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Water from dental unit waterlines (DUWLs) may be a potentialsource of infection for both dental health care personnel andpatients.During dental treatment, both patient and personal are exposedto direct contact with bacteria contaminated water in the formof splatter and with contaminated water aerosol emitted duringwork by units.Many studies have shown that the output water of dental unitsis colonized with microorganisms including environmentalbacteria, opportunistic and true human pathogens andorganisms commonly found in the oral cavity.The center for diseases control and prevention (CDC)recommends that coolant water used in non-surgicalprocedures meets the Environmental ProtectionAgency’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 thelevel from 1.5 × 102 to 1 × 106 cfu/ml.In most cases, the values of bacterial contamination exceed thenorms accepted for potable water both in USA and EuropeanUnion, as well as recommendations for water used inconservative dental treatment. Such recommendation does notexist in Saudi Arabia.Most of the microorganism in dental unit water line are notpathogenic in healthy individuals but, may be of greatimportant in patient with systemic. Although the result of someepidemiologic studies shows that contamination of DUWLscan be dangerous in patients with immune deficiency or otherimmune system problem, it can be true for pregnant women,elderly, graft recipient or even smokers.The organisms can cause pneumonia, other respiratoryinfection, or wound infection in immunocompromised people.Water at the tubing walls is almost stagnant, allowing bacteriato adhere and colonize the tubing surface.Biofilms are imbedded in a polysaccharide slim layer, orglyoxalin, which facilitates adherence and also protects thebiofilm from desiccation the chemical insult. Themicroorganisms are capable of forming biofilm on the surfacesof DUWLs as well as on heart valves, creating endocarditis.A high level of microbial contamination, presence ofopportunistic microorganism and bacterial endotoxinassociated with gram negative bacteria are the most importanthealth risk factors transmitted by water from dental units,which release endotoxin during bacterial growth phase andupon lysis. Despite numerous reports of gram negative bacteriadental water, there are few published reports investigatingendotoxin concentration in DUWLs and the indoor air ofdental clinics.Literature review:Protection of patients and dental personnel needs theappropriate microbiological water quality in dental units.DUWL contamination is universal, and water in dental units isrichly colonized by bacteria and may assume considerablyvarying values.The DUWL contamination was first reported by Shepherd etal.,2001, samples were isolated from the DUWL bacteriatypical for potable water using a R2A agar medium.Human pathogenic microorganisms such as Legionellapneumophila, Mycobacterium sp. and Staphylococcus aureushave already been isolated from DUWLs. Pseudomonasaeruginosa has been isolated from 15–30% of DUWLs.Only a small number of published studies deal with cases ofinfections associated with dental caries. But the obviousconcern is that large numbers of potentially pathogenicmicroorganisms may be swallowed, inhaled or alternativelyinoculated into oral wounds during dental treatment with apotential for both colonization and infection.The differences in biofilm characteristics noted in theintermittent systems at the end of the study, e.g., slight increasein the friability and decrease of bulging, may suggest thatintermittent systems, while reducing the capacity of the biofilmto spread on the surfaces, tends to favor detachment, thusenhancing the dispersion of the microorganisms in the water.In Saudi Arabia, the only published paper showed that the mostcommon bacteria found in the DUWL contamination wereBacillus spp. (29.6%) and Pseudomonas spp. (22.8%).It is not the only presence of bacteria that is important inDUWL contamination monitoring, but their number and thepresence of potential pathogens. Numerous studies emphasizethe need for effective mechanisms to reduce the microbialcontamination in DUWL to control cross-infection in generalpractice, especially increasing number ofimmunocompromised persons who attend dental clinics.

INTRODUCTION

OBJECTIVES

The study was carried out during 14 of May to 6 of December2017. A total of 432 samples of DUWLs (Figure 1) werecollected from 24 clinics in the Government Dental College.The experiments were performed at the laboratories of thePrince Sultan Military Medical City in Riyadh. All testedclinics were connected to municipal water. Those clinics weredivided into two groups, Group A and group B.Group A clinical units hadn't ever been treated with anydisinfectant. While group B clinical units had been useddescaling solution with citric acid.`Samples were collected twice a day: the first one was beforethe 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 firstone in the end of the clinic day. 500 ml of each DUWL samplewas collected in a sterile container using high speed drill handpiece lines (HP), air/water dental- syringe lines(WS) and oralrains main water supply (OR) on each of the dental units. Inaddition to the source of tap water in the dental clinic as acontrol. Samples were then transferred immediately to the labfor samples processing.

