Comparison of antimicrobial efficacy of different disinfectants on the biofilm formation in dental unit water systems using dip slide and conventional methods: A pilot study

Biofilm formation in dental waterlines brings opportunistic infections, especially for immunosuppressive patients. This study aimed to determine biofilm‐forming microorganisms by various methods and investigate disinfectants' effects on biofilm.

Research highlights : • It has been observed that the dip slide method can count bacteria more sensitively than conventional methods in dental water systems without the need for experienced personnel and equipment.
• The difference between biofilm formation in water systems before and after disinfectant exposure in SEM examinations is remarkable.
The effects of ClO2 and HOCl on biofilm were investigated and bacterial growth was inhibited in dental units between 5 and 10 minutes with both disinfectants.

| INTRODUCTION
The water resources of dental units are one of the most important parameters that create the risk of infection during the treatment process to be applied to the patients.The acceptable microorganism limit determined by the American Dental Association in dental unit water systems is 200 CFU/mL (Pullar et al., 2000).However, the old water systems, the lack of periodic cleaning of the water source, and the nature of the materials in the waterway cause many different types of bacteria, fungi, and protozoa to colonize, and these microorganisms can reach unacceptable numbers.Microorganisms that secrete exopolysaccharides adhere to water systems and form a biofilm.The biofilm layer acts as a shield that protects the microorganism from external environmental conditions and prevents the microorganism from being removed (Shajahan et al., 2017).It is known that biofilm-producing microorganisms escape from phagocytosis and are exposed to phenotypic changes that give resistance to antibiotics and disinfectants.
Therefore, the presence of biofilm-producing microorganisms in the dental unit water systems is an important risk factor in the dental treatment of immunocompromised patient groups (Yabune et al., 2008).
Application of disinfectants in the dental unit waterway temporarily reduces the number of microorganisms to the desired level, but cannot eliminate the presence of biofilm (Temel & Eraç, 2018).
Periodic control of the water supply in dental clinics and the addition of disinfectant to dental units, especially prior to invasive procedures, can prevent opportunistic microbial colonization and biofilm formation.
The purpose of this study was to evaluate the effect of hypochlorous acid (HOCl) and chlorine dioxide (ClO 2 ) applications against biofilm-forming microorganisms in dental unit water systems and to compare practical methods for determining microbial contamination with conventional methods.The null hypothesis tested was that there were no differences between solutions and methods.

| MATERIALS AND METHODS
In the study, samples were taken from the water system of six dental units aged between 10 and 15 years in different dental clinics in Anatolia.Ethical approval was obtained for the study.

| Collecting water samples
Six units were randomly divided into two groups, three were treated with 200 ppm HOCl and the other three were treated with 200 ppm chlorine dioxide.While taking water samples to be tested, the tip of the air-water sprayer was wiped with 70% ethyl alcohol prior to each sampling and 0 min samples were taken before disinfectant applications.These samples were considered as the self-control of the study.
A total of 6 samples of 50 mL were taken from each unit at 0, 1, 5, 10, 20, and 30 min of disinfectant application.As a result, a total of 18 water samples were tested for each disinfectant solution.
Before collecting water samples from the polyurethane hoses of the dental unit water inlets and at the end of all procedures, an incision of 1 cm 2 surface width and 1 cm length was taken and prepared for examination in Scanning Electron Microscopy (Zemouri et al., 2020) to observe biofilm formation.

| Detection of bacterial growth and Colony count
In the study, the dipslide method (DSM), which allows rapid colony counting, and surface smear method (SSM), a traditional microbiological method, were used to detect bacterial contamination in water samples.
Eighteen millimeters of each water sample was taken from the apparatus (Dipslides ® , England) containing Heterotrophic Plate Count Agar (HPC Agar) as specified in the product catalog, and the plate on which the cell count was to be made was submerged in water for 30 sec., and then the water sample was discarded from the system.This kit, which does not require an incubator, was kept at room temperature (25 C) for 72 h and then the colonies formed on the counting chamber were counted and the total amount of bacteria in the samples was determined at 0, 1, 5, 10, 20, and 30 min.Figure 1 shows the images of bacterial growth on the counting chamber using the dipslide kit.
Simultaneously with the DSM, 32 mL of water sample was taken into falcon tubes, centrifuged at 2000 rpm and inoculated from the sediment onto Plate Count Agar (Merck ® , Germany) by conventional SSM and incubated at 37 C for 24-48 h.After growth, each colony was counted and calculated in CFU/mL.

