Vol. 34, No. 7
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUlY 1990, P. 1442-1443 0066-4804/90/071442-02$02.00/0 Copyright C) 1990, American Society for Microbiology
In Vitro Susceptibilities of Bordetella pertussis and Bordetella parapertussis to Six New Oral Cephalosporins JORG E. HOPPE* AND JOCHEN MULLER
University Children's Hospital, Rumelinstrasse 23, D-7400 Tubingen, Federal Republic of Germany Received 12 February 1990/Accepted 20 April 1990
Of six new oral cephalosporins, cefixime and cefpodoxime were the most active (MIC for 90% of isolates tested [MIC90], 16 ,ig/ml) against Bordetella pertussis, followed by cefetamet, cefprozil, and loracarbef (LY163892) (MIC90, 64 ,ug/ml) and ceftibuten (MIC90, 128 ,ug/ml). Against Bordetella parapertussis, loracarbef was more active (MIC90, 32 ,ug/ml) than the other compounds tested (MIC90s, 64 to >128 ,ug/ml). The new oral cephalosporins are unlikely to play a role in pertussis treatment.
kindly supplied by B. van Klingeren (Bilthoven, The Netherlands) for a previous study (5) were also included. In addition, B. pertussis ATCC 9797 and B. parapertussis ATCC 15311 were tested. The test strains were cultivated on antibiotic-free IsoSensitest agar (Oxoid, Basingstoke, United Kingdom) supplemented with 5% whole defibrinated horse blood. Several colonies were inoculated into antibiotic-free Iso-Sensitest broth (Oxoid) supplemented with 5% horse blood and incubated at 36°C for 24 h. As determined by cell counts, the broth then contained 0.85 x 108 CFU of B. pertussis and 2.19 x 108 CFU of B. parapertussis per ml. Serial twofold concentrations of antibiotics were incorporated into Iso-Sensitest agar supplemented with 5% horse blood. Inocula were applied with a multipoint inoculator (Scan 400; Mast, Bootle, United Kingdom), resulting in final inocula of 2.6 x 104 CFU of B. pertussis per spot and 6.57 x 104 CFU of B. parapertussis per spot. The plates were incubated for 48 h at 36°C protected from desiccation. MIC endpoint readings were done as recommended by the National Committee for Clinical Laboratory Standards (6). Antibiotic-free plates were inoculated before and after the test plates to check for growth and purity. Escherichia coli ATCC 25922 and, in accordance with the German DIN guidelines (2), Staphylococcus aureus ATCC 25923 served as control strains. All tests were performed in duplicate. Table 1 shows the results of MIC determinations. Against B. pertussis, cefixime and cefpodoxime exhibited the highest degree of activity (MIC for 90% of isolates tested [MIC9], 16 ,ug/ml). The other drugs tested had MIC90s of .64 ,ug/ml. Not surprisingly, B. parapertussis was more resistant to the drugs tested than B. pertussis, the only exception being loracarbef. In general, all compounds were quite inactive against B. parapertussis, with loracarbef showing the lowest MIC90 (32 LLgIml). The susceptibilities of the Dutch B. pertussis isolates and of the two Bordetella ATCC strains were comparable to those of our local isolates, being identical to or one dilution step lower than the MIC%0s. Performance of the tests in duplicate did not show any intrastrain variations. As far as literature data on the in vitro activities of the new oral cephalosporins against B. pertussis are available, they are similar to our results. Simon found MIC90s of >100 ,ug/ml for cefetamet (8) and 12.5 ,ug/ml for cefixime (7). Hagedorn et al. (H.-J. Hagedorn, C. H. Wirsing Von Konig, A. Kraminer-Hagedorn, A. Tacken, and U. Diekmann, Program Abstr. 28th Intersci. Conf. Antimicrob. Agents
