Supplement to Methods

Multiple Combination Bacterial Test Procedures

Antibiotics

When the MCBT test was initiated in 1988, six antibiotics were tested singly and in combinations for bactericidal activity. However, as new antibiotics became available during the 1990s (e.g., cefepime, meropenem, azithromycin) they were added to the panel.

The following antibiotics were used singly and in double and triple combinations for multiple combination bactericidal testing: ceftazidime (32 µg/ml), tobramycin (200 µg/ml) and tobramycin (4 µg/ml), amikacin (32 µg/ml), cloxacillin (20 µg/ml) and chloramphenicol (20 µg/ml) all from Sigma Pharmaceuticals, St. Louis, MO; azithromycin (0.4 µg/ml) from Pfizer Canada Inc.; imipenem (10 µg/ml) from Merck Sharp Dohme Canada Inc.; piperacillin / tazobactam (32/4 µg/ml) from Lederle Cyanamid Canada Inc., Montreal, PQ, Canada; aztreonam (32 µg/ml) and cefepime (32 µg/ml) from Bristol Meyer Squibb Pharmaceutical, St. Laurent, PQ, Canada; trimethoprim/sulfamethoxazole (10/2 µg/ml) from Roche Laboratories Canada; meropenem (32 µg/ml) from Zeneca Pharma Inc, Mississauga, ON, Canada; ticarcillin / clavulanic acid (32/10 µg/ml) from SmithKline Beecham Pharma Inc., Oakville, ON, Canada; minocycline (2.0 µg/ml) from Novopharm Inc.,Toronto, ON, Canada; and ciprofloxacin (2.0 µg/ml) from Miles Laboratories, Etobicoke, ON, Canada.

MCBT Procedure

MCBTs were carried out in four 96-well round-bottomed microtiter plates (Nunc Inc., Roskilde, Denmark). Antibiotic solutions were prepared for each test from stock solutions stored at –80 ° C. The working antibiotic solutions were prepared in Mueller Hinton II Cation Adjusted Broth (MHB II broth, Becton Dickenson Microbiology Systems, Cockeysville, MD) at 10 times the required concentrations. For each MCBT, antibiotic working solutions were made fresh on the day of inoculation. One, two or three antibiotics were added, each in 10 m L volumes, to the appropriate wells. The necessary volume of MHBII was then added to the wells containing 1 or 2 antibiotics so that all the wells had a volume of 30 m L before the addition of organism. The organism inoculum consisted of 70 µL of a 100-fold dilution of a 0.5 McFarland turbidity standard prepared from a culture in Tryptone Soya Broth (Oxoid Laboratories, Basingstoke, UK) in the growth phase. This gave a final inoculum concentration in each well of 5 x10⁵ cfu/ml. Growth and sterility plates (no antibiotics and no organism inoculum respectively) were run with each MCBT procedure as bacteriologic controls. Plates were incubated at 35^OC for 48 h. At 24 and 48 hours the wells were examined for turbidity. The contents of nonturbid wells at 48 hours were subcultured by streaking 10 m L of suspension onto 5% Columbia sheep blood agar plates (PML Microbiologicals, Mississauga, ON, Canada) which were incubated for 24 h at 35 º C and examined for 99.9% kill the next day.

Reproducibility of the MCBT results was confirmed by repeated testing of eight selected isolates over a 2-yr period. Each isolate showed identical MCBT susceptibility results with repeated testing.