Clarithromycin Minimal Inhibitory and Bactericidal Concentrations against Mycobacterium avium 1- 3
LEONID B. HEIFETS, PAMELA J. LINDHOLM-LEVY, and R. DAWN COMSTOCK
Introduction
Clarithromycin (A-56268, TE-031), a new macrolide developed by Abbott Laboratories (Abbott Park, IL), so far is the only antimicrobial agent tested in a double-blind clinicaltrial using patients with Mycobacterium avium infection (1). Although it was a small preliminary trial, it clearly showed that within a period of 6 wk, monotherapy with clarithromycin produced a dramatic decrease in the number of viable bacteria in the blood of patients with acquired immunodeficiency syndrome (AIDS) and a disseminated M. avium infection. This finding is not evidence of eradication even in those patients in whom no bacteria were found in the blood after treatment. Other clinical observations (2, 3) have demonstrated bacteriologic and clinical effect of other drug regimens, but these trials were neither blind nor placebo-controlled. There have been some contradictory reports on the in vitro activity of clarithromycin in studies conducted in the past. On the one hand, it was reported to be inactive against most M. avium strains, having a minimal inhibitory concentration (MIC) of 16.0 ug/ml in an agar-dilution test (4). On the other hand, clarithromycin was found to be the most effective of a group of drugs, including erythromycin, in vitro and in beige mice (5). The MIC lIo of clarithromycin in this and in another report (6) was 4.0 ug/ml, In a recent study, MIC of clarithromycin ranged from 0.25 to 4.0 ug/ml, and they werepH-dependent: for some strains the MIC were 4.0 ug/ml at pH 6.6 and 0.5 ug/ml at pH 7.4 when tested in 7Hll agar plates (1). It is verylikely that differences in the in vitro activity of clarithromycin may have been related to the types of media and particularly the pH conditions used by different investigators. It was reasonable to assume that the effect of clarithromycin on mycobacteria in patients with AIDS may have been a result ofits ability to affect M. avium multiplying.in blood, i.e., at pH 7.4. Therefore, 858
SUMMARY Minimal Inhibitory and bactericidal concentrations (MIC and MBC) of clarlthrornycln were determined with 49 Mycob8c'e,'um 8vlum strains Isolated from patients with acqulrec:llmmunodeflclency syndrome. The Inhibitory activity depended on the pH of the medium: the drug wu more active at pH 7.4 and .... active at pH 5.0, with activity at pH 6.8 In an Intermediate position. The broth-determlned MIC found at pH 7.4 were 0.25 and 0.5 j.1g1ml for moat strains. The aga,. determined MIC for moat atralns ranged from 1.0to 4.0 j.1g1ml. The MBC of the drug were 8- to 64-fold higher than the Mle, which Indicates that the efficacy of clarlthrornycln can be aaaoclated with Ita Inhibitory rather than Its bactericidal activity. AM REV RE8P1R DIS 1112; 145:158-851
it was important to investigate under what pH conditions this drug would be most active in vitro. The aims of this study were: (1) to determine the MIC of clarithromycin against M. avium strains at various pH values in liquid medium, (2) to compare broth- and agar-determined MIC, and (3) to quantitate the bactericidal potency of clarithromycin by determining the minimal bactericidal concentration (MBC) and MBC/MIC ratios. Methods
Ciarithromycin The drug was provided by Abbott Laboratories. According to the manufacturer's instructions, to make 25 ml of a stock solution, 50 mg of clarithromycin were dissolved in 5 ml of methanol and then diluted to a volume of 25 ml with phosphate buffer at pH 6.5 made by combining M/5 solutions ofKH2P04 and K2HP04 • This stock solution, 2 mglml, was diluted further with the phosphate buffer to make appropriate working solutions, which wererefrigerated, according to the manufacturer's instruction, for not more than 2 wk.
Test Strains Fifty strains isolated from the blood of patients with AIDS and identified as M. avium by the Gen-Probes DNA-RNA hybridization technique (Gen-Probe, Ine., San Diego, CA) were selected for this study. Sixteen of these strains were kindly provided to us by Prof. Jacques Grosset, and the remaining 34 strains had been isolated in our laboratory. Smooth transparent colonies (SmT)growing on 7Hll agar were subcultivated in 7H9 broth for 4 to 5 days, and portions of the broth cultures were stored at - 70 0 C until needed for an experiment. A fresh frozen vial was thawed for each experiment.
