Evaluation of Antimicrobial Efficacy of Antibiotics and Calcium Hydroxide against Enterococcus faecalis Biofilm in Dentine

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The objective of this study was to investigate the antimicrobial efficacy of erythromycin, oxytetracycline and calcium hydroxide [Ca(OH)(2)] against Enterococcus faecalis biofilm in dentine. E. faecalis ATCC 29212 (American type culture collection) was inoculated into standard tooth sections and incubated in aerobic atmosphere at 37 degrees C for 21 days. The infected tooth sections were then exposed to the test agents for 5 and 10 min. The colony forming units (CFU) after the exposure periods at three different depths
  Sains Malaysiana 42(1)(2013): 73–80 Evaaton of Antcroba Efcacy of Antbotcs and Cac Hydroxde against  Enterococcus faecalis Bo n Dentne (Kajan Keberkesanan Antbotk Antkrob dan Kas Hdroksda ke AtasBoe  Enterococcus faecalis d Daa Dentn) W . L . C HAi*, H . H AmimAH & M . A BDullAHABSTRACT The objective of this study was to investigate the antimicrobial efcacy of erythromycin, oxytetracycline and calciumhydroxide [Ca(OH) 2 ] against  Enterococcs faecas biolm in dentine. E. faecas ATCC  29212 (American type culture collection) was inoculated into standard tooth sections and incubated in aerobic atmosphere at 37°C for 21 days. Theinfected tooth sections were then exposed to the test agents for 5 and 10 min. The colony forming units ( CFU  ) after the exposure periods at three different depths <100 µm, 100-350 µm and 350-500 µm were enumerated. After 5 min of exposure, both antibiotics had signicantly lower CFU  count than Ca(OH) 2 solution at three dentinal depths. Comparingwith the oxytetracycline, the CFU  count of the erythromycin was signicantly ( p <0.05) lower at the depth of 100-500 μm. Similarly, after 10 min of exposure, erythromycin had signicantly lower CFU  count ( p <0.05) at three dentinaldepths. Oxytetracycline showed signicantly lower CFU  count than Ca(OH) 2 at 100 μm depth. Comparing with the twoexposure times, the erythromycin and Ca(OH) 2 groups showed signicant lower CFU  counts after 10 min of exposure in the antimicrobial agents to 5 min. In conclusion, both antibiotics show better antimicrobial activity than Ca(OH) 2 inremoving the E. faecas biolm in dentine.Keywords: Antimicrobials; biolm; calcium hydroxide; dentinal tubules; Enterococcs faecas ABSTRAK Tujuan kajian ini adalah untuk mengkaji keberkesanan antimikrob eritromisin, okstetrasilisin dan kalsium hidroksida [Ca(OH) 2 ] ke atas biolem Enterococcs faecas di dalam dentin. E. faecas    ATCC  29212 (American Type CultureCollection) diguna untuk menjangkiti keratan piawai akar gigi manusia dalam keadaan atmosfera aerobik pada 37°C  selama 21 hari. Keratan akar gigi yang telah dijangkiti didedahkan kepada ejen ujian selama 5 dan 10 min. Selepas tempoh pendedahan, kiraan unit pembentukan koloni ( CFU  ) pada tiga kedalaman dentin, iaitu <100 µm, 100-350 µm dan 350-500 µm yang berbeza dibandingkan. Selepas pendedahan selama 5 min, kedua-dua jenis antibiotik didapati mempunyai kiraan CFU  yang kurang namun signikan berbanding Ca(OH) 2 pada ketiga-tiga kedalaman dentin. Berbanding denganokstetrasilisin, eritromisin menunjukkan kiraan CFU  yang jauh lebih kurang berbanding okstetrasilisin dan signikan ( p <0.05) pada kedalaman dentin 100-500 μm. Selepas pendedahan selama 10 min, eritromisin mempunyai kiraan CFU     yang paling rendah dan signikan ( p <0.05) pada ketiga-tiga kedalaman dentin.   