Evaluation of bacterial contamination of dental unit waterlines and use of a newly designed measurement device to assess retraction of a dental chair unit

Abstract

Introduction: Dental unit waterline (DUWL) output water is delivered through instruments of a dental chair unit (DCU) to irrigate and cool teeth. However, these waterlines can be heavily contaminated with bacteria. Aim: The purpose of the present study was to assess retraction and investigate the contamination level and prevalence of bacteria in DUWL output water. Methods: Fifty-eight DCUs were randomly selected from 30 hospitals in 10 districts of Tianjin, one of the four special municipalities of China. A unique sampling connector was used in place of the dental handpiece to collect water samples. Evaluation of retraction was accomplished using a retraction measurement device designed in accordance with the International Standard ISO 7494-2:2015(E). Results: A total of 263 water samples were collected, and the highest concentration of bacteria [1.8 × 10⁶ colony-forming units (CFU)/mL] was found in the handpiece group. Thirty (51.72%) water samples in the handpiece group and 21 (36.21%) in the air/water syringe groups were cultured, yielding colony counts of > 500 CFU/mL. Potential infectious agents, such as Bacillus cereus, Kocuria kristinae and Pseudomonas fluorescens, were isolated from the water samples. Thirty (51.72%) DCUs failed the retraction evaluation. There was a significant, positive correlation (P < 0.05) between the concentration of bacteria in the water sample and the retracted volume. Conclusion: It is of paramount importance to increase compliance with the standards for controlling DUWL contamination. Routine microbial monitoring and evaluation of retraction are necessary to provide high-quality water for use in dental treatment.

INTRODUCTION

The dental chair unit (DCU) is one of the most essential and necessary pieces of equipment used in the routine practice of dentistry. The functionality of the DCU has developed rapidly over the past 40 years. At present, the DCU provides all types of services, including water, air, electricity and suction, and a variety of instruments are attached to deliver dental care. Dental unit waterline (DUWL) output water is delivered through dental instruments (e.g. dental handpieces and three-way air/water syringes) in the DCU to irrigate and cool teeth, and these waterlines can be heavily contaminated with numerous species of pathogenic and non-pathogenic microorganisms1., 2., 3., 4.. Researchers first identified this problem in the 1960s, and it has remained significant to date⁵. Many studies have shown that the bacterial count can range from 10⁴ to 10⁶ colony-forming units per milliliter (CFU/mL)3., 4., 6., 7., 8., 9., 10., 11., which represents a potential risk of infection for staff and patients, especially medically compromised or immunocompromised patients12., 13.. Microbial contamination of DUWL output water can be caused by a variety of factors, including narrow-bore waterlines, water stagnation, anti-retraction valve failure, the presence of water heaters and variations in the water supply or type of water, among others4., 13., 14., 15., 16., 17., 18., 19.. These factors are conducive to biofilm formation. Once the biofilm is formed, it serves as a continuous reservoir of bacteria in the DUWLs¹³. Microorganisms in the biofilm may come from the environment or from humans. Gram-negative aerobic heterotrophic species have low pathogenicity and are the most common environmental bacteria found in DUWL output water. In contrast, oral and skin bacteria have higher pathogenicity (e.g. Staphylococcus aureus) and a higher potential for transmission to staff and patients. A recent fatal case of pneumonia caused by Legionella was reported in an 82-year-old Italian woman²⁰. The presence of human-derived bacteria is most likely to be attributed to the retraction of oral fluids into DUWLs during dental treatment¹⁴. Although a considerable amount of research can be found in the literature, most studies are focused on bacterial counts, microbial typing investigations or control methods to improve DUWL output water. However, if the anti-retraction valve of the DCU fails to prevent retraction when the handpiece stops running, oral fluids or contaminated water can be retracted back into the DUWLs, promoting contamination of the waterlines. In the present study, we designed a retraction measurement device (assembled with a water sampling connector, transparent hose and a millimeter measuring scale) to measure the backflow volume when the handpiece stops running. This retraction measurement device has the capacity to monitor the efficacy of the anti-retraction valve of the DCU (note that this feature is not available in the measuring kits currently in use). We also collected water samples from DUWLs to compare the concentration of bacteria in the water with the retracted volume.

