Journal of Antimicrobial Chemotherapy (1990) 25, Suppl. A, 25-28
Bacteriostatic and bactericidal activity of azithromycin against Haemophilus influenzae F. W. Goldstein, M. F. Emirian, A. Cootrot and J. F. Acar
Macrolidc antibiotics, commonly used in upper and lower respiratory tract infections, are inconsistently active against Haemophilus influenzae. The new azalide, azithromycin, was compared with erythromycin and roxithromycin against this pathogen. Azithromycin (MIC range 006-1 mg/1) was four to eight times more potent than erythromycin (MIC range 0-5-8 mg/1) and roxithromycin (MIC range 0-5-16 mg/1). At lmg/1, 100% of the strains of H. influenzae were inhibited by azithromycin compared with 16% with erythromycin and 5% with roxithromycin. Azithromycin exhibited a rapid bactericidal effect, with a 99-9% kill at 4h. The MBC was equal to or up to four-times greater than the MIC. Introduction Macrolidc antibiotics are commonly used in upper and lower respiratory tract infections in adults as well as children but have the disadvantage of inconsistent activity against Haemophilus influenzae, in otitis media (Howie & Ploussard, 1972; Ginsburg & Eichenwald, 1976), sinusitis (Kalm etal., 1975) and acute exacerbations of chronic obstructive pulmonary disease (Williams & Andrews, 1974). The aim of this study was to determine the comparative bacteriostatic and bactericidal activities of azithromycin against strains of H. influenzae that had recently been isolated in France. Materials and methods Bacterial strains Eighty-four strains of H. influenzae, including 13 /Mactamase producers, recently isolated from clinical specimens, were cultured overnight on chocolate-isovitalex agar (Diagnostics Pasteur). Antimicrobial agents Azithromycin (CP-62,993) was obtained from Pfizer Central Research (Sandwich, Kent, UK), erythromycin from Abbott Laboratories (France), and roxithromycin from Roussel Laboratories (France). Minimum inhibitory concentrations MIC determinations were made on Mueller-Hinton agar with the addition of haemin (10 mg/1; Sigma), /J-NAD (10 mg/1; Sigma) and yeast extract (5 g/1; Diagnostics Pasteur, 0305-7453/9O/25A025+O4 S02.00/0
25 © 1990 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at Georgetown University on August 30, 2015
Hopital Saint Joseph, 7 rue Pierre Larousse, 75015 Paris, France
26
F. W. Goldstein et aL
France). The inoculum was 10*cfu/ml and a Steers replicator was used to deliver 10*cfu/spot. Cultures were incubated for 24 h at 37°C in air. Minimum bactericidal concentrations
Results The MIC ranges for azithromycin, erythromycin and roxithromycin were 0-06-1, 0-5-8 and 0-5-16 mg/1, respectively (Table I); MIC^ values were 1, 4, and 8 mg/1, respectively. At a concentration of 1 mg/1, 100% of the strains were inhibited by azithromycin compared with 16% by erythromycin and 5% by roxithromycin. Azithromycin showed the lowest MBC values, with 89 and 100% of the strains being killed at 1 and 4 mg/1, respectively (Table II). This compares with 3 and 79% for erythromycin, and 0 and 9% for roxithromycin. Azithromycin also gave the lowest MBC/MIC ratios (Table HI), with 69% of the strains having a ratio of 1, compared with 61% for erythromycin and 31% for roxithromycin. The MBC was one to four Table L MICs for the 84 strains of H. influenzae Antibiotic
0-06 0-12 0-25
MIC (mg/1) 0-5 1 2
4
8
16
Azithromycin Erythromycin Roxithromycin
1 — —
35 1 1
35 12 2
29 33
3 40
1
13 — —
— 39 7
Table D. MBCs 1(99-9% kill) for 70 strains of H. influenzae Antibiotic
0-25
0-5
Arithromycin Erythromycin Roxithromycin
6 — —
22
1 2 34 2 —
MBC (mg/1) 4 8
16
32
64
1 30 5
1 27
— 5
1
7 23 1
— 14 31
Table m . Ratio of MBC 'to MIC for 70 strains of H. Influenzae Antibiotic Azithromycin Erythromycin Roxithromycin
No. of strains showing MBC/MIC ratios 1 2 4 8 48 43 22
19 20 23
2 6 23
1 1 2
Downloaded from http://jac.oxfordjournals.org/ at Georgetown University on August 30, 2015
MBC and killing curves for 70 strains were carried out simultaneously in MuellerHinton broth, supplemented as above, with an inoculum of approx. 10* cfu/ml. The count for each strain was determined by plating of 0-01 ml amounts of serial ten-fold dilutions from 100 to 0-01% on chocolate-isovitalex agar. Incubation was at 37°C in air, and 0-01 ml was plated on chocolate-isovitalex agar at 4 and 24 h for the MBC and hourly up to 10 h for the killing curves. Incubation of subcultures was for 24 h at 37°C in air. The percentage survival was estimated by comparison with the initial inoculum count.
