Vol. 34, No. 7
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUlY 1990, P. 1366-1370 0066-4804/90/071366-05$02.00/0 Copyright C 1990, American Society for Microbiology
In Vitro Activity of E-1040, a 3-Substituted Cephalosporin, against Pathogens from Cystic Fibrosis Sputum HARRIS R. STUTMAN,l12* JOSEPH C. AKANIRO,1 CAROLINA E. VIDAURRE,l
AND MELVIN I. MARKS"12 Department of Pediatrics, Memorial Miller Children's Hospital, Long Beach, California 90801,1 and University of California, Irvine, College of Medicine, Irvine, California 927172
Received 27 November 1989/Accepted 26 April 1990
On the basis of preliminary in vitro data, we evaluated E-1040, a new cephalosporin, against 188 cystic fibrosis (CF) sputum isolates obtained from 26 CF centers in the United States. These isolates included mucoid and nonmucoid Pseudomonas aeruginosa, Pseudomonas cepacia, Staphylococcus aureus, Haemophilus influenzae, and Escherichia coli. In addition to MICs measured under standard conditions, selected isolates were tested at various pH values, inoculum sizes, and diluent (CF serum and sputum) conditions. E-1040 activities (MICs for 50 and 90% of the strains) against the isolates were as folows: P. aeruginosa (mucoid and nonmucoid), 1 and 4 ,ug/ml; P. cepacia, 4 and 16 ,ug/ml; S. aureus, 8 and 8 ,ug/ml; H. influenzae, 1 and 4 ,ug/ml; and E. coli, .0.12 and 128 0.25 -16.0 1.0 ->128.0 0.125 -64.0 0.062 ->64
32.0 2.0 32.0 16.0 32.0
1.0 -32.0
8.0 0.01/0.30 1.0 4.0 8.0 0.032 1.0 0.063 0.28
50.01/0.30 -0.03/0.60 0.125 -2.0 1.0 ->128.0 0.125 -16.0 64.0 0.125 ->64.0
0.125 -8.0 '0.01/0.30 0.063 -8.0 0.032 -0.25 0.063 -2.0 0.063 -4.0 0.032 -1.0
s0.125 '0.01/0.30 --32.0/608.0 0.25 -128.0
Aztreonam
'0.032 -0.125 '0.032 -0.125 0.125 -0.5 0.125 -1.0 0.5 -32.0 0.25 -1.0
1.0 '0.01/0.30 0.5 0.032 0.063 2.0 0.063
50.125 0.01/0.30 2.0 0.062 0.062 0.125 0.5 1.0
0.5
8.0 0.03/0.60 2.0 8.0 8.0 0.5 1.0 2.0 8.0
4.0
'0.01/0.30 8.0 0.063 0.063 4.0 0.25
64.0 0.25 -64.0 1.0 ->256.0 2.0 -64.0 1.0 ->64.0 0.125 -8.0 0.5 ->64.0
Ampicillin Ceftazidime Imipenem Gentamicin Piperacillin Tobramycin a
Rag Range 0.25 -16.0 0.125 ->64.0 1.0 -64.0 0.5 ->256.0 2.0 -32.0 2.0 ->64.0 0.125 -32.0 1.0 ->64.0
than that of ceftazidime, and 32-fold greater than that of piperacillin. Furthermore, E-1040 demonstrated consistent anti-P. aeruginosa activity against ceftazidime-, imipenem-, piperaciliin-, and aminoglycoside-resistant isolates. This observation is consistent with previous reports with non-CF isolates. Our evaluations showed that E-1040 was more active against P. cepacia than various other beta-lactams, aminoglycosides, and chloramphenicols were. With the exception of one strain, E-1040 inhibited all isolates of P. cepacia at a
E-1040 AND CF PATHOGENS
VOL. 34, 1990
Bacterial Inoculum (Log 10) 10
C
F u p
e r m
Limit of detection
~~------
L 0
2
4
6
8
10
12
14
16
18
Hours of Incubation FIG. 1. Results of exposure of a mucoid strain of P. aeruginosa (no. 31 from CF sputum; MIC, 4 ,ug/ml) to the following concentrations of E-1040 (micrograms per milliliter): 0 (control) (---0---), 2 ( 0-), 16 (+), or 32 (*).
