Effective cleaning and decontamination of the internal air and water channels, heads and 3 head-gears of multiple contra angle dental handpieces using an enzymatic detergent and 4 automated washer disinfection in a dental hospital setting

ABSTRACT


Introduction
Several manufacturers of devices developed to clean DHPs claim that their equipment can 101 ensure adequate cleaning, however little independent direct evidence is available [12]. Spraying a 102 cleaning solution into the channels and transmission components is one of the most widely used 103 approaches to cleaning and disinfection of the internal elements of DHPs. Cleaning fluids often 104 contain alcohols that denature proteins, which are very difficult to remove from metal 105 surfaces [15,16]. Furthermore, the process is very difficult to validate because of the 106 inaccessibility of the internal components of DHPs. One study demonstrated that the use of 70% 107 alcohol to disinfect the external surface of high-speed DHPs was ineffective [16]. DHPs are not 108 suitable for immersion in disinfectants, which can lead to metal corrosion [12]. 109 Washer-disinfection is a reproducible process that can be validated for the external 110 components of medical devices and is the preferred method of cleaning and decontaminating 111 DHPs [17]. Washer disinfectors are not mandatory for dental practices in all countries [17][18][19][20]. 112 Some studies demonstrated the effectiveness of washer disinfectors at cleaning the outside 113 surfaces of DHPs and a few have demonstrated its efficacy at reducing organic contamination on 114 internal components [11,21]. However, little published data is available on the direct effectiveness 115 of washer disinfectors at significantly reducing microbial contamination from the internal 116 components of DHPs, especially in a dental hospital setting where large numbers of DHPs must 117 be decontaminated daily. 118 The purpose of this study was to use contra angle DHPs as a model system to directly 119 investigate the effectiveness of washer-disinfection at reducing microbial bioburden of internal 120 components of multiple DHPs deliberately contaminated with each of four challenge 121 microorganisms in the presence/absence of organic soil in a dental hospital central 122 decontamination unit. 123 124

Methods 125
Dental handpieces 126 127 BienAir Dental SA (Biel/Bienne, Switzerland) CA 1:1 L contra angle DHPs were used 128 and water channels and are powered by an electric motor attached to a flexible arm connected to a 135 dental chair unit (DCU). Three pairs of small water and air outlets surround the dental bur orifice 136 in the DHP head (Figure 1). At the Dublin Dental University Hospital (DDUH) compressed air is 137 provided to each DCU from a central source. Water containing very low levels of microorganisms 138 is provided to DUWLs from a central supply treated continuously with residual 139 electrochemically-generated hypochlorous acid [22]. The parameters for each washer-disinfection cycle were as follows: (i) prewash with 154 mains water at 22 o C for six min, (ii) cleaning with enzymatic detergent at 55 o C for eight min, (iii) 155 rinsing with reverse osmosis purified water for five min, (iv)  Before each experiment, DHPs were sterilized in a vacuum steam sterilizer at 134 o C. Prior 177 to inoculation, the small press button plate sealing the DHP head was removed, followed by 178 removal of the head, head gear and middle gear, providing access to the openings of the air and 179 water channels (Figure 1). The partially disassembled DHP was then positioned horizontally and 180 100 µl of culture inoculum supplemented with 0.3% (w/v) bovine serum albumin (BSA) (clean 181 conditions), 3.0% (w/v) BSA (dirty conditions) or 10% artificial test soil (dirty conditions) 182 (Edinburgh test soil, Cúram Medical, Dublin, Ireland, compliant with ISO-15883-5-2021[25]) 183 was inoculated into both channels using an 0.3 ml insulin syringe with a 30 gauge Micro-Fine TM 184 needle (Becton Dickson and Company, NJ, USA) and allowed to dry for 30 min. The angle in the 185 body of the DHP ensured the head and neck were horizontal, permitting retention of the inocula in 186 the channels. After drying, the inoculated channels were sampled by inserting sterile, tapered 187 periopoints (02 Absorbent Points, Dentsply Sirona, Charlotte, NC). Periopoints are used for 188 sampling periodontal pockets and are ideal for sampling narrow lumens [26]. Periopoints were 189 placed in 1 ml phosphate buffered saline (PBS) (Oxoid) in a sterile 1.5 ml tube and vortexed for 1 190 min to release microorganisms. Serial dilutions were prepared in PBS and 100 µl aliquots spread 191 in triplicate onto TSA agar for bacteria and YPD agar for C. albicans and incubated as described 192 above. Following incubation, the bacterial/yeast colonies were counted and the total number of 193 bacteria/yeasts recovered from the channels determined. 194 For each challenge microorganism, 100 µl of culture inoculum supplemented with 0.3%, 195 3.0% BSA or 10% test soil was inoculated into the head of a non-disassembled DHP placed 196 horizontally through the dental bur orifice and allowed to dry for 30 min. The DHP head was then 197 aseptically removed and the press button plate, the head gear and the head were placed in 5 ml of 198 PBS in a sterile 25 ml tube and agitated for 1 min to release bacterial/yeast cells into solution 199 Additional experiments were undertaken with four DHPs that were lubricated with W&H 203 (Austria) Service Oil F1 MD-500 using an Assistina 301 plus DHP maintenance unit (W&H, 204 Austria) according to the manufacturer's instructions prior to sterilization at 134 o C. Then the 205 heads and channels of three DHPs were inoculated with S. aureus ATCC6538 in the presence of 206 10% test soil as described above, followed by reassembly of the DHPs and washer-disinfection. 207 The fourth DHP was retained as a control. Following washer-disinfection, the DHPs were 208 disassembled and the reduction in bacterial counts and protein recovered from DHP head/head-209 gears and channels calculated relative to the control DHP. Experiments were repeated on three 210 separate occasions. 211

