Ampicillin sodium salt

LB Broth, Miller (BP1426), ampicillin sodium salt (BP1760) and cefotaxime sodium salt (BP29511) were purchased from Fisher Scientific. Meropenem trihydrate (M2574) was purchased from Sigma-Aldrich (Millipore sigma). E. cloni 10G electrocompetent cells (60061) and E. cloni 10G chemically competent cells (60107) were purchased from Lucigen Corp. The KAPA HiFi PCR Kit (KK2102) was purchased from KAPA Biosystems Inc, the E.Z.N.A. Cycle Pure Kit was purchased from OMEGA Bio-tek Inc and the QIAprep Spin Miniprep Kit was purchased from Qiagen. The NextSeq 500/550 High Output Kit (300 cycles) (20024908) was purchased from Illumina Inc.

The medium SP4 was prepared following previous recommendations [36 ] but with some modifications. The modified medium is composed of three parts (A, B and C). Part A is composed of 4.2 g of Difco PPLO broth (BD), 6.4 g of Bacto Peptone (BD), 12 g of Bacto Tryptone (BD) and 724 mL of deionized water. The solid medium includes 7 g of European Bacteriological Agar (Conda-Pronadisa). The pH is adjusted to 7.8 and then part A is autoclaved at 121 °C for 20 min. Part B is composed of 60 mL of RPMI-1640 (Sigma-Aldrich), 21 mL of fresh yeast extract 50% w/v, 2.4 g of yeast extract (Conda-Pronadisa), 4.8 mL of phenol red 0.5%, (Sigma-Aldrich) and 0.642 g of ampicillin sodium salt (Fisher bioreagents). The pH is adjusted to 7.2 and then part B is filter sterilized through a 0.22 μL pore size filter. Part C is composed of 251 mL of heat-inactivated horse serum (Hyclone) for 30 min at 56 °C. Note that, in this study, the SP4 broth medium did not include ampicillin in part B.

Intestinal microbiota were depleted using a cocktail of metronidazole (Acros Organics), neomycin trisulfate (Sigma), and ampicillin sodium salt (Sigma) (all 1 g/l) provided in drinking water for 1 week. Indicated groups received 200 μl fecal suspensions of SFB-monocolonized mice by oral gavage 1 week after ceasing antibiotics. For single antibiotic treatments, ampicillin sodium salt (1 g/l), metronidazole (1 g/l), vancomycin hydrochloride (0.5 g/l, Fisher Scientific), or tobramycin sulfate (1 g/l, Centrafarm) was added to drinking water for 8 weeks and refreshed once a week. Sucrose (6 g/l) was added to drinking water of all groups including controls during treatments.

The pET-6XHis/TEV/EGFP, a plasmid encoding a 6X His tag followed by a TEV protease site and the enhanced green fluorescent protein (EGFP) gene, was purchased from Vector Builder (Chicago, IL). The plasmid was transformed into Escherichia coli BL21(DE3) (Invitrogen, Waltham, MA) for the inducible expression of EGFP. E. coli O157:H7-PC [20 (link)] was also utilized for cell capture studies since the anti-E. coli antibodies used in this study have been shown to be effective for its capture [21 (link)]. Luria-Bertani medium (LB) (BD Difco), LB containing 50 μg/mL ampicillin sodium salt (Fisher Scientific, Fair Lawn, NJ), or LB containing 100 μg/mL of spectinomycin dihydrochloride pentahydrate (GoldBio.com, St. Louis, MO) was used for the growth and maintenance of the E. coli strains within this study. Overnight cultures of E. coli were grown at 37°C with shaking at 180 rpm for ~17 hr.

Chemical, including LC/MS grade methanol and acetonitrile, analytical grade formic acid, Gifu Anaerobic Broth (GAM broth), and ampicillin sodium salt, 3-Nitrophenylhydrazine hydrochloride (3NPH·HCl), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC·HCl), and pyridine were purchased from Fisher Scientific (Pittsburgh, PA, USA). The volatile free fatty acid (VFA) standard mix (certified reference material CRM46975) was purchased from Supelco (Bellefonte, PA, USA).

Antibiotics used in this study included Spectinomycin Sulfate (MP Biomedicals) and Ampicillin Sodium Salt (Fisher).