concentration of 16 ,ug/ml or less, including those resistant to ticarcillin, aminoglycosides, and chloramphenicol. The in vitro activity of E-1040 against E. coli was excellent, although it was similar to activities of other tested antimicrobial agents. E-1040 was less active against H. influenzae than several of the other drugs tested were. While E-1040 is not likely to be first-line therapy against H. influenzae and S. aureus, which are often copathogens in CF infections, this drug demonstrated considerable activity against these organisms. Therre was little or no inoculum effect, and the activity was not silgnificantly influenced by changes in medium pH. therewas no diminution of Furthe rmore, . activity with CF serum
-rmore, or sputum, suggesting that protein binlng and the
1369
E-1040. However, experiments with a nonmucoid isolate of P. aeruginosa did demonstrate regrowth. Additional experiments to determine whether these patterns resulted from selection of resistant clones or from in vitro mutational events are in progress. Unfortunately, brief exposure of P. aeruginosa to E-1040 (mucoid or nonmucoid) did not result in any significant postantibiotic effect, suggesting that bactericidal activity against P. aeruginosa will need to be maintained throughout a clinical dosing interval. These findings are supported by the observations of other investigations (1-4, 8, 14, 16) with beta-lactam agents against P. aeruginosa. Our results lend support to a potential role of E-1040 in the treatment of chronic pulmonary infections due to common pathogens found in patients with CF. With encouraging pharmacokinetic data (9), the broad range of activity of E-1040 against CF pathogens, specifically its activity against P. aeruginosa and P. cepacia, supports further evaluation of this new cephalosporin. ACKNOWLEDGMENTS This work was supported by grants from the Cystic Fibrosis Foundation, Rockville, Md., and the Memorial Medical Center Foundation, Long Beach, Calif., and by Public Health Service grant NIAID R01-24801 from the National Institute of Allergy and Infectious Diseases. We also thank Joyce Bagan and Ellen Hansen for expert editorial and secretarial assistance. LITERATURE CITED 1. Bergan, T., I. B. Carlsen, and I. E. Fuglesang. 1980. An in vitro
model for monitoring bacterial responses to antibiotic agents under simulated in vivo conditions. Infection 8:S96-S102. 2. Bodey, and T. Pan. 1976. Effect of29:1092-1095. cephalothin on growth pattern G.of P., J. Antibiot. microorganisms. 3. Bundtzen, R. W., A. U. Gerber, D. L. Cohn, and W. A. Craig.
ionic s strength of the medium do not adversely affect the
1981. Postantibiotic suppression of bacterial growth. Rev. In-
activit3y of E-1040. The potent antipseudomonal activity of E-1040 has been partly attributed (12, 15) to its resistance to enzymatic hydrol ysis and the low-binding affinity for ,-lactamase of P.
fect. Dis. 3:28-37.
aerugi nosa and P. cepacia. During experiments involving
kill kirnetics and regrowth patterns, selection of resistant mucoi(d P. aeruginosa did not occur in the presence of
Inoculum (Log 10) Bacterial Bacterial_______Inoculum______(Log__10)_ c F
3 .-
--------------------------
--------
U
p e r
s
4
m
2 * 2 t Limit of of detection detectioni Limit
L D
0
2
4
e
8
10
12
14
18
18
Hours of Incubation FIG. 2. Results of exposure of a nonmucoid strain of P. aerugi(rno. 3 from CF sputum; MIC, 1 ,ug/ml) to the following concenitrations of E-1040 (micrograms per milliliter): 0 (control) 0 ), 16 (+), or 32 (*). ), 2 ( (--nosa
4. Gerber, A. U., and W. A. Craig. 1981. Growth kinetics of respiratory pathogens after short exposure to ampicillin and erythromycin in vitro. J. Antimicrob. Chemother. 8:S81-S91. 5. Govan, J. R. W. 1975. Mucoid strains of P. aeruginosa: the influence of culture medium on the stability of mucus production. J. Med. Microbiol. 8:513-522. 6. Jorgensen, J. H., J. S. Redding, L. A. Maher, and A. W. Howell. 1987. Improved medium for antimicrobial susceptibility testing of Haemophilus influenzae. J. Clin. Microbiol. 25:2105-2113. 7. Krieg, D. P., J. A. Bass, and 1. Mattingly. 1986. Aeration selects for mucoid phenotype of Pseudomonas aeruginosa. J. Clin. Microbiol. 24:986-990. 8. McDonald, P. J., P. Hakendorf, and H. Pruul. 1982. Recovery period of bacteria after brief exposure to N-formimidoyl thienamycin and other antibiotics, p. 741-743. In P. Periti and G. G. Grassi (ed.), Current chemotherapy and immunotherapy: proceedings of the 12th International Congress of Chemotherapy. American Society for Microbiology, Washington, D.C. 9. Nakashima, M., T. Uematsu, M. Kanamaru, K. Ueno, T. Setoyama, Y. Tomono, T. Ohno, K. Okano, an N. Morishita. 1989. 1 study of E1040, a new parenteral cephem antibiotic. J. lPhasePharmacol. 29:144-150. ~~~~~~Clin. 10. National Committee for Clinical Laboratory Standards. 1987. Proposed guideline: methods for determining bactericidal activ-
ity of antimicrobial agents. M26-P. National Committee for
Clinical Laboratory Standards, Villanova, Pa.