Media Middlebrook 7H12 broth (Becton Dickinson Diagnostic Instrument Systems,Sparks, MD) was used in three forms that differed only in pH: 5.0, 6.8, and 7.4. These pH conditions remained unchanged during the whole period of incubation. Agar plates were prepared from Mueller-Hinton agar base (Difco Laboratories, Detroit, MI) supplemented with oleicacid-albumin-dextrose complex(OADC); the pH of this medium was 7.4. OADC and appropriate solutions of clarithromycin were added to the autoclaved batches of agar after they were cooled to 45 0 C. Drug-containing and drug-free batches of the agar werepoured into respective sections of quadrant petri dishes. MIC Determination in Agar Plates The inhibitory activity of clarithromycin was tested in twofold doubling concentrations ranging from 16.0 to 0.125 ug/ml. The MIC was defined as the lowest drug concentration that inhibited greater than 99070 of the growth seen on the drug-free control quadrants in the lOQ-mm quadrant plastic plates. The inoculum was prepared from a 7H12 broth culture that reached the radiometric growth index (GI) reading of 999. These seed vials usu-
(Received in original form April 10, 1991 and in revised form October 28, 1991) 1 From the National Jewish Center for Immunology and Respiratory Medicine and the Departments of Microbiology and Medicine, University of Colorado Health Sciences Center, Denver, Colorado. 2 Supported by a grant from Abbott laboratories and by Grant l-AI-72636 from the National Institute of Allergy and Infectious Disease. 3 Correspondence and requests for reprints should be addressed to Leonid B. Heifets, National Jewish Center for Immunology and Respiratory Medicine, 1400Jackson Street, Denver, CO 80206.
857
CLARITHROMYCIN MIC AND MBC AGAINST MYCOBACTERIUM AVIUM
ally contained 1 to 2 x 108 cfu/ml. Each culture was diluted 10-5, and 0.1 ml of this suspension was inoculated into each quadrant. This inoculum usually produced 100 to 200 colonies on the drug-free agar. The plates were incubated in plastic bags in ambient air at 37° C for 14 days.
8.0
4.0
2.0
E
0,
MIC Determination in 7H12 Broth The MIC was considered to be the lowest concentration that inhibited greater than 99070 of the bacterial population within 8 days of cultivation. According to our previous studies on comparison of the viable counts and the radiometric readings in 7H12 broth (7-9), the MIC determined in this broth radiometrically in the BACTEC system (Becton Dickinson) was defined as the lowest drug concentration in the presence of which the final GI reading was no greater than 50 after 8 days of incubation, whereas the growth in the drugfree controls took place according to the established standards described below. Working solutions of clarithromycin were added in a volume of 0.1 ml per vial to achieve doubling concentrations from 16.0to 0.125 ug/ml, A 7H12 broth seed vial culture (at GI 999) was diluted 1:100,and 0.1 ml of this dilution was inoculated into the test vials and one of the drug-free control vials, each containing 4.0 ml of broth, thus bringing the total dilution of the seed culture to 1:4,000, which resulted in final bacterial concentration of 104 to 105 cfu/ml (2.5 x 104 if the seed vial contained exactly 108 cfu/ml). Another drug-free control vial, the 1:100 control, was inoculated with a bacterial suspension diluted further to provide 102 to 103 cfu/ml, representing 1070 of the bacterial population in the drugcontaining vials. The two drug-free control vials were necessary to confirm that the actual inoculum was within the desired limits: the growth in the undiluted control vial should reach a GI reading of 999 no earlier than the fourth day of the test, and the growth in the 1:100 control should have been greater than GI 20 for no less than 3 consecutive days by the eighth day of incubation.
MBC Determination The MBC was defined as the lowest drug concentration that killed more than 99070 of the bacterial population in 7H12 broth within the 15-day duration of the test. Duplicate drugcontaining and drug-free vials were inoculated just as they were for MI C determination, but they were allowed to incubate until the daily GI reached 20 to 80, indicating that they contained 105 to 106 cfu/ml (10-12). Solutions of clarithromycin were then added to achieve doubling concentrations ranging from 64.0 to 4.0 ug/rnl. Samples were taken from one or another of the alternate vials, diluted 10-1 to 10-6 , depending on GI readings and drug concentrations, and inoculated onto duplicate 7HlO agar plates, 0.5 ml per plate, to determine the number of cfu/ml. Dilutions that reduced the concentrations of the drug below the previously determined MIC obviated the possible effect of drug carryover (13, 14).
BROTH 7H12
10
::::L
05
..
025
•
012
o 12
0.25
0.5
1.0
III
..
•
2.0
4.0
8.0
--
)lg/ml Fig. 1. MIC of clarithromycin for 49 strains. determined at pH 7.4 in 7H12 broth and in Mueller-Hinton agar. Strains that have been used for determining MIC at various pHs are shown as open circles.
More technical details are given in our previous publications (9-12).