Okstetrasilisin menunjukkan kiraan CFU    yang ketara kurang daripada Ca(OH) 2 pada kedalaman 100 μm. Dalam perbandingan masa pendedahan, eritromisin dan Ca(OH) 2 menunjukkan CFU  yang ketara kurang selepas pendedahan selama 10 min berbanding dengan 5 min. Secarakesimpulan, kedua-dua antibiotik menunjukkan aktiviti antimikrob yang lebih berkesan daripada Ca(OH) 2 terhadap biolem E. faecas pada dentin.Kata kunci: Antimikrob; biolem; kalsium hidroksida; Enterococcs faecas ; tubul dentin i NTRODuCTiON  Enterococcus faecalis (  E. faecalis ), a factatve anaerobe,has been coony soated fro persstent root cananfectons and post-treatent nfecton (moander et a.1998; Sndqvst et a. 1998). One of the vrence factorsassocated wth ths persstent presence of   E. faecalis in root canas s ts abty to nvade dentna tbes, adhereto coagen n the dentne (love 2001). An in-vitro stdyhas shown that abet wth the presence of ntracanaedcatons, the  E. faecalis was abe to for bo n theroot canas (Dste et a. 2002). Bo s a hghy organzedstrctre wth cps of bactera bond together by acarbohydrate atrx (Costerton et a. 1994). in addton,the  E. faecalis has the abty to srvve wthot dvdngn the bo (Dste et a. 2002) and ndced the apattereprecptaton, especay n a atre bo (Kshen et a.2006). Hence, these featres expan the ow ssceptbty of   E. faecalis bo towards severa antcroba agentswhen copared to ts other orphotypes sch as thepanktonc and peet fors (Abdah et a. 2005). The  74 poysaccharde coatng of the bo cod be the barrerto ost antcroba agents (Dste et a. 2002).Severa stdes have shown that the  E. faecalis is hghy resstant to the antbactera effect of cachydroxde [Ca(OH) 2 ] (Haapasao & Orstavk 1987;Orstavk & Haapasao 1990; Sqera & de uzeda 1996).Evans et a. (2002) reported that  E. faecalis can srvven an akane envronent as hgh as pH11.1 and codony be ked when pH reached hgher than 11.5. However,the pH of the Ca(OH) 2 n the cana cod ony reached pto 10.3 de to the bfferng effect of the radcar dentn(Evans et a. 2002).Knowng the taton of the standard ntracanaedcaton [Ca(OH) 2 ], varos ethods sch as the seof aser (mehrvarzfar et a. 2011; Sahar-Heft et a. 2011),trasonc (Grndng et a. 2011), dfferent nstrentatonsystes (Gordyss et a. 2011; matos Neto et a. 2011)for the eradcaton of   E. faecalis have been reported. Asost root cana nfectons are of bactera orgn, t snot srprsed that there were new antcroba agentscontanng antbotc sch as the mTAD , a xtre of tetracycne soer, an acd and a detergent (Tong et a.2011; Torabnejad et a. 2003) and Tetracean, a xtre of doxycycne, ctrc acd and poypropyengycoe (Gardnoet a. 2006; Nega et a. 2008) have been deveoped.The presence of antbotcs n these new generatons of antcroba agents sggests that the antbotcs ay payan portant roe n the root cana therapy.Severa stdes have nvestgated the effcacy of varos types of antbotcs sch as cndaycn (Gadet a. 1999; la et a. 2001; ln et a. 2003; moanderet a. 1990), erythroycn (moander & Dahen 2003),etrondazoe (la et a. 2001), tetracycne (Gardnoet a. 2006; ln et a. 2003; moander & Dahen 2003;Tong et a. 2011; Torabnejad et a. 2003) and a xtreof cprooxacn, etrondazoe and nocycne (Hoshnoet a. 1996). in addton, the appcaton of antbotcs ndfferent fors, sch as paste (Hoshno et a. 1996; lnet a. 2003; moander & Dahen 2003; moander et a.1990; Sqera & de uzeda 1996), ge (la et a. 2001),soton (Gardno