| Investigation of biofilm presence
Staphylococcus spp.isolates grown in water samples were examined to monitor biofilm formation.
Congo red agar and Christensen tube methods were used to detect the presence of bacterial biofilm.In addition, biofilm formation was investigated by SEM examination of sections taken from the dental unit water drain pipes.

i. Congo red agar method
Staphylococcus spp.isolates were cultivated in Congo Red Agar (KRA) prepared by adding 37 g Brain Hearth Infusion Broth (Merck ® , Germany), 10 g agarose, 50 g sucrose, and 0.8 g Congo red indicator (Merck ® , Germany) to 1 L of distilled water.It was incubated at 37 C for 24-48 h.As a result of reproduction, orange-pinkish colonies were interpreted as negative in terms of the presence of biofilm, and brown-black colonies were interpreted as positive.
ii. Christensen tube method A single colony was taken from Staphylococcus spp.isolates and inoculated into Tryptic Soy Broth (Conda Lab ® , Spain) medium containing 5 mL of 0.25% glucose and incubated at 25 C for 24 h.After the incubation, the contents of the tubes were emptied and kept at room temperature with 1% saffron solution for 30 min, the dye was poured, and the tubes were washed twice with sterile PBS, turned upside down and left to dry on blotting paper.The next day, it was evaluated as (+), (++), (+++), and (À) according to the formation and density of a colored film layer on the inner surface of the tube.

iii. SEM Imaging biofilm formation with SEM
In order to examine the presence of biofilm by SEM, 1-cm 2 surface width and 1-cm long sections were taken from the water inlet hose of the unit before sampling and after disinfectant applications.Electron microscopy examination were carried out at Selçuk University Advanced Research and Application Center (Konya, Turkey).
Samples were soaked for 2 h in 0.1 mol pH = 7.2 cacodylate buffer containing 2.5% glutaraldehyde and washed three times with PBS.Samples were fixed in cacodylate buffer containing 1% osmium tetroxide for 1 h at 4 C.After drying, dehydration and critical drying processes were applied and gold-palladium plating was performed.
The samples were placed in the sample holder with the help of double-sided carbon tape and examined at 15 kVp, 1000Â magnification (Zeiss, EVO LS10, Germany).

| RESULTS
The total colony counts of the water samples at 0 min were evaluated as self-control and the average value was found to be 10.3 Â 10 4 CFU/mL by DSM and 8.25 Â 10 4 CFU/mL by SSM.
In colonies treated with HOCl and counted by DSM; a decrease of approximately 1 log CFU/mL is observed in the first unit; a decrease of 2-logs CFU/mL occurred between the 0th and 1st minute in the third unit, where the growth was most intense.No growth was observed at the 20th minute in the first unit, and at the 10th minute in the second and third units.
When the total colony numbers between the units where ClO 2 is treated with the DSM are compared; a decrease of more than 1 log CFU/mL was observed between the 0th and 1st minute in the third unit, where the growth was most intense.After the 10th minute in the first unit after the ClO 2 application, in other units, growth was not observed after the 5th minute.
Considering the results in the conventional SSM applied groups; in units using both HOCl and chlorine dioxide, the differences between minutes were less than 1-log CFU/mL.While growth was not observed after the 10th minute in only the first unit, in which HOCl was used, growth could not be detected after the 5th minute in all the other five units.
F I G U R E 1 Monitoring bacterial growth in dental unit water samples after 72 h at room temperature with Dipslide ® .
Total colony counts of the water samples were determined by DSM and SSM, and the data obtained in each time period and according to the disinfectant applied are summarized in Table 1.
When the DSM and SSM were compared in both groups (units treated with ClO 2 and HOCl), it was observed that the DSM can detect bacterial growth even during periods of greater exposure to the disinfectant application (Table 2).
According to these results; although a value close to 3% could be obtained even in the 10th minute in the units where HOCl was applied with the DSM; it was determined that the conventional surface spreading method did not show any growth at the same disinfectant exposure and duration.On the other hand, in the units where ClO 2 was applied with the DSM, no growth was observed in the 10th minute, while in the units where HOCl was applied, the 50% growth observed in the first minute could not be detected in the 5th minute (Figure 2).
Staphylococcus spp.isolates were accepted as the predominant microorganism and were identified as S. aureus due to the positivity of catalase, plasma coagulase, and beta-hemolysis formation tests on blood agar, and their golden yellow pigment formation.Methicillin resistance was investigated in all isolates using the Kirby-Bauer disc diffusion method with cefoxitin (30 μg) and oxacillin (1 μg) discs, and all isolates were found to be sensitive to methicillin.
A brownish-black colony forming biofilm was observed on Congo red agar in only one of the S. aureus isolated from four different units.
The biofilm-forming capacity of the same isolate was determined as (+) by the Christensen tube method.Other isolates were evaluated as biofilm negative by this method.The results of these two methods used to investigate the presence of biofilm were found to be 100% compatible with each other.
In the examinations of the sections taken from the hose surfaces of the unit water systems with SEM before and after the disinfectant (Figures 3 and 4), the presence of biofilm-forming bacteria is observed in an intense amount before the disinfectant, and there is a noticeable decrease in the 30 minute exposures (Figure 5a,b).