11. National Committee for Clinical Laboratory Standards. 1988. Tentative standard: methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. M7-T2. National Committee for Clinical Laboratory Standards, Villanova, Pa. 12. Neu, H. C., N.-X. Chin, and A. Novelli. 1988. In vitro activity of
1370
STUTMAN ET AL.
E-1040, a novel cephalosporin with potent activity against Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 32: 1666-1675. 13. Pearson, R. D., R. T. Steigbigel, H. T. Davis, and S. W. Chapman. 1980. Method for reliable determination of minimal lethal antibiotic concentrations. Antimicrob. Agents Chemother. 18:699-708. 14. Shah, P. M. 1981. Bactericidal activity of amoxicillin. J. Anti-
ANTIMICROB. AGENTS CHEMOTHER.
microb. Chemother. 8:S93-S99. 15. Watanabe, N.-A., K. Katsu, M. Moriyama, and K. Kitoh. 1988. In vitro evaluation of E1040, a new cephalosporin with potent antipseudomonal activity. Antimicrob. Agents Chemother. 32: 693-701. 16. Wilson, D. A., and G. N. Rolinson. 1979. The recovery period following exposure of bacteria to penicillins. Chemotherapy (Basel) 25:14-22.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUlY 1990, P. 1366-1370 0066-4804/90/071366-05$02.00/0 Copyright C 1990, American Society for Microbiology
In Vitro Activity of E-1040, a 3-Substituted Cephalosporin, against Pathogens from Cystic Fibrosis Sputum HARRIS R. STUTMAN,l12* JOSEPH C. AKANIRO,1 CAROLINA E. VIDAURRE,l
AND MELVIN I. MARKS"12 Department of Pediatrics, Memorial Miller Children's Hospital, Long Beach, California 90801,1 and University of California, Irvine, College of Medicine, Irvine, California 927172
Received 27 November 1989/Accepted 26 April 1990
On the basis of preliminary in vitro data, we evaluated E-1040, a new cephalosporin, against 188 cystic fibrosis (CF) sputum isolates obtained from 26 CF centers in the United States. These isolates included mucoid and nonmucoid Pseudomonas aeruginosa, Pseudomonas cepacia, Staphylococcus aureus, Haemophilus influenzae, and Escherichia coli. In addition to MICs measured under standard conditions, selected isolates were tested at various pH values, inoculum sizes, and diluent (CF serum and sputum) conditions. E-1040 activities (MICs for 50 and 90% of the strains) against the isolates were as folows: P. aeruginosa (mucoid and nonmucoid), 1 and 4 ,ug/ml; P. cepacia, 4 and 16 ,ug/ml; S. aureus, 8 and 8 ,ug/ml; H. influenzae, 1 and 4 ,ug/ml; and E. coli, .0.12 and 128 0.25 -16.0 1.0 ->128.0 0.125 -64.0 0.062 ->64
32.0 2.0 32.0 16.0 32.0
1.0 -32.0
8.0 0.01/0.30 1.0 4.0 8.0 0.032 1.0 0.063 0.28
50.01/0.30 -0.03/0.60 0.125 -2.0 1.0 ->128.0 0.125 -16.0 64.0 0.125 ->64.0
0.125 -8.0 '0.01/0.30 0.063 -8.0 0.032 -0.25 0.063 -2.0 0.063 -4.0 0.032 -1.0
s0.125 '0.01/0.30 --32.0/608.0 0.25 -128.0
Aztreonam
'0.032 -0.125 '0.032 -0.125 0.125 -0.5 0.125 -1.0 0.5 -32.0 0.25 -1.0
1.0 '0.01/0.30 0.5 0.032 0.063 2.0 0.063
50.125 0.01/0.30 2.0 0.062 0.062 0.125 0.5 1.0
0.5
8.0 0.03/0.60 2.0 8.0 8.0 0.5 1.0 2.0 8.0
4.0
'0.01/0.30 8.0 0.063 0.063 4.0 0.25
64.0 0.25 -64.0 1.0 ->256.0 2.0 -64.0 1.0 ->64.0 0.125 -8.0 0.5 ->64.0
Ampicillin Ceftazidime Imipenem Gentamicin Piperacillin Tobramycin a
Rag Range 0.25 -16.0 0.125 ->64.0 1.0 -64.0 0.5 ->256.0 2.0 -32.0 2.0 ->64.0 0.125 -32.0 1.0 ->64.0
