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Volume 2 | Issue 1/2016 11 Journal of Oral Science & Rehabilitation S ta ph ylo co ccu s au re u s and pe r i i mplant di se ase Table 1 Sites Positive sites/ TBC Mean number of sites bacterial counts GS 1/35 2.11 × 104 5.02 × 102 PISF 1/35 1 positive case, but below 0 level of quantification IIP 1/35 1 positive case, but below 0 level of quantification OF 1/35 1 positive case, but below 0 level of quantification solution in an ultrasonic bath for 10 min. After- ward, a new abutment screw was connected using a torque wrench (Torq Control,Anthogyr, Sallanches, France) until it reached a torque of 30 N cm, according to the manufacturer’s in- structions. In orderto verify properfit between the dental restoration and the implant, stan- dardized digital periapical radiographs were taken using a dental radiographic film holder (Rinn Centrator Bite, DENTSPLY RINN, Elgin, Ill., U.S.) and the paralleling technique. Quanti ta tive rea l-tim e polymerase c h a in rea c tion a s s a ys Quantitative real-time polymerase chain reac- tion(PCR)assayswerecarriedoutfortotalbac- terialcounts (TBCs)foreachtarget species,17,18 in avolume of10 μLcomposed of1× QuantiFast SYBR Green PCR (Qiagen, Hilden, Germany), 2 μL of DNA extract, and 1 μM of each primer. The species-specific PCR primers used in this study were provided by Institut Clinident and manufactured by metabion (Martinsried, Ger- many). Assays were carried out on the Rotor- Gene Q thermal cycling system (Qiagen) with the following program: for TBC, 95 °C for 30 s, followed by 40 cycles of 10 s at 95 °C, 10 s at 60 °C, and 35 s at 72 °C; for S. aureus, 95 °C for 5 min, followed by 40 cycles of 10 s at 95 °C, 10 s at 66 °C, and 35 s at 72 °C. A final melting curve analysis (70–95 °C in 1 °C steps for5 s in- crements) was performed. Fluorescence sig- nals were measured every cycle at the end of the extension step and continuouslyduringthe melting curve analysis. Serial dilutions of stan- dard DNA, provided by Institut Clinident, were used in each reaction as external standards for absolute quantification ofthetarget pathogen. Finally, the data were analyzed using Rotor- Gene Q Series Software (Qiagen). Stati sti cal analysi s The mean and standard deviations for TBCs at each inspected site (PISF, GS, IIP, OF) were recorded and analyzed accordingto a pre-estab- lished analysis plan. A bio-statistician with ex- pertise in dentistry analyzed the data using sta- tistical software (SigmaPlot, Version 13, Systat Software, San Jose, Calif., U.S.). Before running the statistical analysis, the TBCs for each site were transformed (log transformation [log10]) in an attempt to render less skewed distributions, making the data more interpretable and helping to meetthe assumptions ofinferentialstatistics. Asthenormalitytestfailed,anonparametrictest (Kruskal–Wallis) was used. The level of signifi- cancewassetatα = 0.05. Results No implants were lost, and all of the prostheses were in situ atthetime ofexamination.Atthe end ofthestudy,justonesite(outof35)intheGSofthe adjacent teeth presented a TBC of 2.11 × 104 . The mean bacterialcount ofS. aureuswas 5.02 × 102 ; therefore, this value was taken as control. Con- versely, in the PISF of each implant, the IIP and the OF complex, the mean bacterial counts of S. aureuswere0,withonlyonesite(outof35)pos- itive, but below the level of quantification. The dataarereportedinTable 1.Nostatisticallysignifi- cant differences were found among groups regarding site location (Kruskal–Wallis test; p= 0.40). Discussion Currently,thereareneitherstandardizedantibiotic prophylactic regimens for dental implant place- Table 1 Investigated sites and bacterial counts. GS: gingival sulcus of the adjacent teeth; PISF: periimplant sulcular fluid; IIP: inner parts of connection; OF: oropharyngeal complex. Volume 2 | Issue 1/201611 GS 1/352.11 × 104 PISF 1/351 positive case, but below 0 IIP 1/351 positive case, but below 0 OF 1/351 positive case, but below 0