| DISCUSSION
It is important to detect etiologic agents in the dental unit water lines that may not cause problems in healthy individuals but may cause serious infections such as pneumonia and meningitis in immunocompromised individuals (Hussain Akbar et al., 2023).Many studies have reported in many studies that pathogens such as Acinetobacter spp, T A B L E 1 Number of growing colonies determined by dipslide and surface smear methods in chlorine dioxide and hypochlorous acid treated units (CFU/mL).F I G U R E 2 Percentage change in colony counts by dipslide method (DSM) and surface smear method (SSM) after hypochlorous acid (HOCl) and chlorine dioxide (ClO 2 ) applications.
F I G U R E 3 Biofilm layer observed in the dental unit water systems before chlorine dioxide (ClO 2 ) application (0th min.).
F I G U R E 4 Biofilm layer observed in the dental unit water systems before hypochlorous acid (HOCl) application (0th min.).
Legionella pneumophila, Pseudomonas aeruginosa, and Staphylococcus aureus with multi-drug resistant, especially in chronic patients and immunocompromised individuals, colonize the dental water systems and create a wide range of infections ranging from osteomyelitis to infective endocarditis, meningitis to encephalitis (Aprea et al., 2010;Costa et al., 2015).
In dental applications, bioaerosol formation can be observed as a result of the air-water combination used to cool high-speed instruments during treatment with oral biological fluids such as blood and saliva (Cheng et al., 2021;Hussain Akbar et al., 2023).These bioaerosols, which are contaminated with bacteria, viruses, and fungi, can remain suspended in the air for a long periods of time, thus infect people in the environment (AteŞ, 2020).Therefore, dental procedures are considered high-risk procedures that can result in the spread of infection to patients through aerosol formation.Studies examining the dispersal distance of dental aerosol have reported that bacteria can spread up to 1.5 meters, smaller viruses can spread further, and highly virulent pathogens such as SARS-CoV can spread up to 1.8 meters (Kutter et al., 2018;Láng et al., 2021).As a result of direct contact with water contaminated with fungi such as Candida and Aspergillus or inhalation of aerosols emitted by high-speed micromotors during dental treatment, asthma, allergies and various respiratory diseases may develop, especially in immunocompromised patients and in dentists and dental assistants in the occupational risk.
The American Dental Association recommends that the total number of bacteria in the tap water exposed to dental patients should not exceed 200 CFU/mL (Pullar et al., 2000).The present study found contamination far above these levels prior to disinfectant exposure.
For this reason, it is recommended to perform periodic checks to prevent the risk of cross-contamination, especially in older dental units, and to clean them regularly with a non-toxic, biocompatible disinfectant for the patient (Tada et al., 2006).
The antimicrobial effectiveness of the solutions used in the dental unit water system is very important.The disinfectant water in the system comes into contact with the soft and hard tissues in the mouth during dental procedures.Therefore, when selecting the appropriate agent, consideration should be given to issues such as interaction with dental materials, effects on dental hard tissues, effects on adhesive bonding during restorative procedures, and biocompatibility.
In terms of interaction with dental materials, HOCl has been found to increase the bond strength between the tooth and self-adhesive resin cement (Totaro et al., 2022).In terms of biocompatibility, HOCl is an agent with antimicrobial activity, which is naturally produced by the human immune system to fight infection (Zoni et al., 2007) and has been reported to show similar antibacterial activity to NaOCl on E. faecalis, but much lower toxicity than NaOCl (Travis et al., 2022).
It has been reported that the use of ClO 2 as a cavity disinfectant during dental restorative procedures shows high antimicrobial properties against oral pathogens without affecting the bond strength of dentin (Zemouri et al., 2020).In a study evaluating the cytotoxic effect of 0.0025% (25 ppm) ClO 2 on periodontal ligament stem cells at different dilutions compared to conventional mouthwash agents (H 2 O 2 , CHX); It has been observed that ClO 2 can kill bacteria 10 times more powerfully than CHX, but is 21 times less effective in killing human periodontal ligament cells, and there is a difference of about 200 times between the two compounds when their selectivity is taken into account (Rossi-Fedele et al., 2010).These features give ClO 2 a great advantage over conventional disinfectants (Rossi-Fedele et al., 2010).