Results
MIC in Mueller-Hinton Agar Plates and 7H12 Broth Of the 50 M avium cultures, one strain did not grow on the Mueller-Hinton agar, and although it grew in the pH 7.4 broth, this strain was excluded from analysis. The agar-determined MIC of clarithromycin ranged from 1.0 to 4.0 ug/ml, for 41 of 49 tested strains, whereas MIC determined at the same pH 7.4 in 7H12 broth weresubstantially lower, 0.25to 0.5 ug/ml for 43 of the same 49 strains (figure 1). As a result, this difference was fourfold or eightfold for most strains. MIC at Various pH Values MIC determined in 7H12 broth at three different pH values for 25 strains have shown that this macrolide was most active at pH 7.4, under which conditions the MIC ranged from 0.12 to 1.0 ug/ml, whereas they were higher at pH 6.8 and especially at pH 5.0 (table 1). For 900/0 of the strains, the MIC were 0.85 ug/rnl
at pH 7.4,6.0 ug/ml at pH 6.8, and> 16.0 ug/rnl at pH 5.0. The growth of M avium strains at the three pH values wassufficient to meet the above-described standards for MIC determination, although it has to be mentioned that pH 5.0 was more favorable for the growth of M avium strains than was pH 6.8, as seen in our previous observations (12). The relatively least favorable pH was 7.4, slowing the growth for some strains for as long as 2 days in comparison with pH 6.8 in reaching GI 999 in the drug-free control vials. Because of concern that the differences in MIC at different pH values might have been due to the somewhat slowergrowth at higher pH, wecompared the MIC at pH 6.8 and 7.4 for those strains that did not show substantial differences in the growth rates at these pH values (figure 2A), and for those that grew more slowly at pH 7.4 (figure 2B). For both groups of strains the MIC at pH 7.4 were substantially lower than they were at pH 6.8, which is an indication that this difference was related to the mode of action of this macrolide (probably its ability of penetration into cell) rather than to some difference in the growth at the two pH values.
Minimal Bactericidal Concentrations The MBC weredetermined in 7H12broth using four strains at pH 6.8 and three of the same strains at pH 7.4. The MBC ranged from 16.0 to 64.0 ug/ml at pH 6.8, and from ~ 8.0 to 32.0 at pH 7.4, being eight- to 64-fold greater than the MIC found under both conditions (table 2). Discussion
Clarithromycin was found to be an active inhibitory antimicrobial agent against M avium. The in vitro inhibitory activity of this drug in 7H12 broth depended on the pH of the medium: it was higher at pH 7.4 and lower at pH 5.0,
TABLE 1 DISTRIBUTION OF Mycobacterium avium STRAINS BY THE MIC (Ilg/ml) OF CLARITHROMYCIN IN 7H12 BROTH AT THREE pH VALUES Percentage of Strains
MIC Ranges (j.1g/m~
0.13-0.25 0.5-1.0 2.0-4.0 8.0-16.0 > 16.0 MICso MICgo
pH 6.8
pH 7.4
0.0 1.0 8.0 24.0 68.0
4.0 24.0 60.0 12.0 0.0
48.0 52.0 0.0 0.0 0.0
> 16.0 > 16.0
2.1 6.0
pH 5.0
0.28 0.85
858
HEIFETS, LINDHOLM·LEVY, AND COMSTOCK
syndrome. Am Rev Respir Dis 1991; 144:564-9. 2. Hoy J, Mijch A, Sandi and M, Grayson L, Lu4.0 cas R, Dwyer B. Quadruple-drug therapy for M. 011 2.0 avium-intracellulare bacteremia in AIDS patients. Fig. 2. MIC of clarithromycin at pH .,; J Infect Dis 1990; 161:801-5. values 6.8 and 7.4 for two groups of M. i 10 3. Chiu J, Nassbaum J, Bozzette S, et al. TreatU avium strains. A. No substantial differi 05 ment of disseminated M avium complex infection ence in growth rates at these pH. B. in AIDS with amikacin, ethambutol, rifampin and 0.25 Those that showed a 2-day difference ciprofloxacin. Ann Intern Med 1990; 113:358-61. in reaching the maximal growth. 0.12 4. Berlin OGW, Young LS, Floyd-Reising SA, Bruckner DA. Comparative in vitro activity of the 0.25 0.5 10 20 4.0 8.0 012 0.25 0.5 1.0 2.0 4.0 8.0 new macrolide A-56268 (TE-031)against mycobacMIC pH 7.4 teria. Eur J Clin Microbiol 1987; 6:486-7. 5. Fernandes PB, Hardy DJ, McDaniel D, Hanson CW, Swanson RN. In vitro and in vivo activiTABLE 2 ties of clarithromycin against M avium. Antimicrob Agents Chemother 1989; 33:1531-4. BACTERICIDAL EFFECT OF CLARITHROMYCIN IN 7H12 BROTH AGAINST Mycobacterium avium 6. Naik S, Ruck R. In vitro activities of several At pH 7.4 At pH 6.8 new macrolide antibiotics against M. avium complex. Antimicrob Agents Chemother 1989; 33: MBC MIC MBC MBC/MIC MIC MBC/MIC 1614-6. (pg/ml) (pg/ml) Ratio Strain (pg/ml) (pg/ml) Ratio 7. Heifets LB, Iseman MD, Lindholm-Levy PJ, Kanes W. Determination of ansamycin MICs for 1.0 8.0 8 1854 2.0 32.0 16 Mycobacterium avium complex in liquid medium 16 0.5 2.0 64.0 32 8.0 3350 by radiometric and conventional methods. An64 4.0 64.0 0.5 32.0 A 16 timicrob Agents Chemother 1985; 28:570-5. 