ID-YHTO activity against HaemopkUus influnuat
8
10
12
14
ie
is
22
24
Tim* (h)
Figure 1. Killing curves at twice the MIC for the three antibiotics against H. influenzae. A, Azithromycin; O> erythromycin; 0 , rojuthromycin.
times the MIC. Azithromycin had the most rapid bactericidal effect, with 59 and 83% of the strains being killed ( ^ 99.9% kill) at 1 and 4 mg/1,respectively,after only 4 h (Figures 1 and 2). No significant differences in MICs or MBCs were observed when the strains were classified according to serotype, biotype or /J-lactamase production. 100 -
n 10
t.
••
1-0
1 >
•* ::: 0-1
9
-
t:
..
:*:
tn 3*
...
ii
•
0-01 »••»••«•> ••••f••••
I
2 4 Azithromycin (mg/1)
Figure 2. Percentage survival of H. inftuenzae after 4 h incubation with azithromycin at three different concentrations.
Downloaded from http://jac.oxfordjournals.org/ at Georgetown University on August 30, 2015
6
27
28
F. W. Goldstein et aL Discussion
References Barry, A. L., Jones, R. N. & Thornsberry, C. (1988). In vitro activities of azithromycin (CP-62,993), clarithromycin (A-56,268; TE-031), erythromycin, roxithromycin, and clindamycin. Antimicrobial Agents and Chemotherapy 32, 752-4. Dunkin, K. T., Jones, S. & Howard, A J. (1988). The in-vitro activity of CP-62,993 against Haemophilus influenzae, Branhamella catarrhalis. staphylococci and streptococci. Journal of Antimicrobial Chemotherapy 21, 405-11. Fernandes, P. B. & Hardy, D. J. (1988). Comparative in-vitro potencies of nine new macrolides. Drugs Under Experimental end Clinical Research 14, 445-51. Ginsburg, C. M. & Eichenwald, H. F. (1976). Erythromycin: a review of its uses in pediatric practice. Journal of Pediatrics 89, 872-84. Girard, A. E., Girard, D., English, A. R., Gootz, T. D., Cimochowski, C. R., Faiella, J. A. et al. (1987). Pharmacokinetic and in vivo studies with azithromycin (CP-62,993), a new macrolide with an extended half-life and excellent tissue distribution. Antimicrobial Agents and Chemotherapy 31, 1948-54. Hardy, D. W., Hensey, D. M., Beyer, J. M., Vojtko, C , McDonald, E. J. & Fernandes, P. B. (1988). Comparative in vitro activities of new 14-, 15-, and 16-membered macrolides. Antimicrobial Agents and Chemotherapy 32, 1710-9. Howie, V. M. & Ploussard, J. H. (1972). Efficacy offixedcombination antibiotics versus separate components in otitis media. Effectiveness of erythromycin estolate, triple sulfonamide, ampicillin, erythromycin estolate-triple sulfonamide, and placebo in 280 patients with acute otitis media under two and one-half years of age. Clinical Pediatrics 11, 205-14. Kalm, O., Kamme, C , Bergstrom, B., Loftvist, T. & Norman, O. (1975). Erythromycin stearate in acute maxillary sinusitis. Scandinavian Journal of Infectious Diseases 7, 209-17. Retsema, J., Girard, A., Shelkly, W., Manousos, M., Anderson, M., Bright, G. etal. (1987). Spectrum and mode of action of azithromycin (CP-62,993), a new 15-membered-ring macrolide with improved potency against Gram-negative organisms. Antimicrobial Agents and Chemotherapy 31, 1939-47. Williams, J. D. & Andrews, J. (1974). Sensitivity of Haemophilus influenzae to antibiotics. British Medical Journal i, 134-7.