The older oral cephalosporins show very poor activity against Bordetella pertussis. The MICs of cephalexin, cephradine, and cefadroxil range between 31 and >128 ,ug/ml (1, 5), and those of cefaclor range between 12 and 25 ,ug/ml (1). Cephalexin at a concentration of 40 ,ug/ml was chosen by Sutcliffe and Abbott (9) as the selective agent in charcoal horse blood agar for clinical isolation of bordetellae, since the compound is inactive against B. pertussis but inhibitory to most organisms of the normal nasopharyngeal flora. Cefuroxime, a parenteral extended-spectrum cephalosporin, has MICs of 1.5 to 6 ,ug/ml against B. pertussis (1). After oral administration of the new prodrug cefuroxime axetil, cefuroxime concentrations of 3.3 ,ug/ml in respiratory secretions have been measured (R. A. Walstad, J. S. Vilsvik, E. Thurmann-Nielsen, J. V. Griggs, and G. W. Brown, Proc. Cefuroxime Axetil Symp., London, United Kingdom, 1988, p. 65-67). Thus, for the first time an orally applicable cephalosporin has become of interest for the treatment of pertussis. We therefore studied the in vitro activities against B. pertussis and Bordetella parapertussis of six other new oral cephalosporins, some of which show remarkably improved activities against many fastidious and nonfastidious gram-negative bacteria (R. N. Jones, Antimicrob. Newsl. 5:1-8, 1988). The test procedure was as described previously (4). Powders of known potency of the following antimicrobial agents were supplied by the manufacturers or their distributors in the Federal Republic of Germany: cefetamet (Ro 15-8074) (Hoffmann-La Roche, Grenzach, Federal Republic of Germany), cefixime (EMD 49 653; FK-027) (E. Merck AG, Darmstadt, Federal Republic of Germany), cefpodoxime (RU 51764; R-3746) (Albert-Roussel, Wiesbaden, Federal Republic of Germany), cefprozil (BMY-28100) (Bristol, NeuIsenburg, Federal Republic of Germany), ceftibuten (7432-S) (Essex, Munich, Federal Republic of Germany), and loracarbef (LY163892) (Lilly, Bad Homburg, Federal Republic of Germany). Stock solutions at 2,000 ,ug/ml were prepared by following the manufacturers' instructions. An agar dilution procedure patterned after the recommendations of the National Committee for Clinical Laboratory Standards (6) was used. Most of the test strains of B. pertussis (n = 33) and B. parapertussis (n = 33) were recent clinical isolates from children in southern Germany. The isolates had been stored at -70°C in glycerol soon after isolation. Three Dutch strains of B. pertussis which had been *
Corresponding author. 1442
VOL. 34, 1990
NOTES
TABLE 1. Activities of six new oral cephalosporins against 33 isolates each of B. pertussis and B. parapertussis
Organism B. pertussis
B. parapertussis
a
agent
Range
MIC (pLg/ml)a
Cefetamet Cefixime Cefpodoxime Cefprozil Ceftibuten Loracarbef
64 4-32 8-16 16-64 64-128 32-64
64 16 8 64 128 64
64 16 16 64 128 64
Cefetamet Cefixime Cefpodoxime Cefprozil Ceftibuten Loracarbef
>128 16-64 64->128 32-64 128->128 32
>128 32 128 64 >128 32
>128 64 128 64 >128 32
Antimicrobial
50%
9
cephalosporins) against many constituents of the normal nasopharyngeal flora might eventually replace cephalexin as the inhibitory agent in charcoal horse blood agar. Cefetamet and ceftibuten in particular deserve further evaluation in this respect.
50%0 and 90%o, MIC for 50 and 90o of isolates tested, respectively.
