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The postantibiotic effect (PAE) is a pharmacodynamic parameter that refers to the persistent suppression of bacterial growth following short exposure to and subsequent complete extracellular removal of an antibiotic (2, 17). Knowledge of this effect can optimize antibiotic dosage regimen design; however, little pharmacodynamic research has been performed with the Mycobacterium avium complex (MAC) primarily due to its long generation time and the time required to visualize colonies on solid media (3, 4, 11, 16). The aim of this study was to develop a faster radiometric method based upon CO₂ production to determine PAEs by using clinically relevant antibiotics against MAC.

(This work was presented in part at the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 28 September to 1 October 1997.)

Organisms and antibiotics. Cultures of a reference strain of MAC, strain NJ9141, and two clinical isolates, isolates 65319 and 81433, were used. Amikacin (Bristol-Myers Squibb, St. Laurent, Quebec, Canada), azithromycin (Pfizer Canada, Kirkland, Ontario, Canada), clarithromycin (Abbott, Chicago, Ill.), clofazimine (Ciba Geigy, Mississauga, Ontario, Canada), ethambutol (Sigma, St. Louis, Mo.), rifabutin (Pharmacia, Columbus, Ohio), rifampin (Marion Merrell Dow, Laval, Quebec, Canada), and sparfloxacin (Rhone-Poulenc Rorer, Collegeville, Pa.) were tested against MAC. All antimicrobial agents were prepared according to the guidelines of the National Committee for Clinical Laboratory Standards (12).

MIC determinations. BACTEC 12B (Middlebrook 7H12) broth (10) contains ¹⁴C-palmitic acid as a sole source of carbon. Substrate consumption releases ¹⁴CO₂ into the headspace above the medium in the sealed vial. The BACTEC TB-460 instrument detects the amount of radioactivity and records it as a growth index (GI) on a scale of from 0 to 999, simultaneously replacing the evacuated gas with 5 to 10% CO₂ in air (9, 15).

PAE determinations. A BACTEC 7H12 seed vial (GI, 999) was prepared as described above for the MIC determinations. Growth control vials and test vials containing 0.1 ml of the drug at the appropriate concentration were inoculated with 0.1 ml from the seed vial (final inoculum, between 10⁶ and 10⁷ CFU/ml). After 2 h of incubation, 0.1-ml aliquots from each vial were diluted 1:100 in dilution fluid to remove the drug. The dilution fluid was then used to inoculate prewarmed vials with 0.1-ml aliquots. Diluted drug controls containing 1:100 dilutions of antimicrobial agent and MAC were monitored to ensure that the PAEs were not due to residual antibiotic levels. Each vial was evaluated by determining plate counts and GI. Aliquots of 0.1 ml were plated immediately after inoculation (0 h) and then after each GI reading. GI readings were taken daily for the first 48 h and then every 12 h until the GI reached 999. Initial colony counts were also verified by plating the dilution fluid. Except at 0 h, both the growth control and test vials were injected with 0.1 ml from a fresh 12B vial to maintain volume. PAE determinations were performed three to five times on different days for each drug and each strain following a 2-h exposure at 2× the MIC. Experiments were also performed three to five times on different days with each strain following a 2-h exposure at 20× the MIC by using clarithromycin, clofazimine, rifabutin, rifampin, and sparfloxacin. The GIs of the vials were read until the GI reached 999 except for the vials containing rifampin and strain 81433, which were sampled for 14 days without evidence of regrowth.

PAE calculations. PAE by viable counts was calculated by using the formula PAE = T  C, where T and C are the times required for the numbers of CFU per milliliter for the test and control cultures, respectively, to increase 1 log₁₀ above the counts observed immediately after drug removal (2, 17). The PAE obtained by using GI was defined and calculated as the difference in times for the test and control cultures to increase 2 log₁₀, which corresponds to an increase in GI of 100, during the 12-h sampling times. Factorial analyses of variance followed by the post-hoc Scheffe test were conducted with the data.

Results. Differences in MICs for the MAC strains of greater than one twofold dilution occurred with azithromycin, clofazimine, rifampin, and sparfloxacin (Table 1). The PAEs determined by the two methods, viable counting and CO₂ generation (BACTEC) as assessed by determining the GI, after 2-h exposures to antibiotics at 2× the MIC are reported in Table 2. The PAEs determined by the two methods were not significantly different (P > 0.05). The results of tests with rifampin against strain 81433 were so inconsistent that these data are not included in Table 2. Figure 1 depicts a representative determination of the PAE of rifabutin against strain NJ9141 following a 2-h exposure to rifabutin at 2× the MIC. To compare the durations of PAEs induced by the different antibiotics tested, PAEs were calculated by combining data for all three strains and both methods. The PAEs (mean ± standard deviation) of the antibiotics at 2× the MIC against MAC obtained by combining data for all three strains and both methods were as follows: amikacin, 10.3 ± 1.3 h; azithromycin, 4.6 ± 0.9 h; clarithromycin, 6.8 ± 0.7 h; clofazimine, 1.7 ± 0.9 h; ethambutol, 1.1 ± 0.5 h; rifabutin, 9.5 ± 0.6 h; rifampin, 9.7 ± 0.8 h; and sparfloxacin, 4.9 ± 0.9 h. Differences between antibiotics were observed (P < 0.001) with amikacin (mean, 10.3 h), rifampin (mean, 9.7 h), and rifabutin (mean, 9.5 h), resulting in significantly longer PAEs than those for clofazimine (mean, 1.7 h) and ethambutol (mean, 1.1 h). Differences between strains were tested for by combining PAEs determined by both methods. Among all strains, ethambutol and clofazimine resulted in the shortest PAEs, while rifabutin and rifampin resulted in the longest PAEs.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Determination of rifabutin PAE against MAC strain NJ9141 following a 2-h exposure to rifabutin at 2× the MIC. , viable count, growth control; , viable count, rifabutin; , GI, control; , GI, rifabutin. For viable-count curves, arrows indicate the difference in times for control and exposed cultures to increase 1 log10 CFU/ml above the initial counts. For GI curves, arrows indicate the difference in times for GI, for the growth control and exposed cultures to increase 2 log10 above zero.

Discussion. Statistical analysis of our PAE results demonstrated no significant difference between the viable-count method and the CO₂ generation method (BACTEC) as assessed by determining the GI. The GI definition of PAE as the difference in times for control and test GIs to increase 2 log₁₀ was developed because lower GI values were less reliable. In addition, GI values above 400 may be meaningless because the next reading, 12 h later, often reaches the maximum of 999. A GI of 100 (or 2 log₁₀) is a value on the straight portion of the MAC growth curve, with meaningful values occurring both above and below that value, allowing for calculation of the PAE according to the GI. The actual colony count at a GI of 100 is between 10⁵ and 10⁶ CFU/ml.