16.0 N 2.0 8 8. Heifets LB, Iseman MD, Lindholm-Levy PJ. • Not performed. Ethambutol MICs and MBCs for Mycobacterium avium complex and Mycobacterium tuberculosis. Antimicrob Agents Chemother 1986; 30:927-32. with activity at pH 6.8 in an intermedi- possible degradation, the inoculum in the 9. Heifets LB, Lindholm-Levy PJ. Bacteriostatic ate position. These data are in agreement presence of quinolones was not only in- and bactericidal activity of ciprofloxacin against Mycobacterium tuberculosis and M avium comwith the effect of pH on the activity of hibited but killed during the initial phase plex. Tubercle 1987; 68:267-76. other macrolides, being weak bases (15), of cultivation. With drugs that have high 10. Heifets L, Lindholm-Levy P. Comparison of and it is unlikely that these differences MBC/MIC ratios the effect is inhibitory bactericidal activities of streptomycin, amikacin, were due to some differences in the only, and growth on agar plates may kanamycin, and capreornycinagainst M avium and tuberculosis. Antimicrob Agents Chernother growth rates at various pH values. Our emerge when the drug deteriorates to a M 1989; 33:1298-301. observations with other drugs were also concentration below that initially incor- 11. Heifets LB, Lindholm-Levy PJ, Flory MA. indicative that it is unlikely that the more porated into the medium. MBC/MIC ra- Bactericidal activity in vitro of various rifamycins rapid growth at low pH results in less ac- tios for clarithromycin were very high, against M avium and M tuberculosis. Am Rev tivity of clarithromycin. The MIC of oth- and that may explain the 4- to 8-fold Respir Dis 1990; 141:626-30. 12. Heifets LB, Lindholm-Levy PJ. MICs and er drugs, for example most rifamycins greater MIC found by the agar-dilution MBCs of Win 57273 against M. avium and M. (11), were not influenced by pH changes, technique. Clarithromycin had low bac- tuberculosis. Antimicrob Agents Chemother 1990; whereas some drugs were even more ac- tericidal activity in vitro against M. avi- 34:770-4. tive at the low pH (12). It was stated (15) urn, with MBC exceeding the concentra- 13. Pearson RD, Steigbigel RT, Davis HT, Chapman Sw. Method for reliabledetermination of minthat, in general, pH can influence the tion attainable in blood. Therefore, the imallethal antibiotic concentrations. Antimicrob penetration into the bacterial cell of those efficacy of this drug in eliminating the Agents Chemother 1980; 18:699-708. agents that are weak acids or weak bases, bacteria from the blood of patients with 14. Schoenknecht FD, Sabath, LD, Thornsberry though the influence of pH on drug ac- AIDS is likely associated with its inhibi- C. Susceptibility tests: special tests. In: Lennette tivity is poorly understood. tory activity at pH 7.4. At the same time, EH, Balows A, Hausler WJ Jr, Shadomy HJ, eds. Manual of clinical microbiology. 4th ed. WashingAt pH 7.4 the broth-determined MIC one cannot exclude the possibility that ton DC: American Society for Microbiology, 1985; were between 0.13 and 1.0 ug/ml for 49 the clinical efficacy of clarithromycin 1000-8. tested strains, and the MIC go was 0.85 may also be associated with its accumu- 15. Sabath LD, Gerstein DA, Loder PB, Finland ug/ml, These MIC were all below the lation within the phagocytes, where the M. Excretion of erythromycin and its enhanced activity in urine against gram-negative bacilli with Cmax for this drug (16). As with most intracellular concentration may reach ci- alkalization. J Lab Clin Med 1968; 72:916-23. other antimicrobial agents tested against dal levels (18). 16. Ferrero JL, Bopp BA, Marsh KC, et at. MeM. avium previously (8, 10, 17), the tabolism and disposition of clarithromycin in man. Acknowledgment Drug Metab Dispos 1990; 18:441-6. broth-determined MIC were substantial17. Heifets L. MIC as a quantitative measurement The writers thank Abbott Laboratories for ly lower than the MIC determined by the of the susceptibility of Mycobacterium avium agar dilution method. Such a difference providing the clarithromycin, Prof. J. Gros- strains to seven antituberculosis drugs. Antimicrob found previously for other agents was in- set for the M avium strains obtained during Agents Chemother 1988; 32:1131-6. the controlled clinical trial, L. Landskroner terpreted to be the result of substantial for the artwork, and C. Queen for prepara- 18. Ishiguro M, Koga H, Kohno S, Hayashi T, YamaguchiK, Hirota M. Penetration of macrolides absorption of the drug in agar and degra- tion of the manuscript. into human polymorphonuclear leucocytes. J Andation during prolonged periods of intimicrob Chemother 1989; 24:719-29. 0 8.0
A.
B.