MATERIAL AND METHODS

Ethical approvals

The Institutional Review Board of Tianjin Centers for Disease Control and Prevention approved this study design.

Dental units

Fifty-eight DCUs were randomly selected from 30 hospitals in 10 districts of Tianjin. There were a total of approximately 991 DCUs in 18 districts, including 12 in rural counties. Fifty-two of the 58 DCUs were located in a stomatology department in a public comprehensive hospital; the others were located in public dental clinics. The average age of the DCUs was 4.61 ± 0.48 years (range, 0.5–15 years). Of the 58 DCUs, 77.59% (n = 45) used domestic water and 53.45% (n = 31) used water reservoir bottles to provide water to the DUWLs.

Collection of water samples

Water samples were collected at the beginning of the working day. At every sampling occasion, one of the investigators randomly selected one or two DCUs and removed the handpiece aseptically whilst wearing sterile gloves, a single-use mask and a gown. Before obtaining the samples, the water was flushed for 2 minutes. Water samples (20 mL) were obtained with a sterilised sampling connector (Figure 1c). The four-hole connector was connected to the waterlines (Figure 1b) in place of the handpiece (Figure 1a), and it only allowed water flow from the lines (Figure 2). In addition, water samples from air/water syringes, cup filler outlets and water taps were also collected from each DCU. Water samples were enclosed in sterile, airtight test tubes. Considering the scientific nature of the study and the need for confidentiality, the samples were double-coded. The test tubes were stored at 4 °C in a sampling box, transported to the laboratory and processed within 2 hours.

Figure 1.

(a) High-speed handpiece, (b) four-hole connection for handpiece and (c) sampling connector. The dimensions and thread characteristics of the sampling connector were determined according to those of the hose connectors of air-driven dental handpieces (ISO 9168-2009).

Figure 2.

Water samples (20 mL) were obtained from a high-speed handpiece with a sterilised sampling connector (Figure 1) which can be connected to the waterlines in place of the instruments.

Retraction evaluation

The retraction test was performed in accordance with Part 2 (air, water, suction and wastewater systems) of ISO 7494. A retraction measurement device (Figure 3) (assembled with a water sampling connector, a transparent hose with an inner diameter of 1.5 mm and a millimeter measuring scale) was connected to the waterlines in place of the high-speed handpiece. The volume of retraction in the transparent hose was measured. The foot control of the DCU was started up for a few seconds until the water came out freely from the open ends of the transparent hoses; subsequently, it was stopped. The distance between the meniscus of water remaining inside the hoses and the open end of the same hoses was evaluated when the hoses were held vertically, with the open hose end extending upward. The tests were repeated three times for each DCU, and the mean values were calculated. The volume of water retraction was calculated using the formula πd²l/4, where d is the inner diameter of the transparent hose, l is the mean retraction length (as determined by the retraction measurement device) and π is the constant pi. The volume of water retraction should not exceed 40 μL according to the American Dental Association/American National Standard (ADA/ANSI) specification #47 or ISO 7494.

Figure 3.

Retraction detector. A water sampling connector, a transparent hose with an inner diameter of 1.5 mm and a millimeter measuring scale were connected to the waterlines in place of the high-speed handpiece.

Processing of water samples

Water samples were tested and analysed using the methods recommended in the Standard Examination Methods for Drinking Water (GB/T 5750.6-2006)²¹: 1-mL aliquots of water were dispensed, mixed with nutrient agar and incubated at 37 °C for 2 days under aerobic conditions. After incubation, the colonies were counted, and the number of CFU/mL was calculated. Heterotrophic plate counts (HPCs) were the bacteriological indicator used to assess the general microbiological quality of the drinking water. The numbers were then compared with the threshold values established by the Standards for Drinking Water Quality of China (100 CFU/mL)²², the recommendations of the American Dental Association (ADA), and the guidelines of the Centers for Disease Control and Prevention (CDC) (500 CFU/mL)²³. The VITEK 2 (Vitek2 compack30; Biomerieux, USA) analyser was used to identify gram-negative and gram-positive aerobic bacteria.

