Water from dental unit waterlines (DUWLs) may be a potential source of infection for both dental health care personnel and patients. During dental treatment, both patient and personal are exposed to direct contact with bacteria contaminated water in the form of splatter and with contaminated water aerosol emitted during work by units. Many studies have shown that the output water of dental units is colonized with microorganisms including environmental bacteria, opportunistic and true human pathogens and organisms commonly found in the oral cavity. The center for diseases control and prevention (CDC) recommends that coolant water used in non-surgical procedures meets the Environmental Protection Agency’s (EPA), regulatory standards for drinking water, which is less than or equal to 500 colonies forming units (CFUs) of heterotrophic bacteria per milliliter of water. Researchers reported contamination of DUWL water at the level from 1.5 × 10 2 to 1 × 10 6 cfu/ml. In most cases, the values of bacterial contamination exceed the norms accepted for potable water both in USA and European Union, as well as recommendations for water used in conservative dental treatment. Such recommendation does not exist in Saudi Arabia. Most of the microorganism in dental unit water line are not pathogenic in healthy individuals but, may be of great important in patient with systemic. Although the result of some epidemiologic studies shows that contamination of DUWLs can be dangerous in patients with immune deficiency or other immune system problem, it can be true for pregnant women, elderly, graft recipient or even smokers. The organisms can cause pneumonia, other respiratory infection, or wound infection in immunocompromised people. Water at the tubing walls is almost stagnant, allowing bacteria to adhere and colonize the tubing surface. Biofilms are imbedded in a polysaccharide slim layer, or glyoxalin, which facilitates adherence and also protects the biofilm from desiccation the chemical insult. The microorganisms are capable of forming biofilm on the surfaces of DUWLs as well as on heart valves, creating endocarditis. A high level of microbial contamination, presence of opportunistic microorganism and bacterial endotoxin associated with gram negative bacteria are the most important health risk factors transmitted by water from dental units, which release endotoxin during bacterial growth phase and upon lysis. Despite numerous reports of gram negative bacteria dental water, there are few published reports investigating endotoxin concentration in DUWLs and the indoor air of dental clinics. Literature review: Protection of patients and dental personnel needs the appropriate microbiological water quality in dental units. DUWL contamination is universal, and water in dental units is richly colonized by bacteria and may assume considerably varying values. The DUWL contamination was first reported by Shepherd et al.,2001, samples were isolated from the DUWL bacteria typical for potable water using a R2A agar medium. Human pathogenic microorganisms such as Legionella pneumophila, Mycobacterium sp. and Staphylococcus aureus have already been isolated from DUWLs. Pseudomonas aeruginosa has been isolated from 15–30% of DUWLs. Only a small number of published studies deal with cases of infections associated with dental caries. But the obvious concern is that large numbers of potentially pathogenic microorganisms may be swallowed, inhaled or alternatively inoculated into oral wounds during dental treatment with a potential for both colonization and infection. The differences in biofilm characteristics noted in the intermittent systems at the end of the study, e.g., slight increase in the friability and decrease of bulging, may suggest that intermittent systems, while reducing the capacity of the biofilm to spread on the surfaces, tends to favor detachment, thus enhancing the dispersion of the microorganisms