Potassium sulfate

Timelines for all mouse experiments are shown in Additional file 1: Fig. S10. Germ-free mice (four mice per cage) received a single oral gavage of 100 μl thawed donor fecal material (donor No. 41; see below) or sterile PBS. Fecal pellets were collected prior to gavage and at days 3, 7, 14, and 21 following gavage and stored at −20 °C until DNA extraction.
Two cages of ASF mice (five mice per cage) received a single oral gavage of 100 μl thawed donor fecal material from one of two donors (donor No. 23 or a different donor No. 41 preparation lot from donor 41 (41B) than that used in germ-free mice; all mice in the same cage received the same material) with one cage maintained without gavage. Mice were gavaged 2 days following receipt. Fecal samples were collected prior to gavage and at 1, 3, 7, 12, 19, and 26 days following gavage.
For the single-course antibiotic experiment, two cages (five mice each) received systemic antibiotics (see below) and two others received non-absorbable antibiotics for 7 days. One cage for each antibiotic treatment received SUPREP bowel prep solution (Braintree Laboratories, Inc., Braintree, MA, USA), comprised of 262 mM sodium sulfate, 38 mM potassium sulfate, and 28 mM magnesium sulfate in drinking water for 2 days following cessation of the antibiotic regimen while the remaining cages received normal drinking water. Mice then received oral gavage of 100 μl of the same thawed donor fecal material given to germ-free mice (donor 41A). Fecal pellets were collected prior to and following antibiotic exposure (prior to bowel prep), following bowel prep (prior to gavage, T₀), and at days 4, 7, 14, and 21 following gavage.
Mice receiving the three-course antibiotic treatment received non-absorbable antibiotics in drinking water for 7 days, normal drinking water for 2 days, systemic antibiotics in drinking water for 7 days, normal drinking water for 2 days, non-absorbable antibiotics for 7 days, and normal drinking water for 2 days prior to gavage. One cage (five mice) received a single oral gavage of 100 μl of thawed donor fecal material from donor 41A (same used for germ-free), donor 36, or donor 42. Mice in two cages were maintained as no-gavage controls and received the antibiotic regimen without gavage. Mice in two more cages were maintained as no-antibiotic controls and were maintained on normal drinking water for 27 days prior to receiving gavage of 100 μl thawed donor fecal material from either donor 41 or donor 36. Fecal samples were collected prior to antibiotic exposure, following antibiotic exposure (prior to gavage), immediately prior to gavage (T₀), and at days 3, 7, 14, and 21 following gavage. This experiment was performed in two independent studies to determine reproducibility among donors: the first study included the donor No. 41 treated mice, a no-gavage control cage, and a no-antibiotic control cage; the second study included the remaining donors (Nos. 36 and 42) and control cages. Pre- and post-antibiotic fecal samples were not collected during the second study.

Escherichia coli TOP10 (Invitrogen) and E. coli CA434 [39] (link) were cultured in Luria-Bertani (LB) medium, supplemented with chloramphenicol (25 µg/ml), where appropriate. Routine cultures of C. difficile 630 Δerm[40] (link) and C. difficile R20291 were carried out in BHIS medium (brain heart infusion medium supplemented with 5 mg/ml yeast extract and 0.1% [wt/vol] L-cysteine) [41] (link). C. difficile medium was supplemented with D-cycloserine (250 µg/ml), cefoxitin (8 µg/ml), lincomycin (20 µg/ml), and/or thiamphenicol (15 µg/ml) where appropriate. A defined minimal media [18] (link) was used as uracil-free medium when performing genetic selections. A basic nutritive mannitol broth for growth assays of C. difficile strains were prepared as follows : Proteose peptone no. 2 4% [wt/vol] (BD Diagnostics, USA), sodium phosphate dibasic 0.5%[wt/vol], potassium phosphate monobasic 0.1%[wt/vol], sodium chloride, 0.2% [wt/vol], magnesium sulfate, 0.01% [wt/vol], mannitol, 0.6% [wt/vol] with final pH at +/−7.35. For solid medium, agar was added to a final concentration of 1.0% (wt/vol). Clostridium sporogenes ATCC 15579 was cultivated in TYG media [7] (link). All Clostridium cultures were incubated in an anaerobic workstation at 37°C (Don Whitley, Yorkshire, United Kingdom). Uracil was added at 5 µg/ml, and 5-Fluoroorotic acid (5-FOA) at 2 mg/ml. All reagents, unless noted, were purchased from Sigma-Aldrich.

Agarose, BaCl₂ and trichloroacetic acid were purchased from Sigma-Aldrich. Glassware was rinsed several times in deionized H₂0 and then dried before preparing the reagents. A 0.5 % BaCl₂ + 0.01 % Agarose solution was prepared to a final volume of 50 ml using deionized H₂O and a stock solution of 0.1 % Agarose (dissolved by heating in deionized H₂O) and then stored at room temperature from 1 to 11 weeks prior to use. An 8 % trichloroacetic acid solution was prepared in 50 ml deionized H₂O. All reagents were stored away from light for up to 11 weeks. To prepare solutions with known sulfate levels, ranging from 0.05 to 2.0 mM, a stock 100 ml solution of 0.1 M K₂SO₄ (Sigma-Aldrich) was serially diluted in deionized H₂0. These standard solutions were used to generate a standard curve.