...
cubation at 37 C. An exception was found for quinolones, the only drugs that had low MBC/MIC ratios againstM. avium (9, 11). Probably, regardless of the
References
1. Dautzenberg B, Truffot C, Legris S, et al. Activity of clarithromycin against M. avium infection in patients with the acquired immune deficiency
LEONID B. HEIFETS, PAMELA J. LINDHOLM-LEVY, and R. DAWN COMSTOCK
Introduction
Clarithromycin (A-56268, TE-031), a new macrolide developed by Abbott Laboratories (Abbott Park, IL), so far is the only antimicrobial agent tested in a double-blind clinicaltrial using patients with Mycobacterium avium infection (1). Although it was a small preliminary trial, it clearly showed that within a period of 6 wk, monotherapy with clarithromycin produced a dramatic decrease in the number of viable bacteria in the blood of patients with acquired immunodeficiency syndrome (AIDS) and a disseminated M. avium infection. This finding is not evidence of eradication even in those patients in whom no bacteria were found in the blood after treatment. Other clinical observations (2, 3) have demonstrated bacteriologic and clinical effect of other drug regimens, but these trials were neither blind nor placebo-controlled. There have been some contradictory reports on the in vitro activity of clarithromycin in studies conducted in the past. On the one hand, it was reported to be inactive against most M. avium strains, having a minimal inhibitory concentration (MIC) of 16.0 ug/ml in an agar-dilution test (4). On the other hand, clarithromycin was found to be the most effective of a group of drugs, including erythromycin, in vitro and in beige mice (5). The MIC lIo of clarithromycin in this and in another report (6) was 4.0 ug/ml, In a recent study, MIC of clarithromycin ranged from 0.25 to 4.0 ug/ml, and they werepH-dependent: for some strains the MIC were 4.0 ug/ml at pH 6.6 and 0.5 ug/ml at pH 7.4 when tested in 7Hll agar plates (1). It is verylikely that differences in the in vitro activity of clarithromycin may have been related to the types of media and particularly the pH conditions used by different investigators. It was reasonable to assume that the effect of clarithromycin on mycobacteria in patients with AIDS may have been a result ofits ability to affect M. avium multiplying.in blood, i.e., at pH 7.4. Therefore, 858
SUMMARY Minimal Inhibitory and bactericidal concentrations (MIC and MBC) of clarlthrornycln were determined with 49 Mycob8c'e,'um 8vlum strains Isolated from patients with acqulrec:llmmunodeflclency syndrome. The Inhibitory activity depended on the pH of the medium: the drug wu more active at pH 7.4 and .... active at pH 5.0, with activity at pH 6.8 In an Intermediate position. The broth-determlned MIC found at pH 7.4 were 0.25 and 0.5 j.1g1ml for moat strains. The aga,. determined MIC for moat atralns ranged from 1.0to 4.0 j.1g1ml. The MBC of the drug were 8- to 64-fold higher than the Mle, which Indicates that the efficacy of clarlthrornycln can be aaaoclated with Ita Inhibitory rather than Its bactericidal activity. AM REV RE8P1R DIS 1112; 145:158-851
it was important to investigate under what pH conditions this drug would be most active in vitro. The aims of this study were: (1) to determine the MIC of clarithromycin against M. avium strains at various pH values in liquid medium, (2) to compare broth- and agar-determined MIC, and (3) to quantitate the bactericidal potency of clarithromycin by determining the minimal bactericidal concentration (MBC) and MBC/MIC ratios. Methods
Ciarithromycin The drug was provided by Abbott Laboratories. According to the manufacturer's instructions, to make 25 ml of a stock solution, 50 mg of clarithromycin were dissolved in 5 ml of methanol and then diluted to a volume of 25 ml with phosphate buffer at pH 6.5 made by combining M/5 solutions ofKH2P04 and K2HP04 • This stock solution, 2 mglml, was diluted further with the phosphate buffer to make appropriate working solutions, which wererefrigerated, according to the manufacturer's instruction, for not more than 2 wk.
Test Strains Fifty strains isolated from the blood of patients with AIDS and identified as M. avium by the Gen-Probes DNA-RNA hybridization technique (Gen-Probe, Ine., San Diego, CA) were selected for this study. Sixteen of these strains were kindly provided to us by Prof. Jacques Grosset, and the remaining 34 strains had been isolated in our laboratory. Smooth transparent colonies (SmT)growing on 7Hll agar were subcultivated in 7H9 broth for 4 to 5 days, and portions of the broth cultures were stored at - 70 0 C until needed for an experiment. A fresh frozen vial was thawed for each experiment.
