Activity against Pseudomonas aeruginosa - Antimicrobial Agents and ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1988, P. 1666-1675

Vol. 32, No. 11

0066-4804/88/111666-10$02.00/0 Copyright © 1988, American Society for Microbiology

In Vitro Activity of E-1040, a Novel Cephalosporin with Potent Activity against Pseudomonas aeruginosa HAROLD C. NEU,1.2* NAI-XUN CHIN,1 AND ANDREA NOVELLI1t Departments of Medicine1 and Pharmacology,2 College of Physicians & Surgeons, Columbia University, New York, New York 10032 Received 24 May 1988/Accepted 10 August 1988

The in vitro activity of E-1040 [(6R,7R)-3-[(4-carbamoyl-1-quinuclidinio)methyl]-7-[2-(5-amino-1,2,4-thiaa novel cephalosporin, was compared with that of ceftazidime, cefpirome, cefepime, imipenem, and gentamicin. E-1040 inhibited 50% of members of the family Enterobacteriaceae, Pseudomonas aeruginosa, and Haemophilus and Neisseria species at 100 ,uM.

diazol-3-yl)-(Z)-2-methoxyiminoacetoamidoj-8-oxo-5-thia-l-azabicyclo(4,2,0)oct-2-ene-2-carboxylate],

cals, Somerville, N.J.; cefepime from Bristol Laboratories, Wallingford, Conn.; imipenem from Merck Sharp & Dohme, Rahway, N.J.; and the other agents from their respective manufacturers. Organisms. Bacterial strains were obtained from patients hospitalized in the Columbia University hospital system in New York, N.Y., and included some isolates of Pseudomonas and other genera that had been retained because of known 3-lactamase activity and resistance to beta-lactam antibiotics or to aminoglycosides. Antimicrobial susceptibility tests. Antimicrobial activity was measured by an agar dilution method with MuellerHinton agar, unless otherwise specified. A final inoculum of 104 CFU was applied with a replicating device. Broth dilution tests were performed with 5 x 10' CFU in 1-ml tubes. Overnight cultures of organisms to be tested were diluted in Mueller-Hinton broth (BBL Microbiology Systems, Cockeysville, Md.); Haemophilus, Branhamella, and Neisseria spp. in Schaedler broth; and anaerobic species in chopped meat glucose (Scott Laboratories, Inc., Providence, R.I.). The susceptibilities of Neisseria, Branhamella, and Haemophilus spp. were determined with chocolate Mueller-Hinton agar in the presence of 5% CO2. The susceptibilities of streptococci were determined with MuellerHinton agar supplemented with 5% sheep blood, and the susceptibilities of anaerobic species were determined with brucella agar supplemented with sheep blood, hemin, and vitamin K. Anaerobic cultures were incubated for 48 h in GasPak jars (BBL). Susceptibility of methicillin-resistant staphylococci was determined on Mueller-Hinton agar supplemented with 5% NaCl; isolates for which oxacillin MICs were >8 jig/ml were considered resistant. The MIC was defined as the lowest concentration of antibiotic that inhibited development of visible growth on agar or in broth. The MBC was determined by subculture of 0.01-ml samples from clear tubes on antibiotic-free agar plates which indicated a 99.9% reduction in CFU, with calculations based on those of

A large number of new cephalosporin antibiotics have been developed in the past decade, and there has been continued interest in finding new agents in the cephalosporin class that would have activity against Pseudomonas aeruginosa and certain members of the family Enterobacteriaceae, such as Enterobacter species and Citrobacter freundii, which are resistant to many of the broad-spectrum cephalosporins (1, 2, 4, 5). E-1040 [(6R,7R)-3-[(4-carbamoyl-1-quinu-