Downloaded from http://jac.oxfordjournals.org/ at Georgetown University on August 30, 2015
Macrolide antibiotics are very useful for the treatment of upper and lower respiratory tract infection, especially in children. However, their poor activity against H. influenzae has been responsible for clinical failures and had emphasized the need for more potent compounds. Azithromycin has improved potency over erythromycin and other macrolides against H. influenzae (Girard etal., 1987; Retsema etal., 1987; Barry, Jones & Thornsberry, 1988; Dunkin, Jones & Howard, 1988; Fernandes & Hardy, 1988; Hardy etal., 1988). In the present study, azithromycin was up to eight times more effective than erythromycin and roxithromycin against H. influenzae with 100% of the 84 strains being inhibited at 1 mg/1. Moreover, azithromycin has a rapid and bactericidal effect with 82-9% and 100% of the strains killed at 4 mg/1 at 4 and 24 h, respectively. Preliminary pharmacokinetic data indicate that azithromycin exhibits high and sustained tissue levels (Girard etal., 1987; Retsema etal., 1987). These data indicate that azithromycin is a very promising new antimicrobial agent and that it deserves further in-vitro and in-vivo studies.
Bacteriostatic and bactericidal activity of azithromycin against Haemophilus influenzae F. W. Goldstein, M. F. Emirian, A. Cootrot and J. F. Acar
Macrolidc antibiotics, commonly used in upper and lower respiratory tract infections, are inconsistently active against Haemophilus influenzae. The new azalide, azithromycin, was compared with erythromycin and roxithromycin against this pathogen. Azithromycin (MIC range 006-1 mg/1) was four to eight times more potent than erythromycin (MIC range 0-5-8 mg/1) and roxithromycin (MIC range 0-5-16 mg/1). At lmg/1, 100% of the strains of H. influenzae were inhibited by azithromycin compared with 16% with erythromycin and 5% with roxithromycin. Azithromycin exhibited a rapid bactericidal effect, with a 99-9% kill at 4h. The MBC was equal to or up to four-times greater than the MIC. Introduction Macrolidc antibiotics are commonly used in upper and lower respiratory tract infections in adults as well as children but have the disadvantage of inconsistent activity against Haemophilus influenzae, in otitis media (Howie & Ploussard, 1972; Ginsburg & Eichenwald, 1976), sinusitis (Kalm etal., 1975) and acute exacerbations of chronic obstructive pulmonary disease (Williams & Andrews, 1974). The aim of this study was to determine the comparative bacteriostatic and bactericidal activities of azithromycin against strains of H. influenzae that had recently been isolated in France. Materials and methods Bacterial strains Eighty-four strains of H. influenzae, including 13 /Mactamase producers, recently isolated from clinical specimens, were cultured overnight on chocolate-isovitalex agar (Diagnostics Pasteur). Antimicrobial agents Azithromycin (CP-62,993) was obtained from Pfizer Central Research (Sandwich, Kent, UK), erythromycin from Abbott Laboratories (France), and roxithromycin from Roussel Laboratories (France). Minimum inhibitory concentrations MIC determinations were made on Mueller-Hinton agar with the addition of haemin (10 mg/1; Sigma), /J-NAD (10 mg/1; Sigma) and yeast extract (5 g/1; Diagnostics Pasteur, 0305-7453/9O/25A025+O4 S02.00/0
