2.1. Bacteria and media
Bacterial strains (–) used in the spectrum of activity studies were clinical isolates as well as American Type Culture Collection (ATCC) strains. Kill curves were performed using a multidrug-resistant (MDR) strain of Staphylococcus aureus (ONI33) and a MDR strain of Enterococcus faecalis (ONI47), both obtained as fresh clinical isolates from Shands Hospital (Gainesville, FL). These studies were also performed using a strain of Streptococcus pneumoniae (ATCC 49619). Staphylococcus aureus ONI33 and S. pneumoniae ATCC 49619 were also used in the development of resistance study. Staphylococcus aureus strain ONI33 was shown to be resistant to amoxicillin, ampicillin, cefazolin, cefepime, cefotaxime, ceftriaxone, cefuroxime, cefalothin, ciprofloxacin, clindamycin, erythromycin, imipenem, levofloxacin, meropenem, oxacillin, penicillin, sparfloxacin, ticarcillin, azithromycin, amikacin and chloramphenicol. Enterococcus faecalis strain ONI47 was shown to be resistant to ampicillin, ciprofloxacin, erythromycin, levofloxacin, penicillin and vancomycin.
Tier 1 susceptibility study: MU1140 minimum inhibitory concentrations (MICs) for various Gram-positive microorganisms
Tier 3 susceptibility study: minimum inhibitory concentrations (MICs) of MU1140 in comparison with vancomycin against selected clinical isolates
Bacterial strains were stored as 50% glycerol stabs at −80°C. Starter plates of bacterial strains were prepared by inoculation of samples from glycerol stabs onto blood agar plates (BAPs) consisting of 1.5% casein peptone (Remel, Lenexa, KS), 0.5% soy peptone (Remel), 0.5% sodium chloride (Remel), 5% sheep’s blood (Lampire, Everett, PA) and 1.5% agar (Fisher, Fairlawn, NJ). Staphylococcus aureus strain ONI33 and E. faecalis strain ONI47 were grown in cation-adjusted Muller–Hinton broth (Becton Dickinson Biosciences, Franklin Lakes, NJ) at 37°C in a 5% CO2 incubator. Streptococcus pneumoniae strain ATCC 49619 was grown in Todd–Hewitt broth (Becton Dickinson Biosciences) under the same conditions.
2.2. Antimicrobial agents
MU1140 was manufactured by Oragenics Inc. (Alachua, FL). Purity was estimated to be >90% as determined by analytical reverse-phase high-performance liquid chromatography.
2.3. Susceptibility studies
The MICs of MU1140 against target microorganisms were determined by Focus Bio-Inova (Herndon, VA). MU1140 MIC values for aerobes were determined by the broth microdilution method according to Clinical and Laboratory Standard Institute (CLSI) methodology [11
], whilst MU1140 MIC values for anaerobes were determined by the agar dilution according to CLSI methodology [12
2.4. Time–kill studies
MICs for S. aureus strain ONI33, E. faecalis strain ONI47 and S. pneumoniae strain ATCC 49619 used in the time–kill and development of resistance studies were determined using the broth microdilution method. Inocula were prepared from test organisms grown for 4–6 h in the appropriate broth media and diluted in saline to 0.5 McFarland standard to obtain 100 mL of a starting culture containing 106 colony-forming units (CFU)/mL, which was verified by colony counts of replicate samples. Aliquots (10 mL) of the culture were transferred to sterile plastic 25 cm2 culture flasks (Corning Inc., Corning, NY) and MU1140 was added from a sterile stock solution to give final concentrations equal to 0.5, 1, 2, 4, 8 and 16 times the MIC for S. pneumoniae strain ATCC 49619 and S. aureus strain ONI33, and 0.25, 0.5, 1, 2, 4, 8 and 16 times the MIC for E. faecalis strain ONI47. Each assay included a growth control tube with no antibiotic.
The cultures were incubated at 37°C and samples were obtained at 0, 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 h following addition of MU1140. The samples were washed with phosphate-buffered saline and serially diluted 10-fold in ice-cold normal saline and then 10μL samples were spotted onto duplicate BAPs. Following incubation at 37°C for 24 h, colonies that arose on plates with 30–300 colonies were counted.
2.5. Development of resistance
Staphylococcus aureus strain ONI33 and S. pneumoniae strain ATCC 49619 were grown overnight on BAPs. Cells were scraped from the surface and diluted with saline to 0.5 McFarland standard. Cells were then diluted 1:100 in appropriate broth media to give ca. 106 CFU/mL and then 100 μL samples were added to microtitre wells (Corning Inc.) containing 100 μL of doubling concentrations of MU1140 in broth to achieve a final bacterial concentration of 5× 105 CFU/mL. The microtitre plates were incubated overnight at 37°C in an atmosphere of 5% CO2. Wells containing the highest concentration of MU1140 that showed turbidity (equivalent to 0.5× MIC) were diluted to 0.5 McFarland standard and used as the inocula to repeat the above process. This process was repeated daily 21 times and the MIC after each subculture was recorded. After the 7th, 14th and 21st repetition, a sample of cells from the 0.5× MIC well was used to inoculate 1 mL of MU1140-free broth, which was grown overnight to saturation. These cells were subcultured in the absence of MU1140 an additional six times, after which MICs for MU1140 were determined using the broth microdilution method.