Microbiological contamination of dental unit water systems in general practices from Barcelona (Spain)

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1 Microbiological contamination of dental unit water systems in general practices from Barcelona (Spain) R. Araujo* and N. Contreras Departament de Microbiologia, F. Biologia, Universitat de Barcelona, Av. Diagonal 645, Barcelona, Spain ( Abstract Dental Unit Water Systems (DUWS) can be a source of contamination to dentists and patients, especially those who are immunocompromised. Such organisms may originate from incoming water supplies and from oral cavities. The most common cause of dental unit water contamination is believed to be the formation and subsequent sloughing off of microbial biofilms from tubing within DUWS. There are no evidence-based guidelines currently available to dentists for the control of DUWS contamination in Europe. The aim of this study was to investigate the microbial load of water from DUWS, especially the presence of microbial pathogens which can be a source of cross-contamination between patients. Water and tube samples were obtained from 40 dental surgeries in Barcelona. The samples were taken from turbine and from air/water syringe. Twelve of these DUWS were fed with hard water from mains (with chlorine) and 28 dental units with bottle or tank deionized water. Microbial load was ranged from 0 to colonyforming units/ml. Legionella sp., Mycobacterium sp., Pseudomonas aeruginosa and Candida sp. were found in 4, 8, 6, and 2 different surgeries, respectively. Inclusive oral streptococci were found in 2 dental units indicating that there is a back siphonage in the systems analysed. Keywords Biofilms; dental unit; Legionella; Pseudomonas; total viable counts; water Introduction Studies made principally in the EU and in the UK demonstrate that the water delivered by dental units during routine dental practice is highly contaminated by numerous species of pathogenic and nonpathogenic microorganisms (Montebugnoli and Dolci, 2002; Walker et al., 2000; Atlas et al., 1995). These microorganisms enter into dental units delivered by incoming water or retracted up from the oral cavity of patients undergoing dental treatment, afterwards bacteria persist in dental unit waterlines growing as a multispecies biofilm on the inner surface and continuously supplying dental unit water system. In 1996 the American Dental Association (ADA) established a goal for dental water to contain no more than 200 colony forming units (CFU) per ml of heterotrophic bacteria by the year 2000 (Shearer, 1996). The bacterial numbers in DUWS reported by different authors fail ADA recommendations as well as current European Union potable-water guidelines on microbial load (>100 CFU/ml) (Walker et al., 2000). Moreover there are currently no rational evidence-based guidelines available to dentists for the control of DUWS contamination in the European Union. The purpose of this study was to determine, for the first time, existing patterns of microbial contamination in dental unit water systems (DUWS) in private dental clinics in the city of Barcelona. In the study have been included DUWS fed with tap water as well as DUWS fed with deionized water. The tap water in Barcelona is chlorinated and hard; for this reason many dentists use deionized water industrially produced from the tap water of the city. Quantification of heterotrophic bacteria and qualitative assessment of key pathogens, including Legionella, Mycobacterium, Candida, Pseudomonas, Escherichia coli and oral streptoccocci, in dental unit waterlines and tubing, provides useful information to compare Water Science and Technology: Water Supply Vol 4 No 2 pp 1 5 IWA Publishing

