Evaluating the microbial aerosol generated by dental instruments: addressing new challenges for oral healthcare in the hospital infection

Streptococcus mutans was selected as the bacterial tracer to simulate the diffusion of any airborne infective agent which was obtained from the State Key Laboratory of Oral Diseases. S. mutans was grown in Brain Heart Infusion (BHI) broth overnight at 37 °C in a 5%-supplemented carbon dioxide environment. The cells were harvested via centrifugation (4,000 g, 15 min), washed twice with sterile phosphate-buffered saline (PBS), and resuspended and diluted in the same buffer. The bacterial suspension was subjected to vortex (Vortex Genius 3; IKA, Germany; 30 s) to disperse bacterial chains and adjusted to 10⁸ CFU/mL standard for experimental use.

Experiments were conducted in a standard single-chair dental treatment room which meets the requirements of secondary biosafety laboratory located in the West China Hospital of Stomatology, Sichuan University, Chengdu, China. This is a 394 cm by 510 cm by 240 cm (length, width, and height, respectively) operative environment equipped with a dental unit (ESTETICATM E80 Vision; KaVo, Germany), a dental chair, and a 4-door cabinet located behind the dental chair (Fig. 1). The air-conditioning system for the operatory was isolated using sealing the inlet. The mechanical ventilation system was off and the door and windows were closed during the experiment [26]. And the dental unit is exposed to ultra-violet and tested by control agar plates to make sure that there are no S. mutans before the experiment. The temperature remained constant at 24–26 °C and the humidity at 60%—70%.

A dummy head was mounted in a standard working position and was adjusted to make the height of the headrest equal to the operator’s arms with the seat back 45° to the ground. The position of the dummy head’s mouth was 75 cm above the floor. The jaws inside the dummy head oral were taken out and a 50 ml beaker containing 15 ml S. mutans suspension was fixed inside. The operator site is similar to the anterior teeth of the patient when the front part of the operator is immersed in the suspension. A high-speed air turbine handpiece (S609C; KaVo, Germany) was used and connected to the dental unit. The rotating speed of the turbine was measured to be 360,000 r/min. To simulate the clinical procedure of tooth preparation, the handpiece was equipped with a cylindrical bur (TR-12; MANI, Japan) which was 10 mm in working length, and the bur was immersed in the bacterial suspension by approximately 5 mm, while the head of the turbine was about 5 mm away from the surface during the experiment [27]. And a high-speed electric handpiece (Restorative classic CA 1:5, Bien-Air, Swiss) was used with motor control (Optima, Bien-Air, Swiss). The rotating speed is 200,000 r/min and the bur is also a cylindrical bur (TR-12; MANI, Japan). An ultrasonic instrument (P5XS Newtron, Satelec, France) with PerfectMarginShoulder(PMS®) tip 1 was 5 mm in working length, and the tip was immersed in the bacterial suspension by approximately 2.5 mm (Fig. 2). The working power was blue light which means high power and amplitude as recommended. The coolant flow rate is 15 mL per minute and excessive liquid will be removed by an aspiration cannula. Only 1 operator and 2 assistants wearing the same biohazard-protective full suits(4565L; 3 M™, USA), N95 masks without valves(9132; 3 M™, USA), goggles(1621AF; 3 M™, USA), face shields, shoe covers, and gloves conducted the procedures.

The presence of a biological tracer in 252 sites in the operatory was measured for this procedure. According to the length, width, and height of the operatory, 50 cm was set as the unit length, and sampling sites were determined every 50 cm with 4 sampling planes which were set at 0.5 m、1.0 m、1.5 m、2.0 m, 252 sampling sites in totally determined in the whole operatory and the distance between two adjacent sampling sites is 50 cm. Before the beginning of each procedure, 252 mitis salivarius-bacitracin agar plates were suspended by string at the sampling sites and facing upwards while the lids remained closed (Fig. 3a, b). The operator took his position and then 2 assistances opened the lids of every plate. After that, the operator performed a 15-min simulated tooth preparation according to the experimental model. The operator remains in his position and keeps still after the procedure. The plates were closed 15 min after the end of the procedure to allow aerosols to settle and immediately transferred to the microbiological laboratory. The plates were incubated at 37 °C for 48 h in a 5%-supplemented carbon dioxide environment. At the end of the incubation, the colonies were counted and the results were expressed as CFU. Between procedures, the environment was disinfected by spraying 0.5% sodium hypochlorite solution and irradiating ultraviolet rays for 2 h. The same procedure was repeated once using sterile distilled water instead of bacterial suspension as a blank control. This procedure was repeated 20 times; 20 tests and 20 controls.

In the three-dimensional spatial distribution experiment, the area with the highest tracer level was approximately in the middle of the dental unit, 1.5 m above the ground, and 0.8 m away from the operation site, which was determined to be the sampling site of the experiments (Fig. 3a, b) [13]. During each sampling day, air samples were collected using a six-stage Andersen microbial sampler (AMS; 20–600, Thermo, USA), containing 6 mitis salivarius-bacitracin agar plates sampling sites. The operator performed simulated tooth preparation for 15 min. After that, air samples were taken continuously within 2 h and the mitis salivarius-bacitracin agar plates inside were replaced every 10 min. The sample was collected for 10 min before the baseline level was determined. AMS was operated at 28.3 L/min and the sampling time was 10 min. The experiments were carried out under three experimental conditions after using air turbines, electric turbines, or ultrasonic devices. Therefore, this study allows us to freely compare airborne microbial contamination’s dissipation efficiency after different operations. All plates were incubated at 37 °C for 48 h in a 5%-supplemented carbon dioxide environment. The colonies on levels 1–6 of AMS were counted. Then particle size is analyzed as described previously [28] (Table 1). The positive hole method was used to correct the numbers and the airborne microbial contamination at different times was calculated with the formula (1)where \({C}_{i}\) was airborne microbial contamination, CFU m⁻³; \({N}_{i}\) was the sum of 6 grades effective colonies, CFU; \(t\) was the sampling time, min; \(Q\) was the flow rate during sampling, L / min [29].