212
Microorganism counts in DHP channels and heads/head-gears following washer-disinfection 213 214 Each challenge microorganism preparation was inoculated separately into the heads and 215 channels of 11 DHPs as described above. After drying, DHPs were reassembled and 10 were 216 subjected to a washer-disinfection. The remaining inoculated DHP served as a control. Following 217 washer-disinfection, all 11 DHPs were disassembled, sampled as described above and the log 218 reduction in bacterial/yeast counts calculated relative to the control inoculated DHP in each case. 219 Experiments were repeated in triplicate with all 10 DHPs for each challenge organism under clean 220 (0.3% BSA) and two sets of dirty conditions (3.0% BSA and 10% artificial test soil). 221

222
Protein Assay 223 224 Inoculated DHP heads/rotors and channels were tested for residual protein following 225 washer-disinfection. Tests were undertaken on samples recovered as described above. Protein was 226 detected using the QuantiPro BCA assay kit (Sigma-Aldrich/Merck, Arklow, Ireland) according 227 to the manufacturer's instructions. The relative reduction in protein in DHP channels and 228 heads/head-gears from washer-disinfected DHPs was determined relative to unwashed controls. 229 The external surfaces of 10 DHPs were painted with 10% test soil and left to dry for 30 230 min followed by washer-disinfection. One additional painted DHP was retained as a control. The 231 DHPs were visually inspected for residual test soil immediately following washer-disinfection and 232 the surfaces were swabbed with sterile swabs soaked in 1% (w/v) sodium dodecyl sulphate (pH 233 11.0) and tested for protein using the QuantiPro BCA assay kit. Surfaces were also tested using 234 the Pyromol-Test for residual protein (PEREG GmbH, Waldkraiburg, Germany) according to the 235 manufacturer's instructions. 236