Identification and detection of pathogenic bacteria fromsamples of DUWLs by membrane filtration technique(MFT) and culturing pathogenic bacteriaIn order to isolate and identify bacteria, 100 ml of eachselected water sample was filtered through a sterile 0.45µmembrane. Membranes were then removed from the funnelusing sterile forceps and deposited on the surface of thefollowing culture medium blood agar for Bacillus sp.,MacConkey Agar, Xylose lysine deoxycholate agar (XLD) forgram negative rods, cetrimide agar for Pseudomonasaeruginosa and mannitol salt agar for Staphylococcus sp.Duplicate plates were constructed for each sample to verifyaccuracy. After incubation, the initial identification of bacteriawas performed by examine of the bacterial coloniescharacteristics, gram stain, biochemical tests, API system andMicroScan Walkaway automated systems, according tomanufacturer’s instructions.Antibiotic susceptibility of bacteria isolated from DUWLssamplesSusceptibility to antibiotics was tested for selected isolatedbacteria by using standard disc diffusion method (zones ofinhibition were measured and interpreted as recommended bythe National Committee for Clinical Laboratory Standards-NCCLS) and automated MicroSacn.Determination of Endotoxin concentrations using Limulusamebocyte lysate (LAL)In order to evaluate endotoxins concentrations. 10 ml of eachwater samples were analyzed using rapid endotoxin test theLimulus amebocyte lysate (LAL reacts with bacterialendotoxin lipopolysaccharide (LPS), which is a membranecomponent of gram negative bacteria.) according to themanufacturer’s instructions. Separate water samples werecollected into endotoxin- and pyrogen-free containers.Detection the Biofilm using Scanning Electron Microscope(SEM)Selected samples of air/water syringes were obtained forbiofilm detection. 15 mm-length of each tube was cutlongitudinally in center into two pieces. All samples weretransferred immediately in a cool box to the laboratory. Thecollected biofilm samples were fixed by immersion inglutaraldehyde 50% over night at 4°C. Then they were washedby phosphate buffers (pH=7.2). Then the samples wereexposed to Osmium Tetroxide 1% (second fixation) for onehour at 4°C. After that, the samples were dehydrated usingsolutions of ethanol. Prior to SEM analysis, the samples werecoated using gold.

MATERIALS AND METHODSEnumeration of the bacteria in DUWLS samplesAverage of Contamination level for each flushing time intervalfrom different types of DUWLs samples (CFU/ml) frombuilding A before and after work.

HP= High speed hand piece, WS= Air / water dental syringe, OR= Oral rinse Mainwater 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 fromsamples of DUWLs by membrane filtration technique(MFT) and culturing pathogenic bacteriaoccurrence of opportunistic bacteria in water samples fromDUWLS.

Antibiotics Susceptibility Patterns among Isolated bacteriafrom 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 LALEndotoxin levels measurements in water samples of DUWLsafter 3min flushing from building A and B

Detection the Biofilm using Scanning Electron Microscope(SEM)

A B

C D

E FFigure: SEM micrographs represented bulging biofilmformation inside in the inner walls of used tube ( A & B) frombuilding A, (C & D) from building B.E & F showed dispersed rod shape bacteria inside unusedtubes (control).

RESULT CUNCSION The results of this study demonstrated that the highestpercentage of pathogenic bacteria in DUWLsis Pseudomonas aeruginosa. It is also showinga different pattern of antibiotics and we believe thatthis is the reason for biofilm formation inside in theinner walls of the tube and for the bacteria releasing

endotoxin.

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ACKNOWLEDGMENTS

In this project the objectives were:• To determine the qualitative and quantitative contamination of

water in dental unit reservoirs with aerobic and facultativeanaerobic bacteria.

• To provide an overview of bacterial biofilm formation andendotoxin in DUWLs.

• To find out how time dependent waterline flushing affects thelevels of planktonic bacteria, untreated and contaminationDUWLs in large institutional treatment clinics.

• To compare between the contamination level with therecommendation of regulatory standards for drinking water inSaudi Arabia.

Correspondence to Thekra Shouil. E.mail: Thekra.bander660@gmail.com

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 theDeanship of Scientific Research at King SaudUniversity for funding this work through theUndergraduate Research Support Program, Projectno. (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 AF1 min AFBF3 min AF1 min AFBF3 min AF1 min AFBF

A.WB.WA.WB.WA.WB.WA.WB.WA.WB.WA.WB.WA.WB.WA.WB.WA.WB.W

3162.525873515.52971.549204623.530293104.5326835804645.53850.5336136213637.53765.53024.54368.51

20403152.53331.535912541285233283048.5202844642405.53125.527152659.51618.5229431643635.52

3235.53551.5348237194195.541112306210136503718.51447.5138333113396.52346.524983743.537453

3051739.519911344.5859.51289.51001.51589999216115391690601.52342.51622.52024.52046.522694

20412011198530291982.53640.515401564.53129202620962263.52582.5207120712236307831745

29293263.533913727.53299.53395.522823006307735064358.54363.53007.5299833433685.53495.540406

16271183.52333.51634.52945.532601569.52042241122942989.530261539.5242720263575.52532.53984.57

1539995.51568.53054.52675.530191285.51636.5846.52452.52007.540702026201023302617.529703045.58

1993.51883.52285.52280.53500.53290.5196920162865.52700.530552976.59909902270.52225.5221420329

34202996363437813260.5375027952710.521082364.534903045.533302107356030183535360510

1995.51446.52454.516232091.53205.51600.5198620742635399433261989.522471978.531113990362011

205022652457251035663482.51896.5200432033170308521802089.52942.5245032283839349012

200320702903.532672091.526201988.51989274324563467.53228.521442291.5239727373867.52883.513

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

Staphylococcus aureus 55 (83%) 33 (50%) 28 (42%) 116 (58.5%)

Staphylococcus 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.auricularisP.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

Bui

ldin

g A

Site of collection

(numbers)

Mean endotoxin

level

Bui

ldin

g B

Site of collection

(numbers)

Mean

endotoxin

level

WS (3) >5.00 EU/ml WS (3) 1.33 EU/mlHS (20) >5.00 EU/ml HS (14) >5.00EU/ml

OR (4) >5.00 EU/mlHS (6) 2.4 EU/mlOR (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.aeroginosae54 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.baumanii36 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.flourecenses54 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. auricular54 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)