Tryptic soya broth

Vancomycin-resistant Enterococci (VRE) strains (Table 1) were obtained from the American type culture collection (ATCC) and Biodefense and Emerging Infections Research Resources Repository (BEI Resources). All experiments were carried out in accordance with relevant guidelines and regulations and were approved by the Institutional Biosafety Committee of Purdue University. All chemicals and reagents were purchased from commercial vendors. Auranofin, linezolid (Chem-impex International, Wood Dale, IL), ampicillin (Peosta, IA), vancomycin hydrochloride (Gold Biotechnology, St. Louis, MO), gentamicin sulfate (Fisher Bioreagents, Fairlawn, NJ), and ramoplanin (Sigma-Aldrich, St. Louis, MO) were purchased commercially. Brain heart infusion (BHI), tryptic soya broth (TSB), tryptic soya agar (TSA) and enterococcosel broth were purchased from BD (Becton, Dickinson and Company, Cockeysville, MD) and Phosphate buffered saline (PBS) was purchased from corning (Corning, NY).

Two to three isolated colonies from a freshly streaked Trypticase soy agar (Becton Dickinson) plate of a S. sonnei stock culture were transferred to a culture flask containing  approximately 20 mL tryptic soya broth (Becton Dickinson) enriched with 0.6% yeast extract and grown to exponential phase by incubating at 37°C for 2 hours in a shaker incubator. Bacterial cells were pelleted by centrifugation at 7000 rpm for 10 minutes. After 3 washes, bacterial concentration was adjusted to an optical density (OD) at 595 nm of 0.4 with sterile phosphate-buffered saline, which corresponded to 10⁸ colony forming units (CFU) bacteria/mL. A 10-fold serial dilution was made to prepare a 10⁷ CFU/mL bacterial suspension. Serum samples were incubated at 56°C for 30 minutes to inactivate complement. Two-fold dilutions of serum samples (from 1:8 to 1:512) and guinea pig complement (Sigma-Aldrich) were added to the bacterial suspension in Mueller-Hinton broth (MHB; Becton Dickinson) in wells of microtiter plates in a final volume of 200 µL/well. Control wells contained bacteria in MHB without serum. After overnight incubation (at 37°C for approximately 16 hours at 200 rpm), OD of the bacterial suspension in each well was measured at 595 nm. The SBA titer of the serum was defined as the reciprocal of the last dilution in which no growth was evident by visual inspection.