239
Decontamination of DHP internal components by washer-disinfection 240 241 The internal surfaces of the head, press button plate and head-gear (all three hereafter 242 referred to as the head) and air and water channels of contra angle DHPs were used as a model 243 system for monitoring the efficacy of decontamination by washer-disinfection. The internal 244 surfaces of 11 DHP heads and both channels were inoculated with one of four challenge 245 microorganisms under clean and two sets of dirty conditions. Ten of the inoculated DHPs were 246 then inserted into a Míele E919 dental module ( Figure 2a recovered from DHP heads and channels was observed consistently for all 10 DHPs tested (Table  259 I). Similar reductions were observed under both sets of dirty conditions. The average log 260 reduction in S. aureus CFUs from DHP heads was 5.27±0.23 (3% BSA) and 5.11±0.58 (10% test 261 soil) and from channels was 5.57±0.14 (3% BSA) and 5.59±0.16 (10% test soil). The average log 262 reduction in E. hirea CFUs from DHP heads was 5.32±0.38 (3% BSA) and 5.37±0.08 (10% test 263 soil) and from channels was 5.48±0.18 (3% BSA) and 5.58±0.13 (10% test soil). The average log 264 reduction in P. aeruginosa CFUs from DHP heads was 6.07±0.05 (3% BSA) and 5.57±0.48 (10% 265 test soil) and from channels was 5.87±0.22 (3% BSA) and 5.72±0.33 (10% test soil). 266 In the case of the C. albicans strain, on average an approximate 5 log reduction in CFUs 267 recovered from DHP heads was recorded under clean conditions (0.3% BSA) (average 5.25±0.36) 268 with a slightly lower log reduction recorded for DHP channels (average 4.95±0.23) ( Table I). Three DHPs were lubricated using the Assistina 301 plus automated system prior to 292 sterilization and inoculation of the heads and channels with S. aureus ATCC6538 in the presence 293 of 10% test soil followed by washer-disinfection. One additional lubricated and inoculated DPH 294 served as a control. Following washer-disinfection, the log reduction in bacterial CFUs from 295 heads and channels was calculated relative to the control DHP. In three separate experiments, the 296 average log reduction in bacterial CFUs was 5.96±0.1 (heads) and 5.69±0.2 (channels). 297 298 Reduction in protein in DHP heads and channels 299 300 For each of the 10 DHPs inoculated with 10% test soil in the absence of challenge 301 microorganisms, on average a >95% reduction in protein recovered from DHP heads and channels 302 was observed following washer-disinfection relative to unwashed inoculated control DHPs (Table  303 SI). Similar reductions in protein levels in heads and channels were obtained with DHPs 304 inoculated with challenge microorganisms under both sets of dirty conditions following washer-disinfection (Table SI). No protein was detected in heads and channels inoculated under clean 306 conditions following washer-disinfection (data not shown). 307 The average reduction in protein from the 10 DHP heads and channels inoculated with S. 308 aureus and (i)  For all four challenge microorganisms used under both sets of dirty conditions, consistent 318 reductions in protein levels in heads and channels were observed for all 10 DHPs tested regardless 319 of their position in the dental module used to retain the DHPs in the washer disinfector ( Figure  320 2a). Similar results were obtained with the six DHPs inoculated with S. aureus and 10% test soil 321 placed at position 10 in each of six separate Míele E919 dental modules (Table SII, Figure 2). 322 In the case of the three DHPs that were lubricated with oil and sterilized prior to 323 inoculation with S. aureus and 10% test soil followed by washer-disinfection, an average of 324 99.69%±0.1% and 98.98%±0.3% reduction in protein was recorded for DHP heads and channels, 325 respectively, on three separate occasions relative to inoculated but unwashed controls. 