in the water. In Saudi Arabia, the only published paper showed that the most common bacteria found in the DUWL contamination were Bacillus spp. (29.6%) and Pseudomonas spp. (22.8%). It is not the only presence of bacteria that is important in DUWL contamination monitoring, but their number and the presence of potential pathogens. Numerous studies emphasize the need for effective mechanisms to reduce the microbial contamination in DUWL to control cross-infection in general practice, especially increasing number of immunocompromised persons who attend dental clinics. INTRODUCTION OBJECTIVES The study was carried out during 14 of May to 6 of December 2017. A total of 432 samples of DUWLs (Figure 1) were collected from 24 clinics in the Government Dental College. The experiments were performed at the laboratories of the Prince Sultan Military Medical City in Riyadh. All tested clinics were connected to municipal water. Those clinics were divided into two groups, Group A and group B. Group A clinical units hadn't ever been treated with any disinfectant. While group B clinical units had been used descaling solution with citric acid. `Samples were collected twice a day: the first one was before the start of the clinic day, three times (before flushing, after 1- min flushing and after 3-minute flushing) of the DUWL. then samples were collected again three times same as the first one in the end of the clinic day. 500 ml of each DUWL sample was collected in a sterile container using high speed drill hand piece lines (HP), air/water dental- syringe lines(WS) and oral rains main water supply (OR) on each of the dental units. In addition to the source of tap water in the dental clinic as a control. Samples were then transferred immediately to the lab for samples processing. Identification and detection of pathogenic bacteria from samples of DUWLs by membrane filtration technique (MFT) and culturing pathogenic bacteria In order to isolate and identify bacteria, 100 ml of each selected water sample was filtered through a sterile 0.45µ membrane. Membranes were then removed from the funnel using sterile forceps and deposited on the surface of the following culture medium blood agar for Bacillus sp., MacConkey Agar, Xylose lysine deoxycholate agar (XLD) for gram negative rods, cetrimide agar for Pseudomonas aeruginosa and mannitol salt agar for Staphylococcus sp. Duplicate plates were constructed for each sample to verify accuracy. After incubation, the initial identification of bacteria was performed by examine of the bacterial colonies characteristics, gram stain, biochemical tests, API system and MicroScan Walkaway automated systems, according to manufacturer’s instructions. Antibiotic susceptibility of bacteria isolated from DUWLs samples Susceptibility to antibiotics was tested for selected isolated bacteria by using standard disc diffusion method (zones of inhibition were measured and interpreted as recommended by the National Committee for Clinical Laboratory Standards- NCCLS) and automated MicroSacn. Determination of Endotoxin concentrations using Limulus amebocyte lysate (LAL) In order to evaluate endotoxins concentrations. 10 ml of each water samples were analyzed using rapid endotoxin test the Limulus amebocyte lysate (LAL reacts with bacterial endotoxin lipopolysaccharide (LPS), which is a membrane component of gram negative bacteria.) according to the manufacturer’s instructions. Separate water samples were collected into endotoxin- and pyrogen-free containers. Detection the Biofilm using Scanning Electron Microscope (SEM) Selected samples of air/water syringes were obtained for biofilm detection. 