clidinio)methyl]-7-[2-(5-amino- 1,2,4-thiadiazol-3-yl)-(Z)-2methoxyiminoacetoamido]-8-oxo-5-thia-1-azabicyclo(4,2,0) oct-2-ene-2-carboxylate] is a new parenterally administered cephalosporin which has been preliminarily reported to have a broad spectrum of antibacterial activity and to inhibit many P. aeruginosa strains that are resistant to other beta-lactams (N. Watanabe, K. Katsu, M. Moriyama, I. Sugiyama, K. Kitoh, and H. Yamauchi, Program Abstr. 27th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 636 1987; T. Yokota, E. Suzuki, and K. Arai, 27th ICAAC, abstr. no. 639, 1987). E-1040 differs from other oxine cephalosporins in that it has the (4-carbamoyl-1-quinuclidino)methyl side chain at position 3 of the dihydrothiazine ring (Fig. 1). We compared the in vitro activity of E-1040 with those of other cephalosporins, imipenem, and an aminoglycoside and determined the effects of various growth conditions upon the activity of E-1040.

MATERIALS AND METHODS Antibiotics. E-1040 was a gift of Eisai Co., Ltd., Tokyo, Japan. The other drugs were obtained as follows: ceftazidime from Glaxo Pharmaceuticals, Ltd., Research Triangle Park, N.C.; cefpirome from Hoechst-Roussel Pharmaceuti* Corresponding author. t Present address: Department of Pharmacology, University of Florence, Florence, Italy.

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IN VITRO ACTIVITY OF E-1040

VOL. 32, 1988

N

OCH3

coo-

FIG. 1. Structure of E-1040 [(6R,7R)-3-[(4-carbamoyl-1-quinuclidinio)methyl] -7 [2-(5-amino-1,2,4-thiadiazol-3-yl)-(Z)-2-methox-

1667

at concentrations equal to the MIC and four times the MIC. Incubation continued for 2 h. Bacteria were harvested by centrifugation, washed in 0.05 M potassium phosphate buffer (pH 7), and subsequently disrupted by sonication. Cellular debris was removed by centrifugation, and the samples were dialyzed at 4°C for 24 h against phosphate buffer. P-Lactamase activity was determined by using nitrocefin as substrate, with activity based on micromolar substrate hydrolyzed per milligram of protein.

-

yiminoacetoamido]-8-oxo-5-thia-1-azabicyclo(4,2,O)oct-2-ene-2-carboxylate].

Pearson et al. (10) and a 5% pipetting error. All tests were run with American Type Culture Collection control strains of Staphylococcus aureus, Escherichia coli, and P. aeruginosa.

Time-kill studies. Exponentially growing E. coli and P. aeruginosa strains, each at a final inoculum ofapproximately 106 CFU, were exposed to E-1040, ceftazidime, and aztreonam at a concentration onefold above the MBC, as deter-

mined in broth. Organisms were grown in 125-ml flasks, each containing 10 ml. Samples (1 ml) were removed at 2-h intervals for 8 h and at 24 h, and medium was removed by Millipore membrane filtration. Bacteria were suspended in fresh medium to the same volume, and dilutions in MuellerHinton broth were plated on antibiotic-free medium. Organisms which had not been exposed to any drug were processed in a similar manner. Selection of mutants derepressed for production of Ilactamase. Organisms in an inoculum of 109 CFU were placed on agar containing the antibiotics at concentrations ranging from the MIC to 30-fold above the MIC. Organisms growing on the antibiotic-containing medium were isolated, and susceptibility was determined by testing on agar containing the antibiotic in twofold-increasing dilutions. Spontaneous resistance. The development of spontaneous resistance to a concentration of E-1040 eight times the MIC was determined by plating .1010 organisms, obtained by centrifugation, on Mueller-Hinton agar containing the drug, and organisms were picked at 48 h. Two isolates of each species were tested. ,-Lactamase assays. The presence of P-lactamases in clinical isolates was determined by the nitrocefin assay. P-Lactamases used for determination of the stability of the compounds were either purified enzymes or partially purified enzymes, as previously described (3). The stabilities of the compounds to 13-lactamase were determined by spectrophotometric assay by using the change in absorption at the absorption maximum. The A264 for E-1040 was Ae8.64