2 with current standards and to use this information to carry out microbiological risk assessments. This study participates with six other scientific groups from different European countries in a project of the EU to establish the microbial risk of DUWS in Europe. Materials and methods Survey of general dental practices Forty DUWS were randomly selected for this study in 19 private general dental practices in the Metropolitan Area of Barcelona. The selected units included 11 supplied by bottled water, 17 supplied by tank systems (all of them fed with deionized water) and 12 supplied by mains tap water (hard chlorinated water). 2R. Araujo and N. Contreras Sampling of DUWS Water samples were taken from two points in each device at approximately mid-morning: (i) the 3-in-1 syringe s distal outlet (the water line sample) and (ii) the air rotor water line (the air rotor water sample). Bottles for sampling contained sodium thiosulfate to remove any residual chlorine. A section of the water line tubing supplied to the 3-in-1 syringe for biofilm analysis (the biofilm sample) was taken from 11 dental units. All samples were transported in a cool box to the laboratory and tested within 3 h. Water samples were concentrated in the following way. One hundred millilitres of water was filtered through 0.45 µm pore size, 47 mm in diameter, acetate/nitrate cellulose membrane filters (Millipore, HAWG047S3). The membrane was placed in a screw-cap sterile container. Organisms were washed from the membrane by vortexing the container for 1 min in 10 ml of sterile phosphate-buffered saline (PBS). To obtain biofilm samples, approximately 5 cm of the tubing was cut off and the tubing section was then placed in a bottle containing enough PBS to cover the samples. Tubing was sectioned longitudinally and the surfaces were rinsed in nonflowing sterile PBS to removeplanktonic cells. Using a sterile spatula,the surface biofilm was scraped into 2 ml of sterile PBS containing 1 g of glass beads of 0.1 mm in diameter, and then vortexed. Viable counts of selected bacteria Total viable counts (TVC) enumerated onto Yeast Extract (YE) agar plates were used as the measure of total microbial contamination of the water flowing through the DUWS. Samples of appropriate dilutions of biofilm and water samples were plated onto different selective and nonselective agar media. Positive and negative controls were included in every batch. The media were: (i) Blood agar for oral anaerobes, incubated anaerobically at 37 C for up to 10 days; (ii) Mitis Salivarius agar for oral streptococci, incubated at 37 C for up to 10 days; (iii) YE agar for heterotrophic bacteria (Anonymous, 1998), incubated at 37 C for 3 days; (iv) CN supplement SR102 (Oxoid) for Pseudomonas aeruginosa (Anonymous, 1999), incubated at 37 C for 48 h; (v) MacConkey agar for enterobacteria, incubated at 37 C for 48 h; and (vi) Sabouraud chloramphenicol agar for Candida spp., incubated at 37 C for 3 days. For the enumeration of Legionella and Mycobacterium spp., aliquots of the samples were pre-treated either with heat (at 50 C in a water bath for 30 min) or with acid (0.9 ml of sample added to 0.1 ml of a 10 concentrated acid solution for 5 min) and then plated immediately. Portions of untreated and treated samples were plated onto GVPC agar for the enumeration of Legionella spp. (Atlas et al., 1995), and the plates were incubated aerobically at 37 C for up to 10 days. Colonies morphologically typical of Legionella spp. were picked in BCYE without L-cysteine. The serogroup of those colonies that did not growth without L-cysteine was determined using a latex agglutination kit (Oxoid). Similar aliquots were dispensed onto Middlebrook agar plus OADC 7H10 for Mycobacterium spp.

3 (Middlebrook and Cohn, 1958), incubated aerobically at 37 C for up to 30 days. Representative colonies were assessed using the Ziehl-Neelsen technique for the detection of acid-fast bacilli and examined microscopically. Detection of blood The Hemastix Reagent Strips by Bayer Diagnostics was used for testing the presence of blood in water samples. Blood was used as an indicator of cross infection by patients serum. Statistical analysis Statistical analyses were carried out using Excel (Microsoft Office) and SPSS for Windows (The Statistical Package for Social Science; SPSS Inc., Chicago, Illinois). Bacterial loads in different types of water (deionized and mains tap water) were compared using an ANOVA test on log-transformed total viable counts. To log transform the data it was added one to all the results to avoid zero values. Biofilm and planktonic counts from the same surgeries were also analysed for correlation using the nonparametric Kendall rank correlation analysis. Statistical significance was assumed at a p value of <0.05. R. Araujo and N. Contreras Results and discussion Total viable counts in water samples Figure 1 shows the results of heterotrophic bacteria in water samples from DUWS in Barcelona randomly sampled along one year. The data are summarised in Table :1 AR TAP WATER DEIONIZED WATER TVC (CFU/ml) Sample number Figure 1 Total viable counts on yeast extract. Results of water samples from DUWS in Barcelona (3:1 = water sample from the 3-in-1 syringe distal outlet; AR = water sample from the air-rotor water line) Table 1 Comparison of viable counts from the DUWS water samples Sample n Viable count (CFU ml 1 ) from water samples taken from: Water lines Air rotor water lines Geometric Minimum Maximum Geometric Minimum Maximum mean mrsn Water type Tap water Deionized water TOTAL