Statistical analysis

The means and standard deviations of the HPCs were calculated. The values of microbial loads were converted into log₁₀ values to normalise the distributions for correlation analysis. Statistical analyses were performed using spss version 18.0 (IBM, Armonk, NY, USA), and P < 0.05 was considered a significant difference.

RESULTS

The microbial contamination values of water samples from five sampling sites are shown in Table 1.

Table 1.

Microbial contamination of water samples from five sampling sites

	n	Mean	SD	Median	Min.	Max.	25th percentile	75th percentile	Percentage above threshold value (100 CFU/mL)	Percentage above threshold value (500 CFU/mL)
Handpiece	58	81,619	279,944	540	2	1.8 × 106	48.25	12,000	65.52	51.72
Air/water syringes	58	52,371	244,102	99	0	1.7 × 106	8.50	1875	50.00	36.21
Reservoir bottles	31	6749	28,763	43	0	1.6 × 105	2.00	820	35.48	25.81
Cup filler outlet	58	5049	23,936	195	0	1.4 × 105	29.50	865	58.52	38.24
Tap water	58	2526	12,656	37	0	8.1 × 104	1.00	110	25.49	5.88

SD, standard deviation.

The highest concentration was found in the handpiece (1.8 × 10⁶ CFU/mL). The total average concentration of all the bacteria isolated from the air/water syringes was 52,371 CFU/mL, and the maximum was 1.7 × 10⁶ CFU/mL, similar to the concentration found in the handpiece.

The length of the water retraction was measured in millimeters (on a scale of 0 to 200 mm) using the retraction measurement device, and the volume of water retracted was calculated in μL using the following formula: V = π × d² × l/4 = 3.142 × 1.5² × l/4 = 1.767 × l. The results, expressed according to the different ranges of measured retractions and the species identified in each of the volume categories, are shown in Table 2. We also plotted the log₁₀ microbial loads versus the retraction volume (Figure 4). Analysis of variance showed a significant, positive correlation (P < 0.05) between an increased concentration of bacteria in the water sample and the retracted volume. The results also showed that there was a significant, positive correlation (P = 0.004, r = 0.370) between the retracted volumes and the age of the instruments.

Table 2.

Water retracted volume and the species identified for each of the volume categories

Retracted volume (V, μL)	Retraction (l, mm)	Number	Percentage	Genus/species (number of strains)
ISO 7494 (≤40 μL)		28	48.28
0	0	6	10.34	Bacillus cereus (2)
0–20	0.5–11.33	14	24.14	B. cereus (4) Burkholderia gladioli (1)
20–40	12–20	8	13.79	B. cereus (2)
ISO 7494 (>40 μL)		30	51.72
40–100	23.33–55	9	15.52	B. cereus (3) Staphylococcus lentus (1)
100–200	58.33–100	7	12.07	B. cereus (1) Ralstonia pickettii (1) B. gladioli (1) Moraxella catarrhalis (1)
200–300	120–169	8	13.79	B. cereus (1) Pseudomonas fluorescens (2) Kocuria kristinae (1)
300–	170–200	6	10.34	B. cereus (3) K. kristinae (1)

Figure 4.

Comparison of the results of retracted volume and logx concentration of bacteria in the output water of dental unit waterlines (DUWLs) using scatter and line regression. CFU/mL, colony-forming units/mL.

Of the 263 water samples that were tested, 19 contained Bacillus cereus, a spore-forming gram-positive species of bacteria. Kocuria rosea, Kocuria varians, Moraxella catarrhalis, Staphylococcus lentus, Ralstonia pickettii and Pseudomonas alcaligenes were only detected in one sample (Table 3).