25 © 1990 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at Georgetown University on August 30, 2015
Hopital Saint Joseph, 7 rue Pierre Larousse, 75015 Paris, France
26
F. W. Goldstein et aL
France). The inoculum was 10*cfu/ml and a Steers replicator was used to deliver 10*cfu/spot. Cultures were incubated for 24 h at 37°C in air. Minimum bactericidal concentrations
Results The MIC ranges for azithromycin, erythromycin and roxithromycin were 0-06-1, 0-5-8 and 0-5-16 mg/1, respectively (Table I); MIC^ values were 1, 4, and 8 mg/1, respectively. At a concentration of 1 mg/1, 100% of the strains were inhibited by azithromycin compared with 16% by erythromycin and 5% by roxithromycin. Azithromycin showed the lowest MBC values, with 89 and 100% of the strains being killed at 1 and 4 mg/1, respectively (Table II). This compares with 3 and 79% for erythromycin, and 0 and 9% for roxithromycin. Azithromycin also gave the lowest MBC/MIC ratios (Table HI), with 69% of the strains having a ratio of 1, compared with 61% for erythromycin and 31% for roxithromycin. The MBC was one to four Table L MICs for the 84 strains of H. influenzae Antibiotic
0-06 0-12 0-25
MIC (mg/1) 0-5 1 2
4
8
16
Azithromycin Erythromycin Roxithromycin
1 — —
35 1 1
35 12 2
29 33
3 40
1
13 — —
— 39 7
Table D. MBCs 1(99-9% kill) for 70 strains of H. influenzae Antibiotic
0-25
0-5
Arithromycin Erythromycin Roxithromycin
6 — —
22
1 2 34 2 —
MBC (mg/1) 4 8
16
32
64
1 30 5
1 27
— 5
1
7 23 1
— 14 31
Table m . Ratio of MBC 'to MIC for 70 strains of H. Influenzae Antibiotic Azithromycin Erythromycin Roxithromycin
No. of strains showing MBC/MIC ratios 1 2 4 8 48 43 22
19 20 23
2 6 23
1 1 2
Downloaded from http://jac.oxfordjournals.org/ at Georgetown University on August 30, 2015
MBC and killing curves for 70 strains were carried out simultaneously in MuellerHinton broth, supplemented as above, with an inoculum of approx. 10* cfu/ml. The count for each strain was determined by plating of 0-01 ml amounts of serial ten-fold dilutions from 100 to 0-01% on chocolate-isovitalex agar. Incubation was at 37°C in air, and 0-01 ml was plated on chocolate-isovitalex agar at 4 and 24 h for the MBC and hourly up to 10 h for the killing curves. Incubation of subcultures was for 24 h at 37°C in air. The percentage survival was estimated by comparison with the initial inoculum count.
ID-YHTO activity against HaemopkUus influnuat
8
10
12
14
ie
is
22
24
Tim* (h)
Figure 1. Killing curves at twice the MIC for the three antibiotics against H. influenzae. A, Azithromycin; O> erythromycin; 0 , rojuthromycin.
times the MIC. Azithromycin had the most rapid bactericidal effect, with 59 and 83% of the strains being killed ( ^ 99.9% kill) at 1 and 4 mg/1,respectively,after only 4 h (Figures 1 and 2). No significant differences in MICs or MBCs were observed when the strains were classified according to serotype, biotype or /J-lactamase production. 100 -
n 10
t.
••
1-0
1 >
•* ::: 0-1
9
-
t:
..
:*:
tn 3*
...
ii
•
0-01 »••»••«•> ••••f••••
I
2 4 Azithromycin (mg/1)
Figure 2. Percentage survival of H. inftuenzae after 4 h incubation with azithromycin at three different concentrations.