For the three-dimensional spatial distribution experiment, SPSS version 24 (IBM Corp., NY, USA) was used to analyze microbiology data belonging to the tracer presence in the dental treatment room. A Shapiro–Wilk test was applied to check the normality of the data distribution and the Bartlett test was used to check the homogeneity of variances preliminarily. The mean level of each site was used to analyze the three-dimensional spatial distribution of the dentist office. The data were log-transformed to approach a normal distribution. The tracer levels in the planes of different heights were compared through one-way analysis of variance (ANOVA) and Dunnett’s T3 test, at a significance level of 0.05. 3-dimensional graphic models representing the spatial distribution and the biological tracer levels were created using The R Programming Language. 2-dimensional graphic models demonstrating the spatial distribution of biological tracer level in each height plane were created using GraphPad prism 9.

Figures 4, 5 and 6 shows the three-dimensional spatial distribution of bacterial tracers after using a high-speed air turbine, a high-speed electric handpiece, and an ultrasonic device in the dental treatment room. It’s obvious that the bacterial tracer distribution shows higher concentration after using a high-speed air turbine and a high-speed electric handpiece. Although it shows a lower tracer’s concentration with a high-speed electric handpiece. The minimum tracer concentration is in the ultrasonic device. In addition, Figs. 4 and 5 showed the distribution of the tracer in the planes of different heights with high-speed turbines. The bacterial tracer could be detected in nearly the whole space of the dental treatment room after the simulated tooth preparation. Among the 4 different heights, high-speed air turbine handpieces and high-speed electric turbine handpieces showed similar results in the spatial distribution. The 1.5 m plane exhibited the highest mean level of tracer (3.32 ± 0.43 lg CFU, 3.03 ± 0.44 lg CFU), followed by the 0.5 m plane (2.87 ± 0.35 lg CFU, 2.55 ± 0.35 lg CFU), 1.0 m plane (2.34 ± 0.59 lg CFU, 2.01 ± 0.63 lg CFU) and 2.0 m plane (2.31 ± 0.38 lg CFU, 2.00 ± 0.46 lg CFU), with air turbines and electric turbines respectively. The plane of 1.5 m demonstrated a significantly higher tracer level than that of 0.5 m, 1.0 m, and 2,0 m planes (p < 0.05). The plane of 0.5 m showed a significantly higher tracer level than that of 1.0 m, and 2.0 m planes (p < 0.05). But no significant difference was observed between the 1.0 m plane and the 2.0 m plane (Figs. 4e and 5e). With the ultrasonic device, the result was a little different. The 1.5 m plane exhibited the highest mean level of tracer (2.47 ± 0.70 lg CFU), followed by the 1.0 m plane (1.51 ± 1.07 lg CFU), 0.5 m plane (1.33 ± 1.15 lg CFU) and 2.0 m plane (1.17 ± 0.84 lg CFU) (Fig. 6). The plane of 1.5 m demonstrated a significantly higher tracer level than that of 0.5 m, 1.0 m, and 2.0 m planes (p < 0.05). The plane of 1.5 m showed a significantly higher tracer level than that of 2.0 m planes (p < 0.05). But no significant difference was observed between the 0.5 m plane and the 1.0 m or 2.0 m plane (Fig. 6e). Analyzing the spatial distribution map of the biological tracer level in each height plane, the tracer level decreases with the increase of the distance from the infection source. However, the tracer presence at the 2.0 height plane near the ceiling was low and showed a relatively regular distribution, except for the ceiling area just over the dental unit, where we found a high tracer presence. Sampling sites behind the operator showed very low tracer presence.

Additionally, at the four different high planes, the use of the high-speed air turbine showed the highest concentration of the biological tracer, followed by the electric turbine and ultrasonic devices (p < 0.05). As shown in Fig. 7, the error bar of biological tracer concentration with ultrasonic devices is bigger than in high-speed turbines due to no tracer found at the border of the operation unit. However, the tracer concentration of the operation center is higher than rotational instruments in the 0.5 m and 1.0 m heights.

With air turbines, 91% of biological tracers distribute on stage III to V, but 9% in stage I,II and VI. Most of it is on stage V(55%), followed by stage IV(30%), stage VI(7%), stage III (6%), stage II (1%), and stage I (1%) (Fig. 9a). With electric handpieces, 93% of biological tracers distribute on stage III to V, but 1% of it on stage I, 1% on stage II, and 7% on stage VI. Moreover, most tracer distributes in stage IV(56%). 1% of tracer was found in stage I which is the least (Fig. 9b). With ultrasonic devices, the most tracer is found on stage V(44%), followed by stage IV(33%), stage VI (17%), stage III (3%), stage II (2%), and stage I (1%). 94% of it is founded on stage III-IV (Fig. 9c).