Le agarose

pmoA, mxaF, dsrB and narG gene fragments were amplified from the three parallel samples of DNA and cDNA obtained from untreated fracture fluid as well as from the treated samples. PCR was performed in 50 μL reaction volumes containing 4 μL template, 1 × Dynazyme Reaction Buffer (Finnzymes, Vantaa, Finland), 62.5 μM dNTP (final concentration) (Finnzymes, Vantaa, Finland), 0.2 μM (final concentration) of each forward and reverse primer (Eurogentec, Belgium) (Table 1), 1 U Dynazyme II polymerase (Finnzymes, Vantaa, Finland) and nuclease free water (Sigma, St. Louis, MO, USA). All PCR reactions were carried out on a Mastercycler gradient temperature cycler (Eppendorf, Hamburg, Germany) using the following conditions, 95 °C initial denaturation for 5 min followed by 40 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min, with final extension step at 72 °C for 10 min, with the exception of an annealing temperature of 58 °C being used for narG. PCR products were visualized with agarose gel electrophoresis on a 1% agarose gel (LE-agarose, Lonza, Basel, Switzerland) stained with 1 × SyberSafe (Invitrogen, Carlsbad, CA, USA) in 1 × SB-buffer [18 (link)] at 300 V for 15 min.

Salmonella typhimurium (KCCM 12041), Salmonellaenteritidis (KCCM 12021), Escherichiacoli (KCCM 11234), and Staphylococcusaureus (KCCM 12103) were obtained from the Korean Collection of Type Cultures (Daejun, Korea). Tryptic soy (TS) agar, BBL eosin methylene blue (EMB) agar, XLT4 agar base, XLT4 agar supplement, Baird–Parker agar base, and EY tellurite enrichment were purchased from BD Difco (Sparks, MD, USA). The initial ssDNA library and the primers used for amplification were synthesized and purified by polyacrylamide gel electrophoresis (PAGE; Bioneer Co., Ltd, Daejeon, Korea). Phosphate–buffered saline (PBS, pH 7.4) was purchased from Sigma (St. Louis, MO, USA). PCR tubes, reagents, and polymerase were obtained from Takara (Shiga, Japan). LE agarose and TAE buffer were purchased from Lonza (Rockland, ME, USA). The Qiagen MinElute gel extraction kit was obtained from Qiagen (Hilden, Germany). The In-Fusion HD Cloning kit was purchased from Clontech (Mountain View, CA, USA).

The PCR products were subjected to restriction analysis with TaqI, BamHI, and HindIII (New England Biolabs, Massachusetts, United States). Digestion with TaqI was performed at 65°C for 3 hours, and digestion with BamHI and HindIII was performed at 37°C for 4 hours. Restriction products were analysed on a 1.5% agarose gel (SeaKem LE Agarose-Lonza, Maine, United States) electrophoresed in Tris-borate-EDTA (TBE) buffer.

To confirm the selected colonies were E. coli, isolates were screened by PCR of the 16S DNA as described previously by Lamprecht et al. (11 (link)). Amplification was carried out in an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) with the following parameters: 94°C for 3 min; 35 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 1 min; followed by a final extension of 72°C for 10 min. PCR products were subjected to horizontal gel electrophoresis in a 2% agarose gel (LE Agarose, Lonza, Alpharetta, GA) at 100 V for 70 min. A Hi-Lo molecular weight marker (100 bp; Minnesota Molecular, Minneapolis, MN) and negative (sterile water) and positive controls from our lab collections were included on the gel for comparison and confirmation purposes. After electrophoresis, the gel was stained in 0.25% Ethidium Bromide solution (Sigma Aldrich. St. Louis, MO) for 20 min and viewed under UV light using an Omega Lum G imager (Aplegen, San Francisco, CA). Isolates were recorded as positive or negative for the 16S.

The titration of infectious virus was performed as previously described [32 (link)]. Briefly, serial 10-fold dilutions of virus were made in serum-free alpha-MEM medium, and 0.1 ml of each dilution was added per well to a monolayer of BHK-21 cells in a 12-well plate. After 1 hour of absorption at 37°C, the cells were washed with PBS and overlaid with growth medium containing 0.3% LE agarose (Lonza), and the plaque was visualized with naphthol blue–black solution (0.1% naphthol blue–black, 1.36% sodium acetate and 6% glacial acetic acid) at 4 days post-infection. Viral titers were determined as plaque forming units per milliliter.