than that of ceftazidime, and 32-fold greater than that of piperacillin. Furthermore, E-1040 demonstrated consistent anti-P. aeruginosa activity against ceftazidime-, imipenem-, piperaciliin-, and aminoglycoside-resistant isolates. This observation is consistent with previous reports with non-CF isolates. Our evaluations showed that E-1040 was more active against P. cepacia than various other beta-lactams, aminoglycosides, and chloramphenicols were. With the exception of one strain, E-1040 inhibited all isolates of P. cepacia at a
E-1040 AND CF PATHOGENS
VOL. 34, 1990
Bacterial Inoculum (Log 10) 10
C
F u p
e r m
Limit of detection
~~------
L 0
2
4
6
8
10
12
14
16
18
Hours of Incubation FIG. 1. Results of exposure of a mucoid strain of P. aeruginosa (no. 31 from CF sputum; MIC, 4 ,ug/ml) to the following concentrations of E-1040 (micrograms per milliliter): 0 (control) (---0---), 2 ( 0-), 16 (+), or 32 (*).
concentration of 16 ,ug/ml or less, including those resistant to ticarcillin, aminoglycosides, and chloramphenicol. The in vitro activity of E-1040 against E. coli was excellent, although it was similar to activities of other tested antimicrobial agents. E-1040 was less active against H. influenzae than several of the other drugs tested were. While E-1040 is not likely to be first-line therapy against H. influenzae and S. aureus, which are often copathogens in CF infections, this drug demonstrated considerable activity against these organisms. Therre was little or no inoculum effect, and the activity was not silgnificantly influenced by changes in medium pH. therewas no diminution of Furthe rmore, . activity with CF serum
-rmore, or sputum, suggesting that protein binlng and the
1369
E-1040. However, experiments with a nonmucoid isolate of P. aeruginosa did demonstrate regrowth. Additional experiments to determine whether these patterns resulted from selection of resistant clones or from in vitro mutational events are in progress. Unfortunately, brief exposure of P. aeruginosa to E-1040 (mucoid or nonmucoid) did not result in any significant postantibiotic effect, suggesting that bactericidal activity against P. aeruginosa will need to be maintained throughout a clinical dosing interval. These findings are supported by the observations of other investigations (1-4, 8, 14, 16) with beta-lactam agents against P. aeruginosa. Our results lend support to a potential role of E-1040 in the treatment of chronic pulmonary infections due to common pathogens found in patients with CF. With encouraging pharmacokinetic data (9), the broad range of activity of E-1040 against CF pathogens, specifically its activity against P. aeruginosa and P. cepacia, supports further evaluation of this new cephalosporin. ACKNOWLEDGMENTS This work was supported by grants from the Cystic Fibrosis Foundation, Rockville, Md., and the Memorial Medical Center Foundation, Long Beach, Calif., and by Public Health Service grant NIAID R01-24801 from the National Institute of Allergy and Infectious Diseases. We also thank Joyce Bagan and Ellen Hansen for expert editorial and secretarial assistance. LITERATURE CITED 1. Bergan, T., I. B. Carlsen, and I. E. Fuglesang. 1980. An in vitro
model for monitoring bacterial responses to antibiotic agents under simulated in vivo conditions. Infection 8:S96-S102. 2. Bodey, and T. Pan. 1976. Effect of29:1092-1095. cephalothin on growth pattern G.of P., J. Antibiot. microorganisms. 3. Bundtzen, R. W., A. U. Gerber, D. L. Cohn, and W. A. Craig.