et a. 2006; Nega et a. 2008; Tonget a. 2011; Torabnejad et a. 2003) or pregnated n adevery vehce (Gad et a. 1999)   have been nvestgated.it was sggested that when the antbotcs were appedocay nto the nfected root canas nstead of adnsteredsysteatcay, t wod redce the rsk of adverse systeceffects (mohaad 2009). The a of ths stdy was toevaate the antcroba effectveness of oca appcatonof two antbotcs and Ca(OH) 2 against  E. faecalis bon dentn. M ATERiAlS AND M ETHODS The ethod of ths stdy was oded fro a prevosstdy (Haapasao & Orstavk 1987). Extracted hananteror teeth (excdng ower ncsors) were dsnfectedovernght n 0.5% sod hypochorte (NaOC). Theapca 5  of the roots was reoved sng a rotatngdaond saw at 1000 rp (isoet® 1000 PrecsonSectonng Saw, Beher ltd., uSA ) nder water coong(Cooet TM , Beher ltd., uSA ). Sbseqenty, the rootswere ct transversey nto sces of 4  thck. The nternadaeter of the root cana was shaped to a standardzedsze of 1.4  wth an iSO sze 014 (No. 4) rond stee bur ( ASH , Aagaated Denta Trade Dstrbtors ltd,Engand) rn n a sow-speed contra-ange handpece. Theceent ayer of the tooth sectons reaned ntact.The sear ayer of the root cana was reoved bytreatng the tooth sectons n an trasonc bath of 17% EDTA (Ethyenedanetetraacetc acd dsod sat, R& m marketng, uK ) (pH7.8) foowed by 5.25% NaOCfor 4 n each. The tooth sectons ersed n bran-heart infusion broth ( BHi ) ( BHi , Schara Chee SA , Span) werethen sterzed n an atocave at 121°C for 20 n. Thesterty of the tooth sectons was checked by observngany changes of the codness of the broth soton after a24 h of ncbaton n an aerobc ncbator.The stere tooth sectons were then nocated wth 1 × 10 8   CFu /mL  E. faecalis   ATCC 29212 (Aercan typectre coecton) and ncbated nder aerobc condtonat 37°C for 21 days. At the end of the 21 days of ncbatonperod, the tooth sectons were washed 3 tes wth freshstere PBS to reove excess ed and panktonc forof bactera. The nfected tooth sectons were dvdednto 6 grops wth 12 tooth sectons n each grop. Theywere exposed to 10 l of the foowng test agents andncbated n an aerobc ncbator at 37°C: Grop 1 & 2tooth sectons were exposed to cac hydroxde soton[Ca(OH) 2 ] (Cac hydroxde p.a., merck, Gerany) atpH12.3 for 5 and 10 n, respectvey; grop 3 & 4 toothsectons were exposed to erythroycn (Erythroycnlactobonate iV, Abbott laboratores, uSA ) for 5 and 10n, respectvey. The erythroycn soton was preparedby xng the 500 g erythroycn powder wth 10 lof water for njecton and Grop 5 & 6 tooth sectons wereexposed to oxytetracyne (Oxy, Atantc laboratoresCorp ltd., Thaand) for 5 and 10 n, respectvey. Theoxytetracyne was avaabe as 500 g n 10 l sotonfor. in each grop, two tooth sectons were sed as acontro, n whch they were exposed to stere PBS for 5and 10 n, respectvey.At the end of each exposre te, the antcrobaactvtes of the test agents were ceased by washng thetooth sectons wth 10 l of stere PBS for 5 tes. Thedentne sapes of the nner srface of the tooth sectonswere coected sng three dfferent daeters of sterestee rond brs [ iSO szes 016 (#5), 021 (#7), 023 (#8)]rn n a sow speed handpece. These brs excavated thedentne sapes at <100 μ, 100-350 μ and 350-500μ depth of root dentne, respectvey. After each drng,the dentne powder on the srfaces of the tooth sectonsand respectve brs was coected by rnsng wth 5 l of  BHi broth nto a stere botte. The bactera were seraydted and nocated on Coba horse bood agarpates (Boeda, utas maj