Media Middlebrook 7H12 broth (Becton Dickinson Diagnostic Instrument Systems,Sparks, MD) was used in three forms that differed only in pH: 5.0, 6.8, and 7.4. These pH conditions remained unchanged during the whole period of incubation. Agar plates were prepared from Mueller-Hinton agar base (Difco Laboratories, Detroit, MI) supplemented with oleicacid-albumin-dextrose complex(OADC); the pH of this medium was 7.4. OADC and appropriate solutions of clarithromycin were added to the autoclaved batches of agar after they were cooled to 45 0 C. Drug-containing and drug-free batches of the agar werepoured into respective sections of quadrant petri dishes. MIC Determination in Agar Plates The inhibitory activity of clarithromycin was tested in twofold doubling concentrations ranging from 16.0 to 0.125 ug/ml. The MIC was defined as the lowest drug concentration that inhibited greater than 99070 of the growth seen on the drug-free control quadrants in the lOQ-mm quadrant plastic plates. The inoculum was prepared from a 7H12 broth culture that reached the radiometric growth index (GI) reading of 999. These seed vials usu-
(Received in original form April 10, 1991 and in revised form October 28, 1991) 1 From the National Jewish Center for Immunology and Respiratory Medicine and the Departments of Microbiology and Medicine, University of Colorado Health Sciences Center, Denver, Colorado. 2 Supported by a grant from Abbott laboratories and by Grant l-AI-72636 from the National Institute of Allergy and Infectious Disease. 3 Correspondence and requests for reprints should be addressed to Leonid B. Heifets, National Jewish Center for Immunology and Respiratory Medicine, 1400Jackson Street, Denver, CO 80206.
857
CLARITHROMYCIN MIC AND MBC AGAINST MYCOBACTERIUM AVIUM
ally contained 1 to 2 x 108 cfu/ml. Each culture was diluted 10-5, and 0.1 ml of this suspension was inoculated into each quadrant. This inoculum usually produced 100 to 200 colonies on the drug-free agar. The plates were incubated in plastic bags in ambient air at 37° C for 14 days.
8.0
4.0
2.0
E
0,
MIC Determination in 7H12 Broth The MIC was considered to be the lowest concentration that inhibited greater than 99070 of the bacterial population within 8 days of cultivation. According to our previous studies on comparison of the viable counts and the radiometric readings in 7H12 broth (7-9), the MIC determined in this broth radiometrically in the BACTEC system (Becton Dickinson) was defined as the lowest drug concentration in the presence of which the final GI reading was no greater than 50 after 8 days of incubation, whereas the growth in the drugfree controls took place according to the established standards described below. Working solutions of clarithromycin were added in a volume of 0.1 ml per vial to achieve doubling concentrations from 16.0to 0.125 ug/ml, A 7H12 broth seed vial culture (at GI 999) was diluted 1:100,and 0.1 ml of this dilution was inoculated into the test vials and one of the drug-free control vials, each containing 4.0 ml of broth, thus bringing the total dilution of the seed culture to 1:4,000, which resulted in final bacterial concentration of 104 to 105 cfu/ml (2.5 x 104 if the seed vial contained exactly 108 cfu/ml). Another drug-free control vial, the 1:100 control, was inoculated with a bacterial suspension diluted further to provide 102 to 103 cfu/ml, representing 1070 of the bacterial population in the drugcontaining vials. The two drug-free control vials were necessary to confirm that the actual inoculum was within the desired limits: the growth in the undiluted control vial should reach a GI reading of 999 no earlier than the fourth day of the test, and the growth in the 1:100 control should have been greater than GI 20 for no less than 3 consecutive days by the eighth day of incubation.
MBC Determination The MBC was defined as the lowest drug concentration that killed more than 99070 of the bacterial population in 7H12 broth within the 15-day duration of the test. Duplicate drugcontaining and drug-free vials were inoculated just as they were for MI C determination, but they were allowed to incubate until the daily GI reached 20 to 80, indicating that they contained 105 to 106 cfu/ml (10-12). Solutions of clarithromycin were then added to achieve doubling concentrations ranging from 64.0 to 4.0 ug/rnl. Samples were taken from one or another of the alternate vials, diluted 10-1 to 10-6 , depending on GI readings and drug concentrations, and inoculated onto duplicate 7HlO agar plates, 0.5 ml per plate, to determine the number of cfu/ml. Dilutions that reduced the concentrations of the drug below the previously determined MIC obviated the possible effect of drug carryover (13, 14).
BROTH 7H12
10
::::L
05
..
025
•
012
o 12
0.25
0.5
1.0
III
..
•
2.0
4.0
8.0
--
)lg/ml Fig. 1. MIC of clarithromycin for 49 strains. determined at pH 7.4 in 7H12 broth and in Mueller-Hinton agar. Strains that have been used for determining MIC at various pHs are shown as open circles.
More technical details are given in our previous publications (9-12).