mM-1 cm-1. Cephaloridine

was

used

as a

reference

com-

pound at an A260 of As10.2 mM-1 cm-l. Inhibition assays with nitrocefin as the substrate at a 10-4 M concentration were

performed in

a

final volume of 3 ml. Enzyme and

inhibitor were incubated at various concentrations at 35°C for 10 min, and subsequently, nitrocefin was added. Change in the A482 of nitrocefin was monitored for 10 min in a temperature-controlled recording spectrophotometer. As a control, the change in nitrocefin plus enzyme was monitored. Induction of P-lactamases. One strain of P. aeruginosa and strain of Enterobacter cloacae were grown overnight in broth, diluted 20-fold, and incubated for 2 h to achieve exponential growth. The strains had previously been shown to be P-lactamase inducible with both cefoxitin and imipenem (6-9). During incubation at 37°C, inducers were added one

RESULTS The susceptibilities of various bacteria to E-1040 and other agents are shown in Table 1. E-1040 inhibited 50% of members -of the Enterobacteriaceae and P. aeruginosa at s0.25,g/ml. The MIC for 90% of strains (MIC90) of members of the Enterobacteriaceae ranged from 0.06 to 4,ug/ml; the majority of species were inhibited bys0.5,ug/ml with the exception of Enterobacter cloacae (2 ,ug/ml) and Serratia marcescens (4 ,ug/ml). In comparison 'with ceftazidime, E-1040 was equally or two to four times more active against E. coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter diversus, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, Yersinia enterocolitica, and Salmonella, Shigella, and Aeromonas species. E-1040 ats2 jig/ml inhibited Enterobacter species and C. freundii isolates for which ceftazidime MICs were 16 to 64 ,ug/ml. E-1040 was also considerably more active than ceftazidime against Morganella morganii, whereas ceftazidime was more active against some S. marcescens strains. Both cefpirome and cefepime had activity similar to that of E-1040 or were one or two dilutions more active at lower concentrations against members of the Enterobacteriaceae such as some of the Klebsiella spp. and E. coli. Against Enterobacter cloacae and C. freundii, E-1040 had activity equal or superior to that of cefpirome and cefepime. E-1040 was the most active compound tested against P. aeruginosa, with an MIC90 of 0.5 jig/ml. It inhibited isolates resistant to ceftazidime, gentamicin, and amikacin. E-1040 also inhibited at 16 ,ug/ml P. aeruginosa isolates from cystic fibrosis patients which were ceftazidime, imipenem, and tobramycin resistant. Against other Pseudomonas species, such as Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas stutzeri, and Pseudomonas putida, E1040 had MICs that ranged from 0.12 to 64 ,ug/ml, with 90% of the tested strains inhibited by 8 pxg/ml, results similar to those with ceftazidime, cefpirome, and cefepime. However, for most Pseudomonas cepacia strains and for 50% of Xanthomonas maltophilia strains, E-1040 MICs were >16 ,ug/ml. E-1040 was more active than ceftazidime against Acinetobacter spp. and was similar in activity to cefpirome and cefepime but was much less active than irnipenem. Although E-1040 inhibited Haemophilus influenzae, Branhamella catarrhalis, and Neisseria gonorrhoeae, including ,B-lactamase-producing strains, at c0.5 p.g/ml, it was less active than the other three cephalosporins. The activity of E-1040 against Staphylococcus aureus was similar to that of ceftazidime, with an MIC for 50% of strains (MIC50) of 8 jig/ml. Cefpirome and cefepime were considerably more active, with MIC90s of 1 and 4 jig/ml, respectively, compared with 32 pxg/ml for both E-1040 and ceftazidime. E-1040 did not inhibit methicillin-resistant Staphylococcus aureus. Against coagulase-negative staphylococci, the majority of which were Staphylococcus epidermidis, E-1040 had an MIC90 of 8 ,ug/ml, but it did not inhibit methicillin-resistant, coagulase-negative organisms. E-1040

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NEU ET AL.