Chemother., abstr. no. 1219, 1988) tested cefixime and found MIC90 of 8 ,ug/ml. We are not aware of any literature data the activities against B. parapertussis of the compounds tested. The data on the in vitro activities of the new oral cephalosporins have to be interpreted in relation to the concentrations achievable in respiratory secretions. Hayashi (3) measured cefixime concentrations of 0.02 to 0.05 ,ug/ml in sputum. For cefpodoxime, no data are yet available. In conclusion, all of the new oral cephalosporins tested are unlikely to play a role in the treatment of pertussis patients due to the poor in vitro activities of these agents against bordetellae. On the other hand, compounds that are totally inactive against bordetellae but that show high activity (comparable to the activities of parenteral broad-spectrum an on
1443
LITERATURE CITED 1. Bannatyne, R. M., and R. Cheung. 1984. Susceptibility of Bordetella pertussis to cephalosporin derivatives and imipenem. Antimicrob. Agents Chemother. 26:604-605. 2. Deutsches Institut fur Normung. 1979. Methods for the determination of susceptibility of pathogenic bacteria (without mycobacteria) to chemotherapeutic agents; determination of MIC by agar dilution method. DIN 58940, part 6. Deutsches Institut fur Normung, Berlin. 3. Hayashi, I. 1985. Serum and sputum concentration and clinical results of cefixime in respiratory tract infections. Chemotherapy (Tokyo) 33(Suppl. 6):253-267. 4. Hoppe, J. E., and A. Eichhorn. 1989. Activity of new macrolides against Bordetella pertussis and Bordetella parapertussis. Eur. J. Clin. Microbiol. Infect. Dis. 8:653-654. 5. Hoppe, J. E., A. Haug, and K. Botzenhart. 1987. Susceptibility of Bordetella pertussis and Bordetella parapertussis to twenty-four antibiotics. Chemotherapy (Basel) 33:250-254. 6. National Committee for Clinical Laboratory Standards. 1985. Approved standard M7-A. Standard methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. National Committee for Clinical Laboratory Standards, Villanova, Pa. 7. Simon, C. 1987. Zur in-vitro-Aktivitat von Cefixim im Vergleich zu anderen Oralcephalosporinen. Fortschr. Antimikrob. Antineoplast. Chemother. 6:1193-1196. 8. Simon, C. 1987. In vitro activity of Ro 15-8074 and Ro 19-5247 in comparison to cefaclor and cefalexin. Infection 15:122-124. 9. Sutcliffe, E. M., and J. D. Abbott. 1972. Selective medium for the isolation of Bordetella pertussis and parapertussis. J. Clin. Pathol. 25:732-733.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUlY 1990, P. 1442-1443 0066-4804/90/071442-02$02.00/0 Copyright C) 1990, American Society for Microbiology
In Vitro Susceptibilities of Bordetella pertussis and Bordetella parapertussis to Six New Oral Cephalosporins JORG E. HOPPE* AND JOCHEN MULLER
University Children's Hospital, Rumelinstrasse 23, D-7400 Tubingen, Federal Republic of Germany Received 12 February 1990/Accepted 20 April 1990
Of six new oral cephalosporins, cefixime and cefpodoxime were the most active (MIC for 90% of isolates tested [MIC90], 16 ,ig/ml) against Bordetella pertussis, followed by cefetamet, cefprozil, and loracarbef (LY163892) (MIC90, 64 ,ug/ml) and ceftibuten (MIC90, 128 ,ug/ml). Against Bordetella parapertussis, loracarbef was more active (MIC90, 32 ,ug/ml) than the other compounds tested (MIC90s, 64 to >128 ,ug/ml). The new oral cephalosporins are unlikely to play a role in pertussis treatment.