Results
MIC in Mueller-Hinton Agar Plates and 7H12 Broth Of the 50 M avium cultures, one strain did not grow on the Mueller-Hinton agar, and although it grew in the pH 7.4 broth, this strain was excluded from analysis. The agar-determined MIC of clarithromycin ranged from 1.0 to 4.0 ug/ml, for 41 of 49 tested strains, whereas MIC determined at the same pH 7.4 in 7H12 broth weresubstantially lower, 0.25to 0.5 ug/ml for 43 of the same 49 strains (figure 1). As a result, this difference was fourfold or eightfold for most strains. MIC at Various pH Values MIC determined in 7H12 broth at three different pH values for 25 strains have shown that this macrolide was most active at pH 7.4, under which conditions the MIC ranged from 0.12 to 1.0 ug/ml, whereas they were higher at pH 6.8 and especially at pH 5.0 (table 1). For 900/0 of the strains, the MIC were 0.85 ug/rnl
at pH 7.4,6.0 ug/ml at pH 6.8, and> 16.0 ug/rnl at pH 5.0. The growth of M avium strains at the three pH values wassufficient to meet the above-described standards for MIC determination, although it has to be mentioned that pH 5.0 was more favorable for the growth of M avium strains than was pH 6.8, as seen in our previous observations (12). The relatively least favorable pH was 7.4, slowing the growth for some strains for as long as 2 days in comparison with pH 6.8 in reaching GI 999 in the drug-free control vials. Because of concern that the differences in MIC at different pH values might have been due to the somewhat slowergrowth at higher pH, wecompared the MIC at pH 6.8 and 7.4 for those strains that did not show substantial differences in the growth rates at these pH values (figure 2A), and for those that grew more slowly at pH 7.4 (figure 2B). For both groups of strains the MIC at pH 7.4 were substantially lower than they were at pH 6.8, which is an indication that this difference was related to the mode of action of this macrolide (probably its ability of penetration into cell) rather than to some difference in the growth at the two pH values.
Minimal Bactericidal Concentrations The MBC weredetermined in 7H12broth using four strains at pH 6.8 and three of the same strains at pH 7.4. The MBC ranged from 16.0 to 64.0 ug/ml at pH 6.8, and from ~ 8.0 to 32.0 at pH 7.4, being eight- to 64-fold greater than the MIC found under both conditions (table 2). Discussion
Clarithromycin was found to be an active inhibitory antimicrobial agent against M avium. The in vitro inhibitory activity of this drug in 7H12 broth depended on the pH of the medium: it was higher at pH 7.4 and lower at pH 5.0,
TABLE 1 DISTRIBUTION OF Mycobacterium avium STRAINS BY THE MIC (Ilg/ml) OF CLARITHROMYCIN IN 7H12 BROTH AT THREE pH VALUES Percentage of Strains
MIC Ranges (j.1g/m~
0.13-0.25 0.5-1.0 2.0-4.0 8.0-16.0 > 16.0 MICso MICgo
pH 6.8
pH 7.4
0.0 1.0 8.0 24.0 68.0
4.0 24.0 60.0 12.0 0.0
48.0 52.0 0.0 0.0 0.0
> 16.0 > 16.0
2.1 6.0
pH 5.0
0.28 0.85
858
HEIFETS, LINDHOLM·LEVY, AND COMSTOCK
syndrome. Am Rev Respir Dis 1991; 144:564-9. 2. Hoy J, Mijch A, Sandi and M, Grayson L, Lu4.0 cas R, Dwyer B. Quadruple-drug therapy for M. 011 2.0 avium-intracellulare bacteremia in AIDS patients. Fig. 2. MIC of clarithromycin at pH .,; J Infect Dis 1990; 161:801-5. values 6.8 and 7.4 for two groups of M. i 10 3. Chiu J, Nassbaum J, Bozzette S, et al. TreatU avium strains. A. No substantial differi 05 ment of disseminated M avium complex infection ence in growth rates at these pH. B. in AIDS with amikacin, ethambutol, rifampin and 0.25 Those that showed a 2-day difference ciprofloxacin. Ann Intern Med 1990; 113:358-61. in reaching the maximal growth. 0.12 4. Berlin OGW, Young LS, Floyd-Reising SA, Bruckner DA. Comparative in vitro activity of the 0.25 0.5 10 20 4.0 8.0 012 0.25 0.5 1.0 2.0 4.0 8.0 new macrolide A-56268 (TE-031)against mycobacMIC pH 7.4 teria. Eur J Clin Microbiol 1987; 6:486-7. 5. Fernandes PB, Hardy DJ, McDaniel D, Hanson CW, Swanson RN. In vitro and in vivo activiTABLE 2 ties of clarithromycin against M avium. Antimicrob Agents Chemother 1989; 33:1531-4. BACTERICIDAL EFFECT OF CLARITHROMYCIN IN 7H12 BROTH AGAINST Mycobacterium avium 6. Naik S, Ruck R. In vitro activities of several At pH 7.4 At pH 6.8 new macrolide antibiotics against M. avium complex. Antimicrob Agents Chemother 1989; 33: MBC MIC MBC MBC/MIC MIC MBC/MIC 1614-6. (pg/ml) (pg/ml) Ratio Strain (pg/ml) (pg/ml) Ratio 7. Heifets LB, Iseman MD, Lindholm-Levy PJ, Kanes W. Determination of ansamycin MICs for 1.0 8.0 8 1854 2.0 32.0 16 Mycobacterium avium complex in liquid medium 16 0.5 2.0 64.0 32 8.0 3350 by radiometric and conventional methods. An64 4.0 64.0 0.5 32.0 A 16 timicrob Agents Chemother 1985; 28:570-5. 