TABLE 1. Comparative in vitro activities of E-1040 and other agents against gram-negative organisms MIC (pLg/ml) Organism (no. of isolates)

Escherichia colia (30)

Klebsiella pneumoniaea (30)

K. oxytocaa (20)

Antibiotic

E-1040 Ceftazidime Cefpirome Cefepime Imipenem

Hafnia alveia (10)

Citrobacterfreundiia (30)

Proteus mirabilis (30)

0.25 0.25 0.12 0.12 0.5 1

0.03-1 0.03-16 0.03-0.5 0.03-0.5 0.12-2 0.25->16

0.12 0.25 0.06 0.03 0.25 16

E-1040

0.064 0.12-4 0.03-4 0.03-4 0.25-4 0.5->32

0.12 0.12 0.12 0.25 2

0.25 0.25 0.25 0.25 0.5 32

0.06-32

0.12 0.25 0.06 0.5 2 2

0.12 16 0.5 2 4 8

Ceftazidime Cefpirome Cefepime

E-1040 Ceftazidime Cefpirome Cefepime

E-1040 Ceftazidime Cefpirome Cefepime Imipenem Gentamicin

0.06->64 0.015-1 0.05-2 0.25-4 0.5->16

0.06-0.25 0.12-1

Q.015-4 0.015-0.5 0.12-2 0.06->16

0.06-4 0.12-64 0.015-8 0.015-4 0.12-2 0.25->16

0.06 0.5 0.03 0.03 0.5 1

0.5 64 2 0.5 2 2

0.03-0.06

0.06 0.12 0.03 0.015 0.25 0.5

0.06 0.12 0.06 0.03 0.25 0.5

0.25 0.06 0.06 0.06 0.5 2

0.25 0.25 0.12 0.06 1 4

0.03-0.12 0.5-32 0.03-0.25 0.03-0.25

0.25-0.5 0.25-1

0.06-0.25

0.015-0.5

Cefepime Imipenem Gentamicin

0.015-0.06

E-1040 Ceftazidime

0.06-0.25

Cefpirome Cefepime Imipenem Gentamicin

0.12 0.25 0.06 0.03 0.5 0.12

0.5

E-1040

E-1040 Ceftazidime Cefpirome

0.5 >16

0.12 16 0.25 0.25 0.5 1

0.06-32 0.03-16 0.06-8 0.5-128

E-1040

0,12

0.25 1 0.25 0.06

0.06 4 0.06 0.06 0.5

0.06-64

Ceftazidime Cefpirome Cefepime

0.5

2 64 4 2 4 64

0.06-4

Ceftazidime Cefpirome Cefepime Imipenem Gentamicin

Ceftazidime Cefpirome Cefepime Imipenem Gentamicin

0.12

0.25 0.5 0.12 0.25 1 2

E-1040

Imipenem Gentamicin

C. diversus (20)

0.12 0.12 0.06 0.06


Imipenem Gentamicin

E. cloacaea (25)

0.06-0.5

9o0

Gentamicin

Gentamicin

E. agglomeransa (6)

509o

0.03-0.5 0.015-0.25 0.03-0.12 0.06-1 0.25-2

Imipenem Enterobacter aerogenesa (25)

Range

0.12-0.5 0.25-2

0.06-0.5 0.03-0.25 0.03-0.12 0.5-2 1-8

Continued on following page

IN VITRO ACTIVITY OF E-1040

VOL. 32, 1988

1669

TABLE 1-Continued Organism (no. of isolates)

Antibiotic

MIC (,g/ml)