Table 3.

Frequency of detection of pathogenic and non-pathogenic bacteria and heterotrophic plate counts (HPCs) in water samples

Genus/species	Gram staining	Air/water syringes		Handpiece		Reservoir bottles		Tap water		Total number of strains
		Number of strains	CFU/mL	Number of strains	CFU/mL	Number of strains	CFU/mL	Number of strains	CFU/mL
Bacillus cereus	P	5	4.3 × 105	6	6.9 × 105	5	3.9 × 102	3	1.9 × 102	19
			6.2 × 102		1.5 × 104		2.2 × 102		1.6 × 102
			1.8 × 102		7.5 × 103		62		54
			73		1.3 × 103		59
			59		7.0 × 102		10
					78
Kocuria kristinae	P	2	1.3 × 104	0		0		0		2
			5
Kocuria rosea	P	0		0		0		1	15	1
Kocuria varians	P	0		0		0		1	1	1
Moraxella catarrhalis	N	0		0		1	1.6 × 104	0		1
Staphylococcus lentus	N	1	2.7 × 103	0		0		0		1
Ralstonia pickettii	N	1	1.0 × 103	0		0		0		1
Burkholderia gladioli	N					2	3.1 × 103			2
							36
Pseudomonas fluorescens	N	1	3.7 × 105	1	1.3 × 103	0		0		2
Pseudomonas alcaligenes	N	0		0		0		1	1.2 × 102	1
Total		10		7		8		6		31

N, gram-negative stain; P, gram-positive stain.

DISCUSSION

Water for testing was taken from four sites of the DUWLs, including handpieces, air/water syringes, the reservoir bottle, the cup filler outlet and one water tap. According to the literature, contamination of DUWLs has been documented in scientific reports13., 15., and microbial levels of 10⁴–10⁶ CFU/mL9., 11., 24., 25. have been reported in DUWL water samples. In our study, the maximum concentration of bacteria in water from the 58 handpieces was 1.8 × 10⁶ CFU/mL, which is higher than that reported by other studies. This result could be explained by the fact that, at present, China has not enacted any national or industry standards for DUWL output water. Either the ADA recommendations or the CDC guidelines for Infection Control in Dental Health-Care Settings are used (≤500 CFU/mL)²³, which reflect the US standards for safe drinking water set by the Environmental Protection Agency (EPA). However, in China, the standards for bacteria in drinking water are more restrictive (≤100 CFU/mL); thus, the output water from DUWLs is unsuitable. This is due to a lack of knowledge about, and lack of attention to, DUWL contamination among oral health-care workers.

The lack of standards is one of the main reasons for the current serious contamination of DUWL output water in China. With economic development, the need for all types of oral health services for Chinese people will increase²⁶. The DCU is one of the most essential and necessary components for dental care, and the output water of DUWLs is a potential source of infection for dental staff and patients, especially those who are medically compromised or immunocompromised¹². It will be meaningful for China to create DUWL output water standards by following the CDC guidelines for Infection Control in Dental Health-Care Settings.