15 mm-length of each tube was cut longitudinally in center into two pieces. All samples were transferred immediately in a cool box to the laboratory. The collected biofilm samples were fixed by immersion in glutaraldehyde 50% over night at 4°C. Then they were washed by phosphate buffers (pH=7.2). Then the samples were exposed to Osmium Tetroxide 1% (second fixation) for one hour at 4°C. After that, the samples were dehydrated using solutions of ethanol. Prior to SEM analysis, the samples were coated using gold. MATERIALS AND METHODS Enumeration of the bacteria in DUWLS samples Average of Contamination level for each flushing time interval from different types of DUWLs samples (CFU/ml) from building A before and after work. HP= High speed hand piece, WS= Air / water dental syringe, OR= Oral rinse Main water supply, BW= Before work, AW= After work, BF= Before flushing, 1 min AF= 1 min After flushing, 3 min AF= 3 min After flushing. Identification and detection of pathogenic bacteria from samples of DUWLs by membrane filtration technique (MFT) and culturing pathogenic bacteria occurrence of opportunistic bacteria in water samples from DUWLS. Antibiotics Susceptibility Patterns among Isolated bacteria from different water samples of DUWLS from building A S= Sensitive to all tested antibiotics, R to 1= resistant to one antibiotic, MR= multi- resistant to 2 antibiotics or more. Determination of Endotoxin concentrations using LAL Endotoxin levels measurements in water samples of DUWLs after 3min flushing from building A and B Detection the Biofilm using Scanning Electron Microscope (SEM) A B C D E F Figure: SEM micrographs represented bulging biofilm formation inside in the inner walls of used tube ( A & B) from building A, (C & D) from building B. E & F showed dispersed rod shape bacteria inside unused tubes (control). RESULT CUNCSION The results of this study demonstrated that the highest percentage of pathogenic bacteria in DUWLs is Pseudomonas aeruginosa. It is also showing a different pattern of antibiotics and we believe that this is the reason for biofilm formation inside in the inner walls of the tube and for the bacteria releasing endotoxin. REFRENCES 1. Al-Saif KM, Assery M, Nahas MA: Microbial contamination of dental unit water systems in Saudi Arabia. Saudi Dent J 2007, 19, 110-114. 2. Atlas, Ronald M., Jeffrey F. Williams, and Mark K. Huntington. 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"Waterline contamination and role of flushing dental water unit lines in private dental clinics of Mangalore." Original research. Ha(2010)l: 121-125. 18.Singh, T., and M. M. Coogan. "Isolation of pathogenic Legionella species and legionella-laden amoebae in dental unit waterlines." Journal of Hospital Infection 61.3 (2005): 257- 262. 19.Singh, Tanusha S., et al. "Workplace determinants of endotoxin exposure in dental healthcare facilities in South Africa." Annals of occupational hygiene 54.3 (2009): 299-308. 20.Szymańska, Jolanta, Jolanta Sitkowska, and Jacek Dutkiewicz. "Microbial contamination of dental unit waterlines." Ann Agric Environ Med 15.2 (2008): 173-179. 21.Szymańska, Jolanta, Jolanta Sitkowska, and Jacek Dutkiewicz. "Microbial contamination of dental unit waterlines." Ann Agric Environ Med 15.2 (2008): 173-179. 22.Szymańska, Jolanta. "Exposure to bacterial endotoxin during conservative dental treatment." Annals of agricultural and environmental medicine: AAEM 12.1 (2005): 137-139. 23.Tambekar, D. H., et al. "Prevalence of Pseudomonas aeruginosa in dental unit water-lines." Res J Microbiol 2 (2007): 983-987. ACKNOWLEDGMENTS In this project the objectives were: • To determine the qualitative and quantitative contamination of water in dental unit reservoirs with aerobic and facultative anaerobic bacteria. • To provide an overview of bacterial biofilm formation and endotoxin in DUWLs. • To find out how time dependent waterline flushing affects the levels of planktonic bacteria, untreated and contamination DUWLs in large institutional treatment clinics. • To compare between the contamination level with the recommendation of regulatory standards for drinking water in Saudi Arabia. Correspondence to Thekra Shouil. E.mail: [email