kindly supplied by B. van Klingeren (Bilthoven, The Netherlands) for a previous study (5) were also included. In addition, B. pertussis ATCC 9797 and B. parapertussis ATCC 15311 were tested. The test strains were cultivated on antibiotic-free IsoSensitest agar (Oxoid, Basingstoke, United Kingdom) supplemented with 5% whole defibrinated horse blood. Several colonies were inoculated into antibiotic-free Iso-Sensitest broth (Oxoid) supplemented with 5% horse blood and incubated at 36°C for 24 h. As determined by cell counts, the broth then contained 0.85 x 108 CFU of B. pertussis and 2.19 x 108 CFU of B. parapertussis per ml. Serial twofold concentrations of antibiotics were incorporated into Iso-Sensitest agar supplemented with 5% horse blood. Inocula were applied with a multipoint inoculator (Scan 400; Mast, Bootle, United Kingdom), resulting in final inocula of 2.6 x 104 CFU of B. pertussis per spot and 6.57 x 104 CFU of B. parapertussis per spot. The plates were incubated for 48 h at 36°C protected from desiccation. MIC endpoint readings were done as recommended by the National Committee for Clinical Laboratory Standards (6). Antibiotic-free plates were inoculated before and after the test plates to check for growth and purity. Escherichia coli ATCC 25922 and, in accordance with the German DIN guidelines (2), Staphylococcus aureus ATCC 25923 served as control strains. All tests were performed in duplicate. Table 1 shows the results of MIC determinations. Against B. pertussis, cefixime and cefpodoxime exhibited the highest degree of activity (MIC for 90% of isolates tested [MIC9], 16 ,ug/ml). The other drugs tested had MIC90s of .64 ,ug/ml. Not surprisingly, B. parapertussis was more resistant to the drugs tested than B. pertussis, the only exception being loracarbef. In general, all compounds were quite inactive against B. parapertussis, with loracarbef showing the lowest MIC90 (32 LLgIml). The susceptibilities of the Dutch B. pertussis isolates and of the two Bordetella ATCC strains were comparable to those of our local isolates, being identical to or one dilution step lower than the MIC%0s. Performance of the tests in duplicate did not show any intrastrain variations. As far as literature data on the in vitro activities of the new oral cephalosporins against B. pertussis are available, they are similar to our results. Simon found MIC90s of >100 ,ug/ml for cefetamet (8) and 12.5 ,ug/ml for cefixime (7). Hagedorn et al. (H.-J. Hagedorn, C. H. Wirsing Von Konig, A. Kraminer-Hagedorn, A. Tacken, and U. Diekmann, Program Abstr. 28th Intersci. Conf. Antimicrob. Agents
The older oral cephalosporins show very poor activity against Bordetella pertussis. The MICs of cephalexin, cephradine, and cefadroxil range between 31 and >128 ,ug/ml (1, 5), and those of cefaclor range between 12 and 25 ,ug/ml (1). Cephalexin at a concentration of 40 ,ug/ml was chosen by Sutcliffe and Abbott (9) as the selective agent in charcoal horse blood agar for clinical isolation of bordetellae, since the compound is inactive against B. pertussis but inhibitory to most organisms of the normal nasopharyngeal flora. Cefuroxime, a parenteral extended-spectrum cephalosporin, has MICs of 1.5 to 6 ,ug/ml against B. pertussis (1). After oral administration of the new prodrug cefuroxime axetil, cefuroxime concentrations of 3.3 ,ug/ml in respiratory secretions have been measured (R. A. Walstad, J. S. Vilsvik, E. Thurmann-Nielsen, J. V. Griggs, and G. W. Brown, Proc. Cefuroxime Axetil Symp., London, United Kingdom, 1988, p. 65-67). Thus, for the first time an orally applicable cephalosporin has become of interest for the treatment of pertussis. We therefore studied the in vitro activities against B. pertussis and Bordetella parapertussis of six other new oral cephalosporins, some of which show remarkably improved activities against many fastidious and nonfastidious gram-negative bacteria (R. N. Jones, Antimicrob. Newsl. 