4 4R. Araujo and N. Contreras The viable counts from water samples were low, either considering water lines or air rotor water lines and either considering the origin of water, tap water or deionized water. This idea is based on the fact that only 17 out of 80 samples were superior to 200 CFU/ml (ADA recommendations for DUWS water quality), and from these only 6 samples exceeded 1000 CFU/ml. The geometric mean of water lines was 28 CFU/ml and 45 CFU/ml for the air rotor water lines. These numbers were much lower than those published in the literature (Montebugnoli and Dolci, 2002; Walker et al., 2000; Meiller et al., 2000; Pankhurst and Johnson, 1998; Barbeau et al., 1996). However, it was difficult to compare the data of this study with many others published before, because the total viable counts could be measured using different parameters as media, incubation temperature, incubation time and even water sample could be directly tested or concentrated as we did. All these differences had to be avoided and the method should be standardised to compare the data of different DUWS. The statistical analysis of these numbers, using an ANOVA test, indicated that the differences between water lines and air rotor water lines were not significant. In addition the differences between water type (tap water and deionized water) were not significant, neither in water lines nor in air rotor water lines. Total viable counts in biofilm samples Table 2 shows the results of heterotrophic bacteria from the DUWS biofilm samples compared with bacteria in the same DUWS water lines. The results of viable counts from biofilm samples showed low numbers. Even so, the numbers of bacteria obtained from biofilm tubing were two logs higher than the number of bacteria obtained from water lines. The numbers of bacteria in biofilms were significantly associated with the numbers in the corresponding planktonic phase (Kendall rank correlation = 0.852; p = 0.001). Presence of pathogens in DUWS from Barcelona Table 3 shows the distribution of pathogens found in the DUWS water samples. Fifty per cent of DUWS (20/40) presented potential pathogens for human health. The pathogens were isolated from 14 3-in-1 syringes and 13 air rotor water lines out of 40 DUWS, and from 3 biofilms out of 11 biofilm samples. Presence of blood in water samples from DUWS obtained in Barcelona The water line and the air rotor water line samples were negative for the Hemastix test. Table 2 Comparison of viable counts from DUWS water samples with biofilm samples. Sample n Viable count Geometric mean Minimum Maximum Water lines (CFU ml 1 ) Biofilm (CFU cm 2 ) Table 3 Medically important bacteria detected in water samples from dental units analysed in Barcelona n = number of Streptococci Enterobacteria Escherichia Pseudomonas Legionella Mycobacterium Candida dental units aeruginosa spp. spp. spp. DUWS supplied Tap water 8% 0% 0% 17% 17% 42% 0% (n = 12) (1/12) (0/12) (0/12) (2/12) (2/12) (5/12) (0/12) Deionized water 4% 0% 0% 18% 11% 4% 7% (n = 28) (1/28) (0/28) (0/28) (5/28) (3/28) (1/28) (2/28)

5 Conclusions DUWS are colonised by bacteria derived from incoming water, either mains water or independent bottle/tank water systems, and, to a lesser extent, from patients mouths. Although counts of CFU/ml are common in the literature, the bacterial numbers reported here are lower and they could be considered in the most part of cases within the recommended EU drinking water limits and the ADA recommendations (Walker et al., 2000). Notwithstanding low total numbers of viable counts, the analysed DUWS from the area of Barcelona presented pathogens. Most of these opportunistic and true human pathogens could be considered from water origin except for oral streptococci, which would have an oral origin, indicating a back siphonage in those two DUWS where they were isolated. One of the equipment had more than 10 years and did not have anti-retrieval valve. The presence of these microorganisms may be a potential source of infection for both practice staff (by inhalation) and patients (by contact, ingestion or inhalation). In conclusion, a microbiological control applying standardised methods and regular disinfection are need to ensure a high microbiological quality of DUWS and a low health risk either for patients as well as for staff. R. Araujo and N. Contreras Acknowledgements This study was carried out with financial support from the Commission of the European Communities, specific RTD programme Quality of Life and Management of Living Resources, Key Action 4, Environment and Health. It does not necessarily reflect its views and in no way anticipates the Commission s future policy in this area. We thank the dentists for their colaboration. We also acknowledge the support of the Generalitat de Catalunya (001 SGR00099) and the Centre de Referència en Biotecnologia (CeRBa). References Anonymous (1998). PrEN ISO 6222: E. Water quality Enumeration of culturable micro-organisms. Colony count by inoculation in a nutrient agar culture medium. Anonymous (1999). PrEN 12780: E. Water quality Detection and enumeration of Pseudomonas aeruginosa by membrane filtration. Atlas, R.M., Williams, J.F. and Huntington, M.K. (1995). Legionella contamination of dental-unit waters. Appl. Environ. Microbiol., 61, Barbeau, J., Tanguay, R., Faucher, E., Avezard, C., Trudel, L., Côté, L. and Prévost, A.P. (1996). Multiparametric analysis of waterline contamination in dental units. Appl. Environ. Microbiol., 62(11), Meiller, T.F., Kelley, J.I., Baqui, A.A. and DePaola, L.G. (2000). Disinfection of dental unit waterlines with an oral antiseptic. Journal of Clinical Dentistry, 11(1), Middlebrook, G., and M. Cohn, M. (1958). Bacteriology of tuberculosis: laboratory methods. Am. J. Public Health, 48, Montebugnoli, L. and Dolci, G. (2002). A new chemical formulation for control of dental unit water line contamination: An in vitro and clinical study. BMC Oral Health, 2, 1 Pankhurst, C.L. and Johnson, N.W. (1998). Microbial contamination of dental unit waterlines: the scientific argument. International Dental Journal, 48(4), Shearer, B.G. (1996). Biofilm and the dental office. JADA, 127, Walker, J.T., Bradshaw, D.J, Bennett, A.M., Fulford, M.R.,Martin, M.V. and Marsh, P.D. (2000). Microbial biofilm formation and contamination of dental unit water systems in general dental practice. Appl. Environ. Microbiol., 66(8),