protected] Thekra Shouil, M Alkhulifi, D Alotaibi, H Alajlan Detection the presence of pathogenic bacteria from Dental waterline systems Deanship of Scientific Research “The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through the Undergraduate Research Support Program, Project no. (URSP – 17-89)” Site of collection Clinic ID OR (before work/after work) WS (before work/after work) HP (before work/after work) 3 min AF 1 min AF BF 3 min AF 1 min AF BF 3 min AF 1 min AF BF A.W B.W A.W B.W A.W B.W A.W B.W A.W B.W A.W B.W A.W B.W A.W B.W A.W B.W 3162.5 2587 3515.5 2971.5 4920 4623.5 3029 3104.5 3268 3580 4645.5 3850.5 3361 3621 3637.5 3765.5 3024.5 4368.5 1 2040 3152.5 3331.5 3591 2541 2852 3328 3048.5 2028 4464 2405.5 3125.5 2715 2659.5 1618.5 2294 3164 3635.5 2 3235.5 3551.5 3482 3719 4195.5 4111 2306 2101 3650 3718.5 1447.5 1383 3311 3396.5 2346.5 2498 3743.5 3745 3 305 1739.5 1991 1344.5 859.5 1289.5 1001.5 1589 999 2161 1539 1690 601.5 2342.5 1622.5 2024.5 2046.5 2269 4 2041 2011 1985 3029 1982.5 3640.5 1540 1564.5 3129 2026 2096 2263.5 2582.5 2071 2071 2236 3078 3174 5 2929 3263.5 3391 3727.5 3299.5 3395.5 2282 3006 3077 3506 4358.5 4363.5 3007.5 2998 3343 3685.5 3495.5 4040 6 1627 1183.5 2333.5 1634.5 2945.5 3260 1569.5 2042 2411 2294 2989.5 3026 1539.5 2427 2026 3575.5 2532.5 3984.5 7 1539 995.5 1568.5 3054.5 2675.5 3019 1285.5 1636.5 846.5 2452.5 2007.5 4070 2026 2010 2330 2617.5 2970 3045.5 8 1993.5 1883.5 2285.5 2280.5 3500.5 3290.5 1969 2016 2865.5 2700.5 3055 2976.5 990 990 2270.5 2225.5 2214 2032 9 3420 2996 3634 3781 3260.5 3750 2795 2710.5 2108 2364.5 3490 3045.5 3330 2107 3560 3018 3535 3605 10 1995.5 1446.5 2454.5 1623 2091.5 3205.5 1600.5 1986 2074 2635 3994 3326 1989.5 2247 1978.5 3111 3990 3620 11 2050 2265 2457 2510 3566 3482.5 1896.5 2004 3203 3170 3085 2180 2089.5 2942.5 2450 3228 3839 3490 12 2003 2070 2903.5 3267 2091.5 2620 1988.5 1989 2743 2456 3467.5 3228.5 2144 2291.5 2397 2737 3867.5 2883.5 13 Bacterial Site of collection NO. of isolated bacterial (%) among 66 HP NO. of isolated bacterial (%) among 66 WS NO. of isolated bacterial (%) among 66 OR Total NO. of bacterial isolates (%) out of 198 DUWLs Gram positive Staphylococcu s aureus 55 (83%) 33 (50%) 28 (42%) 116 (58.5%) Staphylococcu s auricularis 42 (63%) 27 (40%) 29 (43%) 98 (49.4%) Gram negative bacterial Pseudomonas aeroginosae 62 (93%) 65 (98%) 62 (93%) 189 (95.4%) Pseudomonas fluorescens 21 (31%) 42 (63%) 25. 25 88 (44.4%) Acintobacter baumanii 20 (30%) 21 (31%) 0 41 (20.7%) S.aureus S.auricularis P.aeroginosae P.fluorescens A.baumanii NO. OF ISOLATED BACTERIAL (%) AMONG 66 HP S.aureus S.auricularis P.aeroginosae P.fluorescens NO. OF ISOLATED BACTERIAL (%) AMONG 66 OR S.aureus S.auricularis P.aeroginosae P.fluorescens A.baumanii NO. OF ISOLATED BACTERIAL (%) AMONG 66 WS S.aureus S.auricularis P.aeroginosae P.fluorescens A.baumanii TOTAL NO. OF BACTERIAL ISOLATES (%) OUT OF 198 DUWLS Building A Site of collection (numbers) Mean endotoxin level Building B Site of collection (numbers) Mean endotoxin level WS (3) > 5.00 EU/ml WS (3) 1.33 EU/ml HS (20) > 5.00 EU/ml HS (14) > 5.00EU/ml OR (4) > 5.00 EU/ml HS (6) 2.4 EU/ml OR (4) 1.35 EU/ml No. of bacterial isolates Oral rinse Main water supply NO. (%) Air / water dental syringe No. (%) High speed hand piece No. (%) OR S* R to 1 MR WS S R to 1 MR HP S R to 1 MR P.aeroginosae 54 18 2 (11.1) 0 16 (88.8%) 18 11 (61.1%) 1 (5.5%) 6 (33.3) 18 7 (38.8) 2 (11.1) 9 (50) A.baumanii 36 0 0 0 0 18 0 5 (27.7) 13(72.2 ) 18 2 (11.1) 2(11.1) 14 (77.7) S. aureus 54 18 4 (22.2) 2 (11.1) 12 (66.6) 18 2 (11.1) 2 (11.1) 14 (77.7) 18 8(44.4) 1 (5.5) 9(50) Bacillus sp. 54 18 6 (33.3) 2 (11.1) 10 (55.5) 18 10(55.5 ) 2 (11.1) 6 (33.3) 18 4 (22.2) 1(5.5) 13 (72.2) P.flourecenses 54 18 4 (22.2) 4(22.2) 10 (55.5) 18 2 (11.1) 4 (22.2) 12(66.6 ) 18 5 (27.7) 3 (16.6) 10(55.5 ) S. auricular 54 18 3 (16.6) 6 (33.3) 9 (50) 18 0 3 (16.6) 15 (83.3) 18 5 (27.7) 5 ( 27.7) 8 (44.4)