévost, A P. Multiparametric analysis of waterline contamination in dental units. Appl Environ Microbiol. 1996;62:3954–3959. [PubMed]7. Barer, M R; Harwood, C R. Bacterial viability and culturability. Adv Microb Physiol. 1999;41:93–137. [PubMed]8. Challacombe, S J; Fernandes, L L. Detecting Legionella pneumophila in water systems: a comparison of various dental units. J Am Dent Assoc. 1995;126:603–608. [PubMed]9. Costerton, J W; Cheng, K J; Geesey, G G; Ladd, T I; Nickel, J C; Dasgupta, M; Marrie, T J. Bacterial biofilms in nature and disease. Annu Rev Microbiol. 1987;41:435–464. [PubMed]10. Daeschel, M A; McGuire, J. Interrelationships between protein surface adsorption and bacterial adhesion. Biotechnol Genet Eng Rev. 1998;15:413–438. [PubMed]11. Dennis, P J; Bartlett, C L R; Wright, A E. Comparison of isolation methods for Legionella spp. In: Thornsberry C, Balows A, Feeley J C, Jakubowski W. , editors; Thornsberry C, Balows A, Feeley J C, Jakubowski W. , editors. Legionella. Washington, D.C.: American Society for Microbiology; 1984. pp. 294–296. 12. Dibdin, G H; Assinder, S J; Nichols, W W; Lambert, P A. Mathematical model of beta-lactam penetration into a biofilm of Pseudomonas aeruginosa while undergoing simultaneous inactivation by released beta-lactamases. J Antimicrob Chemother. 1996;38:757–769. [PubMed]13. Donoghue, H D; Overend, E; Stanford, J L. A longitudinal study of environmental mycobacteria on a farm in south-west England. J Appl Microbiol. 1997;82:57–67. [PubMed]14. Douglas, C W I; van Noort, R. Control of bacteria in dental water supplies. Br Dent J. 1993;174:167–174. [PubMed]15. Farmer, J J, III; Davis, B R; Hickman-Brenner, F W; McWhorter, A; Huntley-Carter, G P; Asbury, M A; Riddle, C; Wathen-Grady, H G; Elias, C; Fanning, G R; Steigerwalt, A G; O'Hara, C M; Morris, G K; Smith, P B; Brenner, D J. Biochemical identification of new species and biogroups of Enterobacteriaceae isolated from clinical specimens. J Clin Microbiol. 1985;21:46–76. [PubMed]16. Fayle, S A; Pollard, M A. Decontamination of dental unit water systems: a review of current recommendations. Br Dent J. 1996;181:369–372. [PubMed]17. Gilbert, P; Das, J; Foley, I. Biofilm susceptibility to antimicrobials. Adv Dent Res. 1997;11:160–167. [PubMed]18. Goslee, S; Wolinsky, E. Water as a source of potentially pathogenic mycobacteria. Am Rev Respir Dis. 1976;113:287–292. [PubMed]19. Karpay, R I; Plamondon, T J; Mills, S E. Comparison of methods to enumerate bacteria in dental unit water lines. Curr Microbiol. 1999;38:132–134. [PubMed]20. Korber, D R; Choi, A; Wolfaardt, G M; Caldwell, D E. Bacterial plasmolysis as a physical indicator of viability. Appl Environ Microbiol. 1996;62:3939–3947. [PubMed]21. Martin, M V. The significance of the bacterial contamination of dental unit water systems. Br Dent J. 1987;163:152–154. [PubMed]22. Mayo, J A; Oertling, K M; Andrieu, S C. Bacterial biofilm: a source of contamination in dental air-water syringes. Clin Prev Dent. 1990;12:13–20. 23. Meiller, T F; Depaola, L G; Kelley, J I; Baqui, A A; Turng, B F; Falkler, W A. Dental unit waterlines: biofilms, disinfection and recurrence. J Am Dent Assoc. 1999;130:65–72. [PubMed]24. Middlebrook, G; Cohn, M. Bacteriology of tuberculosis: laboratory methods. Am J Public Health. 1958;48:844–853. [PubMed]25. Nichols, W W; Evans, M J; Slack, M P; Walmsley, H L. The penetration of antibiotics into aggregates of mucoid and non-mucoid Pseudomonas aeruginosa. J Gen Microbiol. 1989;135:1291–1303. [PubMed]26. Pagan, E F. Isolation of human pathogenic fungi from river water. Ph.D. dissertation. Columbus: Ohio State University; 1970. 27. Palleroni, N J. Introduction to the family Pseudomonadaceae. In: Starr M P, Stolp H, Trüper H G, Balows A, Schlegel H G. , editors; Starr M P, Stolp H, Trüper H G, Balows A, Schlegel H G. , editors. The prokaryotes: a handbook on habitats, isolation, and identification of bacteria. I. New York, N.Y: Springer-Verlag; 1981. pp. 655–665. 28. Pankhurst, C L; Philpott-Howard, J N; Hewitt, J H; Casewell, M W. The efficacy of chlorination and filtration in the control and eradication of Legionella from dental chair water systems. J Hosp Infect. 1990;16:9–18. [PubMed]29. Reasoner, D J; Geldreich, E E. A new medium for the enumeration and subculture of bacteria from potable water. Appl Environ Microbiol. 1985;49:1–7. [PubMed]30. Schulze-Robbecke, R; Feldmann, C; Fischeder, R; Janning, B; Exner, M; Wahl, G. Dental units: an environmental study of sources of potentially pathogenic mycobacteria. Tuber Lung Dis. 1995;76:318–323. [PubMed]31. Smith, A J; Hood, J; Bagg, J; Burke, F T. Water, water everywhere but not a drop to drink? Br Dent J. 1999;186:12–14. [PubMed]32. Walker, J T; Mackerness, C W; Mallon, D; Makin, T; Williets, T; Keevil, C W. Control of Legionella pneumophila in a hospital water system by chlorine dioxide. J Ind Microbiol. 1995;15:384–390. [PubMed]33. Walker, J T; Roberts, A D G; Lucas, V J; Roper, M M; Brown, R. Quantitative assessment of biocide control of biofilms and Legionella using total viable counts, fluorescent microscopy and image analysis. Methods Enzymol. 1999;310:629–637. [PubMed]34. Williams, H N; Johnson, A; Kelley, J I; Baer, M L; King, T S; Mitchell, B; Hasler, J F. Bacterial contamination of the water supply in newly installed dental units. Quintessence Int. 1995;26:331–337. [PubMed]35. Williams, H N; Kelley, J; Folineo, D; Williams, G C; Hawley, C L; Sibiski, J. Assessing microbial contamination in clean water dental units and compliance with disinfection protocol. J Am Dent Assoc. 1994;125:1205–1211. [PubMed]36. Williams, J F; Johnston, A M; Johnson, B; Huntington, M K; Mackenzie, C D. Microbial contamination of dental unit waterlines: prevalence, intensity and microbiological characteristics. J Am Dent Assoc. 1993;124:59–65. 37. Williams, J F; Molinari, J A; Andrews, N. Microbial contamination of dental unit waterlines: origins and characteristics. Compend Contin Educ Dent. 1996;17:538–542. [PubMed]38. Witt, S; Hart, P. Cross infection hazards associated with the use of pumice in dental laboratories. J Dent. 1990;18:281–283. [PubMed] |
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