ionic s strength of the medium do not adversely affect the
1981. Postantibiotic suppression of bacterial growth. Rev. In-
activit3y of E-1040. The potent antipseudomonal activity of E-1040 has been partly attributed (12, 15) to its resistance to enzymatic hydrol ysis and the low-binding affinity for ,-lactamase of P.
fect. Dis. 3:28-37.
aerugi nosa and P. cepacia. During experiments involving
kill kirnetics and regrowth patterns, selection of resistant mucoi(d P. aeruginosa did not occur in the presence of
Inoculum (Log 10) Bacterial Bacterial_______Inoculum______(Log__10)_ c F
3 .-
--------------------------
--------
U
p e r
s
4
m
2 * 2 t Limit of of detection detectioni Limit
L D
0
2
4
e
8
10
12
14
18
18
Hours of Incubation FIG. 2. Results of exposure of a nonmucoid strain of P. aerugi(rno. 3 from CF sputum; MIC, 1 ,ug/ml) to the following concenitrations of E-1040 (micrograms per milliliter): 0 (control) 0 ), 16 (+), or 32 (*). ), 2 ( (--nosa
4. Gerber, A. U., and W. A. Craig. 1981. Growth kinetics of respiratory pathogens after short exposure to ampicillin and erythromycin in vitro. J. Antimicrob. Chemother. 8:S81-S91. 5. Govan, J. R. W. 1975. Mucoid strains of P. aeruginosa: the influence of culture medium on the stability of mucus production. J. Med. Microbiol. 8:513-522. 6. Jorgensen, J. H., J. S. Redding, L. A. Maher, and A. W. Howell. 1987. Improved medium for antimicrobial susceptibility testing of Haemophilus influenzae. J. Clin. Microbiol. 25:2105-2113. 7. Krieg, D. P., J. A. Bass, and 1. Mattingly. 1986. Aeration selects for mucoid phenotype of Pseudomonas aeruginosa. J. Clin. Microbiol. 24:986-990. 8. McDonald, P. J., P. Hakendorf, and H. Pruul. 1982. Recovery period of bacteria after brief exposure to N-formimidoyl thienamycin and other antibiotics, p. 741-743. In P. Periti and G. G. Grassi (ed.), Current chemotherapy and immunotherapy: proceedings of the 12th International Congress of Chemotherapy. American Society for Microbiology, Washington, D.C. 9. Nakashima, M., T. Uematsu, M. Kanamaru, K. Ueno, T. Setoyama, Y. Tomono, T. Ohno, K. Okano, an N. Morishita. 1989. 1 study of E1040, a new parenteral cephem antibiotic. J. lPhasePharmacol. 29:144-150. ~~~~~~Clin. 10. National Committee for Clinical Laboratory Standards. 1987. Proposed guideline: methods for determining bactericidal activ-
ity of antimicrobial agents. M26-P. National Committee for
Clinical Laboratory Standards, Villanova, Pa.
11. National Committee for Clinical Laboratory Standards. 1988. Tentative standard: methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. M7-T2. National Committee for Clinical Laboratory Standards, Villanova, Pa. 12. Neu, H. C., N.-X. Chin, and A. Novelli. 1988. In vitro activity of
1370
STUTMAN ET AL.
E-1040, a novel cephalosporin with potent activity against Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 32: 1666-1675. 13. Pearson, R. D., R. T. Steigbigel, H. T. Davis, and S. W. Chapman. 1980. Method for reliable determination of minimal lethal antibiotic concentrations. Antimicrob. Agents Chemother. 18:699-708. 14. Shah, P. M. 1981. Bactericidal activity of amoxicillin. J. Anti-
ANTIMICROB. AGENTS CHEMOTHER.
microb. Chemother. 8:S93-S99. 15. Watanabe, N.-A., K. Katsu, M. Moriyama, and K. Kitoh. 1988. In vitro evaluation of E1040, a new cephalosporin with potent antipseudomonal activity. Antimicrob. Agents Chemother. 32: 693-701. 16. Wilson, D. A., and G. N. Rolinson. 1979. The recovery period following exposure of bacteria to penicillins. Chemotherapy (Basel) 25:14-22.