The antimicrobial and disinfection efficiency of the water in the dental unit system is important in terms of protecting both the patient and the dental team from aerosol-induced infections.In the present study, the effects of HOCl and ClO 2 solutions on biofilm were investigated.HOCl is recommended for the disinfection of endoscopes, dental unit water systems of dental units, and dental impression materials (Jones & Brosseau, 2015).Like HOCl produced by human immunity by the myeloperoxidase-H 2 O 2 -Cl system of phagocytic cells, HOCl produced artificially for disinfection can fight invading pathogens and infections (Hortac Istar et al., 2020;Tazawa et al., 2023;Zemouri et al., 2020;Zoni et al., 2007).In one study, it has been reported that HOCl had a toxic effect on bacteria by completely disrupting bacterial ATP production (Shajahan et al., 2016), and in another study, HOCl solution was effective to reduce the risk of oral pathogens and SARS-CoV-2 viruses even after the dental unit had passed through the waterline, and airborne infection in dental practice (Liebers et al., 2015).Control of microbiological contamination in dental unit water lines; It is often achieved by the chlorine-based disinfectants, but short-term exposures are known to reduce bacterial colonization but not remove biofilm (Watamoto et al., 2013).
In this study, the effects of ClO 2 and HOCl on biofilm were investigated and bacterial growth was inhibited in dental units between 5 and 10 min with both disinfectants.These times were determined as 10 min in HOCl and 5 min in ClO 2 .In similar studies, it has been reported that the 10 min effectiveness of 200 ppm ClO 2 in the disinfection of dental instruments provides bacterial inhibition and can be an alternative to toxic disinfectants such as glutaraldehyde and sodium hypochlorite (Barnhart et al., 2005;Hsieh et al., 2020).In the current study, it was observed that it inhibited bacterial growth of 200 ppm ClO 2 application for 10 min.In a similar study, it was determined that 10 and 40 ppm derivatives of HOCl were treated in polyethylene dental unit water systems for 10 days and inhibited biofilm-forming bacterial growth by 98% (Hatanaka et al., 2021).It has been reported that the inhibitory effect of HOCl on E. coli is approximately 10 times greater than that of hydrogen peroxide, and its 100 ppm solution reduces Staphylococcus growth by more than 99% (Zemouri et al., 2020).
ClO 2 is a biocompatible disinfectant that is widely used in water treatment, surface disinfection, mouthwashes, oxidation, and bleaching (Kadaifciler et al., 2013).It also has strong bactericidal and virucidal activity against enveloped/non-enveloped viruses (Barrette Jr. et al., 1989;Soysal et al., 2020).It is stated that ClO 2 inactivates ≥99.9% of viruses in 15 s with effective antiviral activity at concentrations ranging from 1 to 100 ppm, and its antiviral activity is approximately 10 times higher than NaOCl (Kettering et al., 2002;Totaro et al., 2022).
In a study by Costa et al., in which they investigated the effectiveness of hydrogen peroxide and EDTA-based disinfectants on the biofilm layer in dental units, a disinfection protocol was created based on two approaches: curative and prophylactic.In the curative approach, disinfectant treatment was applied to the biofilm layer artificially produced on well plates for 15 min, and in the prophylactic approach, disinfectant treatment was applied for 30 min for 7 days (Costa et al., 2016).In this study, inhibition of bacterial growth was achieved within 10 min with both disinfectant treatments on the biofilm layer that formed naturally and over time and this period was found to be sufficient to adapt the protocol to be practically applied in the daily maintenance of the units.
In a study investigating the effects of disinfectant applications with manual and automated methods on biofilm eradication in old and new units, positive results were obtained after manual and automated applications for biofilm inhibition in both groups (Tirali et al., 2016).In the study, it was emphasized that if the disinfectants applied periodically manually cannot be done regularly by the personnel, an automated system can be used.In our study, disinfectant applications were made manually, and initial inhibition applications were investigated.Regular disinfectant treatment and growth control with rapid dip slide tests to monitor biofilm formation can ensure effective use of units, even if they are old.