16.0 N 2.0 8 8. Heifets LB, Iseman MD, Lindholm-Levy PJ. • Not performed. Ethambutol MICs and MBCs for Mycobacterium avium complex and Mycobacterium tuberculosis. Antimicrob Agents Chemother 1986; 30:927-32. with activity at pH 6.8 in an intermedi- possible degradation, the inoculum in the 9. Heifets LB, Lindholm-Levy PJ. Bacteriostatic ate position. These data are in agreement presence of quinolones was not only in- and bactericidal activity of ciprofloxacin against Mycobacterium tuberculosis and M avium comwith the effect of pH on the activity of hibited but killed during the initial phase plex. Tubercle 1987; 68:267-76. other macrolides, being weak bases (15), of cultivation. With drugs that have high 10. Heifets L, Lindholm-Levy P. Comparison of and it is unlikely that these differences MBC/MIC ratios the effect is inhibitory bactericidal activities of streptomycin, amikacin, were due to some differences in the only, and growth on agar plates may kanamycin, and capreornycinagainst M avium and tuberculosis. Antimicrob Agents Chernother growth rates at various pH values. Our emerge when the drug deteriorates to a M 1989; 33:1298-301. observations with other drugs were also concentration below that initially incor- 11. Heifets LB, Lindholm-Levy PJ, Flory MA. indicative that it is unlikely that the more porated into the medium. MBC/MIC ra- Bactericidal activity in vitro of various rifamycins rapid growth at low pH results in less ac- tios for clarithromycin were very high, against M avium and M tuberculosis. Am Rev tivity of clarithromycin. The MIC of oth- and that may explain the 4- to 8-fold Respir Dis 1990; 141:626-30. 12. Heifets LB, Lindholm-Levy PJ. MICs and er drugs, for example most rifamycins greater MIC found by the agar-dilution MBCs of Win 57273 against M. avium and M. (11), were not influenced by pH changes, technique. Clarithromycin had low bac- tuberculosis. Antimicrob Agents Chemother 1990; whereas some drugs were even more ac- tericidal activity in vitro against M. avi- 34:770-4. tive at the low pH (12). It was stated (15) urn, with MBC exceeding the concentra- 13. Pearson RD, Steigbigel RT, Davis HT, Chapman Sw. Method for reliabledetermination of minthat, in general, pH can influence the tion attainable in blood. Therefore, the imallethal antibiotic concentrations. Antimicrob penetration into the bacterial cell of those efficacy of this drug in eliminating the Agents Chemother 1980; 18:699-708. agents that are weak acids or weak bases, bacteria from the blood of patients with 14. Schoenknecht FD, Sabath, LD, Thornsberry though the influence of pH on drug ac- AIDS is likely associated with its inhibi- C. Susceptibility tests: special tests. In: Lennette tivity is poorly understood. tory activity at pH 7.4. At the same time, EH, Balows A, Hausler WJ Jr, Shadomy HJ, eds. Manual of clinical microbiology. 4th ed. WashingAt pH 7.4 the broth-determined MIC one cannot exclude the possibility that ton DC: American Society for Microbiology, 1985; were between 0.13 and 1.0 ug/ml for 49 the clinical efficacy of clarithromycin 1000-8. tested strains, and the MIC go was 0.85 may also be associated with its accumu- 15. Sabath LD, Gerstein DA, Loder PB, Finland ug/ml, These MIC were all below the lation within the phagocytes, where the M. Excretion of erythromycin and its enhanced activity in urine against gram-negative bacilli with Cmax for this drug (16). As with most intracellular concentration may reach ci- alkalization. J Lab Clin Med 1968; 72:916-23. other antimicrobial agents tested against dal levels (18). 16. Ferrero JL, Bopp BA, Marsh KC, et at. MeM. avium previously (8, 10, 17), the tabolism and disposition of clarithromycin in man. Acknowledgment Drug Metab Dispos 1990; 18:441-6. broth-determined MIC were substantial17. Heifets L. MIC as a quantitative measurement The writers thank Abbott Laboratories for ly lower than the MIC determined by the of the susceptibility of Mycobacterium avium agar dilution method. Such a difference providing the clarithromycin, Prof. J. Gros- strains to seven antituberculosis drugs. Antimicrob found previously for other agents was in- set for the M avium strains obtained during Agents Chemother 1988; 32:1131-6. the controlled clinical trial, L. Landskroner terpreted to be the result of substantial for the artwork, and C. Queen for prepara- 18. Ishiguro M, Koga H, Kohno S, Hayashi T, YamaguchiK, Hirota M. Penetration of macrolides absorption of the drug in agar and degra- tion of the manuscript. into human polymorphonuclear leucocytes. J Andation during prolonged periods of intimicrob Chemother 1989; 24:719-29. 0 8.0
A.
B.
...
cubation at 37 C. An exception was found for quinolones, the only drugs that had low MBC/MIC ratios againstM. avium (9, 11). Probably, regardless of the
References
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