Sdn. Bhd., maaysa) and    75 ncbated n aerobc atosphere at 37°C. The coony-forng nt per tooth secton were enerated after the24 h of ncbaton perod.To deterne the foraton of bo, two nfectedtooth sectons were prepared and exaned nder scannngeectron croscope ( SEM ). Brey, at the end of the21days ncbaton, the tooth sectons were xed n 4%gtaradehyde 0.1 m cacodyate bffer soton (AgarScentc ltd., unted Kngdo) and sbseqenty post-xed for 2 h n 2% os tetroxde (Agar Scentcltd., unted Kngdo). Dehydraton was perfored na seres of ascendng ethano concentraton (30%, 50%,70%, 95% and 100%) for 15 n each. The tooth sectonswere then frther dred n a crtca pont dryer (Poaron CPD 7501, Qor Technoogy ltd., unted Kngdo).Foowng that, the tooth sectons were spt nto two andsptter coated wth a ayer of 200Å god paad ( BiO-RAD , Bo-rad mcroscence Dvson, unted Kngdo)before exanaton nder SEM (Phps SEM 515, Phpsindstra & Eectro-acostc Systes, Netherands).The data were entered nto a Statstca Package forSoca Scence ( SPSS ) software (Verson 11.0; SPSS inc,Chcago, il , uSA ). One way ANOVA was sed to coparethe CFu conts of the test grops. independent t-test wassed to anayse the dfference of  CFu conts at the twoexposre tes. A  p -vae of <0.05 was regarded asstatstcay sgncant. R ESulTS There was evdence of coonzaton of   E. faecalis boon the tooth sectons nder SEM exanaton (Fgre 1).The bactera coones encopassed n a dense atrx,appeared as a shroo-shaped coones on the root canasrface were dented.An overa coparson of the antcroba efcacyof the three test agents after 5 and 10 n of exposre tewere shown n Fgres 2 and 3, respectvey.After 5 n of exposre te, the Ca(OH) 2 has thesgncant (  p <0.05) hghest reanng vabe bacteracont at the three dentne depths copared wth theerythroycn and oxytetracyne grops (Fgre 2).Ths sggests that the Ca(OH) 2 was the east effectveantcroba actvty aganst the  E. faecalis bo onthe tooth sectons copared wth the antbotc grops. incontrast, the erythroycn showed a better antcrobaactvty. it had the owest bactera conts and wassgncanty ower copared wth the other 2 test agents nthe dentne depth of 100-500 μ (brs #7 and #8) (Fgre 2).   Sary, after 10 n of exposre, the hghestand owest reanng vabe bactera conts werethe Ca(OH) 2 and erythroycn grops, respectvey(Fgre 3). The CFu conts of Ca(OH) 2 was sgncantyhgher than the erythroycn grop at a three dentnedepths (  p <0.05) bt was not statstcay dfferent when FiGuRE 1. A SEM vew of the root cana srface n an nfected tooth secton. Notea shroo-shaped coony of   E. faecalis ebedded n poysaccharde atrx   andpenetraton of bactera coony nto dentna tbe (Scae bar = 0.1 )  76 copared wth the oxytetracycne n deeper ayers (brs#7 & #8).in genera, the CFu cont was ower after a ongerexposre perod to the test agents (Fgre 4). There werestatstcay ower CFu conts after 10 n of exposren Ca(OH) 2 (Fgre 4(a)) and erythroycn (Fgre4(b)) grops, at sapes taken wth brs #7 and #5,respectvey. No statstcay dfferent of  CFu count in FiGuRE   2. mean og 10 ( CFu +1) of   E. faecalis at three dentne depths after 5 n of exposre te to three test agentsBr sze    m  e  a  n   l  o  g   (   C   F   u   +   1   ) Ca(OH)2 Erythromycin Oxytetracycne FiGuRE 3. mean og 10 ( CFu +1) of   E. faecalis at three dentne depths after 10 n of exposre te to three test agentsBr sze    m  e  a  n   l  o  g   (   C   F   u   +   1   ) Ca(OH)2 Erythromycin Oxytetracycne
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