Broth microdilution method for aerobes and anaerobes.

The comparator drugs were reconstituted according to the manufacturers' instructions. For all drugs against aerobic organisms, Mueller-Hinton broth supplemented with 5% lysed horse blood was used for testing. For anaerobes only, pexiganan was reconstituted in water and serially diluted in brucella broth with or without 5% lysed horse blood. Concentrations for pexiganan were serial 2-fold dilutions from 512 to 0.25 μg/ml. Concentrations for penicillin were from 8 to 0.03 μg/ml, for piperacillin-tazobactam from 32 to 0.03 μg/ml, and for the remaining drugs from 16 to 0.03 μg/ml. The Quick Spense apparatus was used to dispense 100 μl of the dilutions into 96-well microtiter trays, which were immediately placed in the −70°C freezer for storage. On the day of the test, they were removed from the freezer and thawed at room temperature.

On the day of testing, aerobic strains were suspended in saline to equal the 0.5 McFarland standard, further diluted 1:30 in saline, and added to the trays using a 96-pronged inoculation device that delivered 10 μl to each well, for a final concentration of approximately 5 × 10⁴ CFU/well. The plates were incubated in an ambient atmosphere at 35°C for 20 h. Anaerobic isolates were suspended in brucella broth to equal the turbidity of the 0.5 McFarland standard and further diluted 1:15 in saline before adding 10 μl to each well, for a final concentration of approximately 1 × 10⁵ CFU per well. The anaerobic isolates were incubated in the anaerobic chamber at 35°C for 44 h, and the Eikenella corrodens plates were incubated in 5% CO₂ for 48 h. After incubation, the trays were examined for growth using an inverted mirror. The MIC was considered the lowest concentration that completely inhibited growth or caused a marked reduction of growth compared to the drug-free control well. The susceptibility of test isolates to the comparator agents was determined per the interpretive breakpoints (6). Interpretive breakpoints for pexiganan have not been defined.

The quality control strains Bacteroides fragilis ATCC 25825 and Clostridium difficile ATCC 700057 were included each day of testing anaerobes, and Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 were included with the aerobic testing.