326 327 Test soil removal from the outside surfaces of DHPs by washer-disinfection 328 329 The exterior surfaces of 10 DHPs that were painted with 10% test soil and left to dry were 330 free from visible contamination following washer-disinfection. All the DHPs were negative for 331 residual protein using the Pyromol-Test. There was a 99.98%±0.02% reduction in protein on the 332 DHP surfaces using the DHPs QuantiPro BCA assay relative to controls. An approximate five log reduction in S. aureus, E. hirea and P. aeruginosa CFUs 353 recovered from DHP heads and channels was consistently observed for all 10 DHPs tested 354 following washer-disinfection under clean and both sets of dirty conditions (Table I). On average 355 a >93% reduction in protein was recorded for DHP heads and channels under all test conditions 356 (Table SI). Similar reductions in microbial CFUs and protein were obtained with C. albicans 357 SC5314 (Tables I and SI Tables I & SI). DHP10, which was furthest away from the water inlet in 360 the washer-disinfector module, yielded similar results to DHP1 (closest to the water inlet). A 361 series of experiments with six DHPs in which the channels and heads were inoculated with S. 362 aureus and 10% test soil were undertaken with six separate dental modules, with each DHP 363 located at position 10 (i.e., furthest away from the water inlet of each module) ( Figure 2) followed 364 by washer-disinfection. In each case, a >5 log reduction in bacterial count and a >93% reduction 365 in protein recovered from heads and channels was consistently recorded, regardless of dental 366 module (Table SII). Only one of the 10 adapters for DHPs was occupied in each of the six dental 367 modules used and water freely discharged from the nine unoccupied adapters in each module 368 during washer-disinfection. 369 All these findings demonstrated that the Míele PG8528 washer disinfector with the 370 enzymatic detergent used was consistently effective at significantly reducing microbial and to 60 individual DHPS can be decontaminated simultaneously using the Míele PG8528 washer 373 disinfector, which is ideal for dental hospitals where large numbers of DHPs must be 374 decontaminated daily. 375 The internal components of DHPs must be lubricated regularly.   Míele PG8528 washer-disinfector can accommodate up to six E919 dental modules, each of 557 which contains adapters for up to 10 DHPs. The dental module in the foreground is fitted with 558 W&H DHP adapters. The other five modules are fitted with other DHP adapters that were not 559 used in this study. Table I: Reduction in the density of four challenge microorganisms recovered from internal components of 10 contra angle dental handpieces (DHPs) under clean and dirty conditions following washer disinfection relative to inoculated DHPs not subjected to washer-disinfection Each challenge microorganism was inoculated separately into the head and air and water channels of 11 DHPs. Ten of these DHPs were processed by washer-disinfection. For each washer-disinfector cycle, one inoculated DHP was left untreated as a control. Following washer-disinfection, all 11 DHPs were tested for recovery of microorganisms using periopoints as described in the Methods and the log reduction in bacterial/yeast counts calculated relative to the untreated control inoculated DHP in each case.    SI: Reduction in protein recovered from the internal components of 10 contra angle dental handpieces (DHPs) under two different sets of dirty conditions following washerdisinfection relative to inoculated DHPs not subjected to washer-disinfection Each challenge microorganism was inoculated separately into the head and air and water channels of 11 DHPs. Ten of these DHPs were processed by washer-disinfection. For each washer-disinfector cycle, one inoculated DHP was left untreated as a control. Following washer-disinfection, all 11 DHPs were tested for recovery of protein as described in the Methods and the percentage reduction in protein calculated relative to the untreated control inoculated DHP in each case.  Table SII: Reduction in the protein recovered and density of Staphylococcus aureus ATCC 6538 under dirty conditions recovered from internal components of six contra angle dental handpieces (DHPs) in each of six different washer-disinfector dental modules following washer disinfection relative to inoculated DHPs not subjected to washer-disinfection a Bacterial recovery and protein data shown for channels represent the average recovery data from both air and water channels for each DHP tested with each challenge microorganism in three separate experiments. Bacteria recovery data from heads includes bacteria recovered from the DHP head, press button plate and head gear of each DHP (see Figure 1).

Log Reduction in Bacterial Count a (+/-Standard deviation)
Percentage protein reduction a (+/-Standard deviation)