It is stated that due to these properties, it may be useful in reducing the coronavirus pandemic (COVID-19) and the use of chlorine dioxide-containing mouthwashes before high-risk dental procedures may be beneficial in minimizing the possibility of COVID-19 transmission (Kadaifciler et al., 2013;Kalay et al., 2022).It has been reported that ClO 2 is useful in preventing contamination of dental unit water systems by waterborne pathogens, and residual chlorine activity is effective against airborne bacteria at least 1 m away from the aerosol source (Yerliyurt & Hatirli, 2023).Considering the current literature, the antimicrobial effect of the solution in the form of an aerosol, which is preferred for disinfection of units, can reduce the risk of infectious diseases for both patients and staff in dental practices.
In the present study, bacteria were not measured in dental aerosol, but it was observed that both solutions were effective in biofilm formation and ClO 2 had a faster effect.According to these findings, the use of ClO 2 can be recommended for disinfection of dental unit water systems.
Two methods were used in the current study to compare the antimicrobial efficacy of these disinfectants and to evaluate the practical methods that will provide contamination control along with the conventional methods.In the determination of the bacterial colony count, the DSM and the conventional SSM were compared, and in the reproduction studies carried out with the dipslide kit, a growth rate of 3% was observed in the units treated with HOCl even at the 10th minute, while no growth was observed in the SSM.
Similarly, a faster and more effective result was observed in units treated with ClO 2 compared to HOCl, with 12.27% growth after 5 min in DSM, while no growth was detected after 1 min in SSM.
These results suggest that the dipslide kit can be considered a more sensitive method for detecting bacterial growth compared to conventional methods.
Although rapid methods such as ATP activity, endotoxin content, and metabolic by-product determination have been used to detect microbial contamination of drinking and potable water, such tests require a laboratory and experienced personnel (Fulford et al., 2004;Ra'fat, 2019).There is no doubt that the DSM used in this study can be used without the need for a laboratory environment and equipment and will facilitate dentists and their assistants in controlling the contamination of tap water in dental clinics.Of course, this method does not include classical identification methods in terms of microbiological evaluation and does not provide information about colony morphology.However, when evaluated independently of species identification, it provides a sensitive and very practical determination of contamination and becomes preferable for periodic maintenance of the units.
The limitation of the current study; molecular-based methods such as 16 s rRNA sequencing methods are not used and not all genera and species can be detected as contaminants in water.However, a biofilm-forming strain of Staphylococcus aureus, which is a serious clinical threat, was isolated.Methicillin resistance was also tested in the S. aureus isolates obtained in the study and all isolates were found to be susceptible to methicillin.It is an inescapable fact that methicillinresistant S. aureus (MRSA) infections pose a serious threat, especially to the elderly and immunocompromised patients (Bulut & Kızılkaya, 2007).Another limitation in our study; is the small number of units.Chambered and old units were preferred as the unit criteria we determined in testing disinfectant exposure, and the number of units providing these criteria in the center where the study was conducted was six.
The compatibility of the classical methods (Congo red method, Christensen tube method) and the SEM results used in the present study for the determination of biofilm shows that the research has reached its experimental goal.It is highly probable that the biofilmforming microorganisms shown in the SEM belong to the species that we could not isolate as water contaminants in the study.The relationship between the (+) result in the Christensen tube method used to detect biofilm in the study and the growth pattern in Congo red agar and the methicillin susceptibility of S. aureus we isolated; is consistent with the results of the previous study (Hortac Istar et al., 2020).
In the current study, the presence of weakly positive biofilm was observed in MSSA isolates using conventional methods; strong biofilm positivity was found at a higher rate in MRSAs.

| CONCLUSION
Considering the limitations of the present study, it was concluded that it is important and advisable to routinely disinfect the water systems of dental units with non-toxic doses of ClO 2 application before treating patients in clinics, and also to periodically check the contamination by the dipslide method, which is a fast, sensitive, and very practical method.

F
I G U R E 5 (a, b) SEM images of unit sections after hypochlorous acid (a) and chlorine dioxide (b) administration (30th min.).
T A B L E 2 Average values of total colony numbers (CFU/mL) determined by DSM and SSM methods according to the application times of ClO 2 and HOCl.