Range

0.5-4 0.25-16

0.06 0.06 0.03 0.015 0.5 1

0.12 2 0.12 0.12 2 4

0.12-2 0.03-8 0.06-16 0.03-8 0.5-8 0.12->16

0.25 0.12 0.12 0.03 0.5 0.5

0.5 1 0.5 0.25 2 8


0.06-0.25 0.12-4 0.015-0.12

Proteus vulgarisa (30)


P. rettgeria (20)


Providencia stuartija (30)

E-1040 Ceftazidime

Morganella morganiia (30)

90%o

50%

0.015-0.25

0.25 1 0.06 0.03 1 4

0.5 2 1 0.5 2 >16

Gentamicin

0.015-0.25 1-8 4->16

0.12 0.12 0.12 0.12 4 16

0.12 0.5 0.25 0.25 4 >16

Serratia marcescensa (30)


0.25-8 0.12-8 0.06-4 0.06-4 0.5-4 0.5-16

0.25 0.5 0.25 0.25 1 2

4 1 2 1 2 8

Pseudomonas aeruginosaa (60)

E-1040 Ceftazidime Cefpirome Cefepime Amikacin Gentamicin Imipenem Ticarcillin Piperacillin

0.12-8 0.5-32 2-32 0.5-16 1->64 0.5->32 0.5-8 16->128 8->128

0.25 2 2 4 8 4 2 >128 64

0.5 8 16 16 32 >32 4 >128 >128

P. aeruginosa, imipenem, ceftazidime, and tobramycin resistant (11)

E-1040

0.5-16

4

16

P. cepacia (15)


1->128 0.5->128 2->128 4-128 1->32 2->32

32 2 8 16 32 >32

128 128 >128 64 >32 >32

Xanthomonas maltophilia (20)

E-1040 Ceftazidime

2-64

16 >128 64 16 16 >32

32 >128 >128 32 32 >32

0.12-64 0.25-32 0.5-32 0.12-16

1 4 4 4

4-32

32

8 8 8 8 >32

Cefpirome Cefepime Imipenem

Cefpirome Cefepime Imipenem Gentamicin Pseudomonas

spp.b (30)

E-1040 Ceftazidime Cefpirome Cefepime Imipenem

-0.03-2 0.03-2 0.015-2 0.015-2 0.12-4 0.12-16

0.06-0.12 0.12-4

0.015-0.5

8->128 64->128 2->128 2->128 >32

Continued on following page

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ANTIMICROB. AGENTS CHEMOTHER.

NEU ET AL. TABLE 1-Continued Organism (no. of isolates)

Antibiotic

MIC Range

MIC

(pg/m1)

50/l990%o 50%o

Acinetobacter spp.c (32)


0.5-32 0.25->64 1-32 1-16 0.06-1 0.25->64

2 8 2 4 0.25 1

8 64 8 8 0.25 2

Salmonella spp.a (29)


003-0.25 0.06-0.25 0.03-0.25 0.03-0.25 0.12-0.25 0.25-4

0.12 0.25 0.06 0.06 0.25 0.5

0.25

Shigella spp.' (30)


0.03-0.25 0.06-0.5 0.015-0.25 0.015-0.25 0.06-0.25 0.25-2

0.06 0.06 0.015 0.015 0.06 1

0.06 0.25 0.06 0.06 0.25 2

Aeromonas spp.' (15)


0.03-0.5 0.06-0.25 0.015-0.06 0.015-0.12 0.25-8 1-4

0.06 0.12 0.06 0.03 2 2

0.12 0.25 0,06 0.12 8 4

Yersinia enterocoliticaa (15)


0.615-0.12

0.06 0.12 0.03 0.015 0.25 1

0.12 0.5 0.03 0.03 0.5 2

E-1040 Ceftazidime

.'0003-0.5

Cefpirome Cefepime

50.03-0.5

0.25 0.06 0.06 0.06

0.5 0.12 0.06 0.06

0.5-1 0.03-0.25 50.03-0.25 "0.03-0.25

0.5 0.06 0.06 0.06

0.5 0.25 0.12 0.12

0.03-0.5