Antimicrobial microdilution assay

Glycerol stocks of S. aureus, doxycycline/clindamycin-resistant S. aureus, and vancomycin-resistant E. faecalis were thawed and streaked on Nutrient agar (NA) plates. Methicillin-resistant S. aureus, on the other hand, was streaked on Nutrient agar supplemented with 8μg/mL oxacillin. E. coli was streaked on Luria-Bertani agar plates. Plates were incubated at 37°C for 8 – 12h. A single colony from the NA plates was transferred into 7mL Mueller Hinton broth (MHB) (with additional 2% NaCl for MRSA)(LB broth for E. coli) and incubated for 6-8h at 30°C, 150 rpm. The turbidity of the broth culture was then adjusted to match the 0.5 McFarland standard (1 x 10⁸ cells/mL). The adjusted broth culture was then further diluted 200-fold and used as the final inoculum for the assay. 200 μL of the test organism was dispensed to each well of a 96 well plate. PYRC, EQI, positive control and 1% DMSO as solvent control were tested using a two-fold dilution scheme starting at 32μg/mL with 10 dilutions each. For E. coli, PYRC (2–128 μg/mL), EQI (2–128 μg/mL), polymyxin B (0.016–1 μg/mL) and polymyxin B nonapeptide (0.125–64 μg/mL) were tested singly, and in combination using a two-fold dilution scheme. Following an 18 – 20h incubation period, ten microliters of MTT (5mg/mL) were added to wells and incubated for 2h. One hundred microliters of solubilizing reagent (50% DMF/20% SDS) was added to wells and incubated for 1 hour. The A₅₇₀ was measured using a Biotek–Synergy 2 Microplate Reader (Biotek;Winooski, VT).