Downloaded from http://jac.oxfordjournals.org/ at Georgetown University on August 30, 2015
6
27
28
F. W. Goldstein et aL Discussion
References Barry, A. L., Jones, R. N. & Thornsberry, C. (1988). In vitro activities of azithromycin (CP-62,993), clarithromycin (A-56,268; TE-031), erythromycin, roxithromycin, and clindamycin. Antimicrobial Agents and Chemotherapy 32, 752-4. Dunkin, K. T., Jones, S. & Howard, A J. (1988). The in-vitro activity of CP-62,993 against Haemophilus influenzae, Branhamella catarrhalis. staphylococci and streptococci. Journal of Antimicrobial Chemotherapy 21, 405-11. Fernandes, P. B. & Hardy, D. J. (1988). Comparative in-vitro potencies of nine new macrolides. Drugs Under Experimental end Clinical Research 14, 445-51. Ginsburg, C. M. & Eichenwald, H. F. (1976). Erythromycin: a review of its uses in pediatric practice. Journal of Pediatrics 89, 872-84. Girard, A. E., Girard, D., English, A. R., Gootz, T. D., Cimochowski, C. R., Faiella, J. A. et al. (1987). Pharmacokinetic and in vivo studies with azithromycin (CP-62,993), a new macrolide with an extended half-life and excellent tissue distribution. Antimicrobial Agents and Chemotherapy 31, 1948-54. Hardy, D. W., Hensey, D. M., Beyer, J. M., Vojtko, C , McDonald, E. J. & Fernandes, P. B. (1988). Comparative in vitro activities of new 14-, 15-, and 16-membered macrolides. Antimicrobial Agents and Chemotherapy 32, 1710-9. Howie, V. M. & Ploussard, J. H. (1972). Efficacy offixedcombination antibiotics versus separate components in otitis media. Effectiveness of erythromycin estolate, triple sulfonamide, ampicillin, erythromycin estolate-triple sulfonamide, and placebo in 280 patients with acute otitis media under two and one-half years of age. Clinical Pediatrics 11, 205-14. Kalm, O., Kamme, C , Bergstrom, B., Loftvist, T. & Norman, O. (1975). Erythromycin stearate in acute maxillary sinusitis. Scandinavian Journal of Infectious Diseases 7, 209-17. Retsema, J., Girard, A., Shelkly, W., Manousos, M., Anderson, M., Bright, G. etal. (1987). Spectrum and mode of action of azithromycin (CP-62,993), a new 15-membered-ring macrolide with improved potency against Gram-negative organisms. Antimicrobial Agents and Chemotherapy 31, 1939-47. Williams, J. D. & Andrews, J. (1974). Sensitivity of Haemophilus influenzae to antibiotics. British Medical Journal i, 134-7.
Downloaded from http://jac.oxfordjournals.org/ at Georgetown University on August 30, 2015
Macrolide antibiotics are very useful for the treatment of upper and lower respiratory tract infection, especially in children. However, their poor activity against H. influenzae has been responsible for clinical failures and had emphasized the need for more potent compounds. Azithromycin has improved potency over erythromycin and other macrolides against H. influenzae (Girard etal., 1987; Retsema etal., 1987; Barry, Jones & Thornsberry, 1988; Dunkin, Jones & Howard, 1988; Fernandes & Hardy, 1988; Hardy etal., 1988). In the present study, azithromycin was up to eight times more effective than erythromycin and roxithromycin against H. influenzae with 100% of the 84 strains being inhibited at 1 mg/1. Moreover, azithromycin has a rapid and bactericidal effect with 82-9% and 100% of the strains killed at 4 mg/1 at 4 and 24 h, respectively. Preliminary pharmacokinetic data indicate that azithromycin exhibits high and sustained tissue levels (Girard etal., 1987; Retsema etal., 1987). These data indicate that azithromycin is a very promising new antimicrobial agent and that it deserves further in-vitro and in-vivo studies.
