faecalis in the blood and to reveal its resistance type to vancomycin.
˚ Material and Methods We used a standard strain VRE (PTCC 1447, and PTCC 1237) prepared by the division of Bacteria and Fungi Collections, Iranian Institute of Industrial and Scientific Researches, Tehran, Iran). A suspension 108 cfu/ml was made in normal saline by adding some single colonies, which were grown on TSA by adjusting its optical density to half McFarland solution and checking their absorbance in 700 nm with spectrophotometer. Inhibitors,research,lifescience,medical Then, diluted solutions with different bacterial contents (106 cfu/ml, 104 cfu/ml and 102 cfu/ml) were made by diluting it in normal saline. They are used for inoculating to blood. By adding certain amount of each bacterial solution to certain amount of defibrinated sheep blood, some blood samples with different bacterial Inhibitors,research,lifescience,medical content (104 cfu/ml, 103 cfu/ml, 102 cfu/ml, 101 cfu/ml, 5 cfu/ml and zero as control) were prepared. Ten-milliliter-samples of each dilution were prepared to be used in ten
experiments of each of the PCR and Inhibitors,research,lifescience,medical routine assays. For routine assay, we used initial enrichment procedure for each specimen by inoculating to TSB and this website incubation at 37˚C for 24 hours, passage to TSA and incubation in 37˚C for more 24 hours, identifying by catalase test, PYR test, growth on TSA with 6.5% Nacl, and hydrolysis Inhibitors,research,lifescience,medical esculin
in the presence of bile on BEA. Differentiation of E. faecalis from E. faecium was done by three tests including ability to use pyruvate, fermentation of sorbitol, and reduction of tellurite.15,24 For screening VRE, we used BEA including Inhibitors,research,lifescience,medical 6 µg/ml vancomycin.10,15,24 The extraction of DNA was achieved using the following procedure. Transferring 100 µl of each blood sample to a 2 ml ependorf vial contain 400 µl sterile double distilled H2O and incubation in 37˚C for 30 minutes, adding 500 µl red cell lysis buffer (NAHCO3 10 mM, NH4CL 0.155M, pH=7) and incubation at 37˚C for one hour, centrifugation at 10,000 rpm for 15 minutes, discarding the supernatant, adding 200 µl lysis buffer Amisulpride for bacteria (Tris 10 mM, sucrose 0.3 M, MgCl2 5 mM) to the pellet with 10 µl lysozyme (0.1 mg/ml, Sinagen, Lot: MR7735) and incubation at 37˚C for one hour, adding 4 µl proteinase K (900 u/ml, Fermentaze, Lot: 00022411) and incubation at 65˚C for one hour, extraction of DNA by standard phenol-chloroform method and precipitation of DNA by cold isopropanol. PCR mix was prepared as 3 µl of 10x PCR buffer, 2 µl of MgCl2 (25 mM), 0.5 µl of dNTP 10 mM, 100 pM of each primer, 0.2 µl DNA pol (5 u/µl), 2 µl DNA, and double distilled H20 to final volume of 25 µl. Special features of primers that were used in this study are shown in table 1.