In addition to the formation of biofilm formed by bacteria present in the water supply, the retraction of oral fluids into DUWLs during dental instrument use can expand the range of microorganisms present in both DUWL biofilms and output water¹³. A number of studies have demonstrated that oral fluids can be retracted into DUWLs during dental instrument use, especially when the handpiece is used. Lewis et al. and Bagga et al. have reported that up to 1 mL of oral fluids could be drawn back into the DUWLs in old equipment, as well as in some new DCUs, under experimental conditions27., 28.. Although this aspiration can be limited by anti-retraction valves fitted distally to the handpieces and air/water syringes, anti-retraction valves in DCUs may fail to prevent microbial contamination of DUWLs after a few months of use¹³. To our knowledge, the first study on the efficacy of anti-retraction valves for preventing microbial contamination of DUWLs in DCUs that are currently in use was performed by Berlutti et al. This group showed that the overwhelming majority (74%) of anti-retraction devices did not prevent retraction when the turbine stopped running, leading to contamination of the waterlines and consequently to a potential cross-contamination of patients¹⁶. The retraction of DUWLs during dental instrument use is rarely reported, for various reasons. Awareness of the standards described in Part 2 (air, water, suction and wastewater systems) of ISO 7494 is low among oral health-care workers. The lack of proper test kits that refer to the standard stipulated method for evaluating the retraction is another important reason. There are several problems associated with testing the retraction in accordance with the method provided by the international standards. One is that removal of the handpiece connector from the waterline requires special tools and skill. Additionally, removal and reinstallation of the handpiece connector to the waterline is relatively time-consuming. Thus, it is difficult to test a large number of DCUs using this method. Moreover, when the handpiece is disconnected, compressed air and DUWL output water will be emitted at the same time when the foot control is operated, causing splashing to occur. Finally, there are no regularly available kits for retraction testing in China. We solved this problem by designing our measurement device according to ISO 7494. If this newly designed measurement device is used, the handpiece connector does not need to be removed from waterline. The measurement device can be connected to the handpiece connector via a water sampling connector that is a component of the detector.

In this work, 30 (51.72%) DCUs failed the evaluation, and 21 (36.21%) DCUs retracted more than 100 μL of fluid. This volume is twice as high as the threshold for DCU retraction described in the American Dental Association/American National Standard (ADA/ANSI) specification #47 or ISO 7494. Retracted volumes that exceed 40 μL indicate failure of the anti-retraction valves to prevent retraction when the turbine stops running, leading to contamination of the waterlines. In this study, analysis of variance showed that there is a significant, positive correlation (P < 0.05) between an increased concentration of bacteria in the water sample and the retracted volume. Therefore, DCUs equipped with anti-retraction valves should be periodically monitored and preventive maintenance should be performed to minimise instances of anti-retraction valve failure¹³.

The bacterial species found in the present study were mostly environmental organisms that were also present at low levels in the tap water. Some of the species isolated (e.g. Kocuria kristinae, Burkholderia gladioli and Pseudomonas fluorescens) are known opportunistic pathogens. They have also been found in many other studies and have been previously associated with hospital-acquired infections and infections in immunocompromised patients29., 30., 31.. These findings suggest that the bacteria present in the DUWLs are derived from the input water supply, in agreement with previous studies32., 33.. However, of the 19 isolates, B. cereus was that most frequently detected. Bacillus cereus is also occasionally isolated from human dental plaque; thus, its presence could indicate a defect in retraction³⁴. However, based on the data currently available, we do not propose a correlation between the species detected and the retracted volume. It is possible that the number of strains was relatively small and the distribution was relatively concentrated, mainly for Bacillus subtilis. However, it is important to note that several B. cereus strains can enhance biofilm formation. Within the established biofilms, B. cereus is able to form spores, which may lead to contamination of the output water of DUWLs; however, B. cereus is uncommon in oral infections34., 35..

CONCLUSION

The results of this study show that, in Tianjin (one of the four special municipalities of China), DUWL output water is heavily contaminated. A series of policies and approaches should be developed for dental health-care workers to prevent cross-infection. Most importantly, DUWL output water standards should be created. Also, the concentration of bacteria in DUWL output water and the efficacy of anti-retraction valves should be periodically monitored. Furthermore, improved awareness of DUWL output water contamination should be promoted among dental staff through training and increased compliance with the various approaches available for controlling DUWL biofilm (e.g. flushing, use of sterile, deionised or distilled water and use of periodic or intermittent DUWL treatment agents for regular DUWL treatment).

Acknowledgements

This study was supported by (i) the technology fund (no. CDCKY1302) of the Tianjin Center for Disease Control and Prevention and (ii) a scientific research project (no. 2014KZ043) from the Tianjin Health and Family Planning Commission of Tianjin, China. The authors declare that they have no competing financial interests.