5:1-8, 1988). The test procedure was as described previously (4). Powders of known potency of the following antimicrobial agents were supplied by the manufacturers or their distributors in the Federal Republic of Germany: cefetamet (Ro 15-8074) (Hoffmann-La Roche, Grenzach, Federal Republic of Germany), cefixime (EMD 49 653; FK-027) (E. Merck AG, Darmstadt, Federal Republic of Germany), cefpodoxime (RU 51764; R-3746) (Albert-Roussel, Wiesbaden, Federal Republic of Germany), cefprozil (BMY-28100) (Bristol, NeuIsenburg, Federal Republic of Germany), ceftibuten (7432-S) (Essex, Munich, Federal Republic of Germany), and loracarbef (LY163892) (Lilly, Bad Homburg, Federal Republic of Germany). Stock solutions at 2,000 ,ug/ml were prepared by following the manufacturers' instructions. An agar dilution procedure patterned after the recommendations of the National Committee for Clinical Laboratory Standards (6) was used. Most of the test strains of B. pertussis (n = 33) and B. parapertussis (n = 33) were recent clinical isolates from children in southern Germany. The isolates had been stored at -70°C in glycerol soon after isolation. Three Dutch strains of B. pertussis which had been *
Corresponding author. 1442
VOL. 34, 1990
NOTES
TABLE 1. Activities of six new oral cephalosporins against 33 isolates each of B. pertussis and B. parapertussis
Organism B. pertussis
B. parapertussis
a
agent
Range
MIC (pLg/ml)a
Cefetamet Cefixime Cefpodoxime Cefprozil Ceftibuten Loracarbef
64 4-32 8-16 16-64 64-128 32-64
64 16 8 64 128 64
64 16 16 64 128 64
Cefetamet Cefixime Cefpodoxime Cefprozil Ceftibuten Loracarbef
>128 16-64 64->128 32-64 128->128 32
>128 32 128 64 >128 32
>128 64 128 64 >128 32
Antimicrobial
50%
9
cephalosporins) against many constituents of the normal nasopharyngeal flora might eventually replace cephalexin as the inhibitory agent in charcoal horse blood agar. Cefetamet and ceftibuten in particular deserve further evaluation in this respect.
50%0 and 90%o, MIC for 50 and 90o of isolates tested, respectively.
Chemother., abstr. no. 1219, 1988) tested cefixime and found MIC90 of 8 ,ug/ml. We are not aware of any literature data the activities against B. parapertussis of the compounds tested. The data on the in vitro activities of the new oral cephalosporins have to be interpreted in relation to the concentrations achievable in respiratory secretions. Hayashi (3) measured cefixime concentrations of 0.02 to 0.05 ,ug/ml in sputum. For cefpodoxime, no data are yet available. In conclusion, all of the new oral cephalosporins tested are unlikely to play a role in the treatment of pertussis patients due to the poor in vitro activities of these agents against bordetellae. On the other hand, compounds that are totally inactive against bordetellae but that show high activity (comparable to the activities of parenteral broad-spectrum an on
1443
LITERATURE CITED 1. Bannatyne, R. M., and R. Cheung. 1984. Susceptibility of Bordetella pertussis to cephalosporin derivatives and imipenem. Antimicrob. Agents Chemother. 26:604-605. 2. Deutsches Institut fur Normung. 1979. Methods for the determination of susceptibility of pathogenic bacteria (without mycobacteria) to chemotherapeutic agents; determination of MIC by agar dilution method. DIN 58940, part 6. Deutsches Institut fur Normung, Berlin. 3. Hayashi, I. 1985. Serum and sputum concentration and clinical results of cefixime in respiratory tract infections. Chemotherapy (Tokyo) 33(Suppl. 6):253-267. 4. Hoppe, J. E., and A. Eichhorn. 1989. Activity of new macrolides against Bordetella pertussis and Bordetella parapertussis. Eur. J. Clin. Microbiol. Infect. Dis. 8:653-654. 5. Hoppe, J. E., A. Haug, and K. Botzenhart. 1987. Susceptibility of Bordetella pertussis and Bordetella parapertussis to twenty-four antibiotics. Chemotherapy (Basel) 33:250-254. 6. National Committee for Clinical Laboratory Standards. 1985. Approved standard M7-A. Standard methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. National Committee for Clinical Laboratory Standards, Villanova, Pa. 7. Simon, C. 1987. Zur in-vitro-Aktivitat von Cefixim im Vergleich zu anderen Oralcephalosporinen. Fortschr. Antimikrob. Antineoplast. Chemother. 6:1193-1196. 8. Simon, C. 1987. In vitro activity of Ro 15-8074 and Ro 19-5247 in comparison to cefaclor and cefalexin. Infection 15:122-124. 9. Sutcliffe, E. M., and J. D. Abbott. 1972. Selective medium for the isolation of Bordetella pertussis and parapertussis. J. Clin. Pathol. 25:732-733.
