Vol. 36, No. 3

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1992, p. 589-597

0066-4804/92/030589-09$02.00/0 Copyright © 1992, American Society for Microbiology

In Vitro Evaluation of E1077, a New Cephalosporin with a Broad Antibacterial Spectrum NAO-AKI WATANABE,* RYOICHI HIRUMA, AND KANEMASA KATSU

Department of Microbiology and Infectious Diseases, Tsukuba Research Laboratories, Eisai Co. Ltd., Tsukuba, Ibaraki 300-26, Japan Received 5 September 1991/Accepted 2 January 1992

E1077 is a novel parenteral cephalosporin with a wide spectrum of potent antibacterial activity against aerobic and anaerobic gram-positive and gram-negative bacteria. Against methicillin-susceptible Staphylococcus aureus, E1077 was twice as active as cefpirome, with an MIC for 90% of strains tested (MIC90) of 0.78 ,Lg/ml. Methicillin-resistant S. aureus was moderately to highly resistant to E1077, but E1077 was at least twice as active as other 13-lactams tested. Against Enterococcus faecalis, E1077 was the most active of the cephalosporins tested (MIC90, 12.5 pg/ml) and was at least fourfold more active than cefpirome and ceftazidime. At concentrations of .0.78 ,ug/ml, E1077 inhibited 90%o of streptococci and most of the members of the family Enterobacteriaceae tested, with the exceptions of Serratia marcescens and Proteus vulgaris, for which the MIC%0s of E1077 were both 3.13 ,ug/ml. Against Pseudomonas aeruginosa, E1077 was two- to fourfold more active than cefpirome and ceftazidime. For the anaerobes, E1077 was as active against Bacteroides fragilis as was cefuzonam, and its activity was fourfold higher than those of cefpirome and ceftazidime. E1077 was at least as resistant as cefpirome to hydrolysis by various 13-lactamases, and these enzymes had a low affinity for E1077. Pharmaceutical Co., Tokyo, Japan), cefoperazone and piperacillin (Toyama Chemical Co., Tokyo, Japan), cephalothin and cephaloridine (Shionogi Pharmaceutical Co., Osaka, Japan), penicillin G (Meiji Seika Co., Tokyo, Japan), and imipenem-cilastatin (Banyu Pharmaceutical Co., Tokyo, Japan). Imipenem was separated from cilastatin and purified by high-pressure liquid chromatography. Organisms. The bacterial strains used in this study were originally isolated from clinical specimens from humans. The strains used for susceptibility testing were random clinical isolates obtained from various hospitals in Japan since 1985 and were unselected, but many were resistant to ,B-lactam antibiotics. Other strains, including methicillin-resistant S. aureus, were chosen for testing because of certain characteristics, such as antibiotic resistance, 13-lactamase production, and ,-lactamase induction. Included in this group were nine laboratory strains elaborating characterized 13-lactamases and five derepressed mutants overproducing group i ,B-lactamase, kindly provided by M. Mitsuhashi (Episome Institute, Gunma, Japan) and T. Sawai (Chiba University, Chiba, Japan), respectively. All isolates were maintained as stock cultures in our laboratory. Determination of MICs. MICs were determined as described previously (25). Mueller-Hinton agar (BBL Microbiology Systems, Cockeysville, Md.) was used for all organisms but the following: streptococci (Mueller-Hinton agar supplemented with 5% defibrinated sheep blood), Haemophilus influenzae (Mueller-Hinton agar supplemented with 5% Fildes enrichment), Moraxella catarrhalis (chocolate agar or Mueller-Hinton agar supplemented with 5% defibrinated sheep blood), Neisseria gonorrhoeae (gonococcal medium supplemented with 1% hemoglobin and 1% IsoVitaleX; BBL), and obligate anaerobes (GAM medium; Nissui Pharmaceutical Co., Tokyo, Japan). Trypticase soy agar (BBL), heart infusion agar (Difco Laboratories, Detroit, Mich.), nutrient agar (Difco), and sensitivity test agar (Nissui) were used to test for the effects of media. The inocula were prepared by diluting fresh overnight broth cultures,

Infections caused by methicillin-resistant strains of Staphylococcus aureus or by Pseudomonas aeruginosa remain clinical problems, despite the availability of extendedspectrum cephalosporins and the new quinolones. Over the past few years, new cephalosporins with broad spectra of antibacterial activities and with improved activities against both S. aureus and P. aeruginosa, such as cefpirome (22), have been developed. However, the activities of these compounds are still insufficient, especially against S. aureus and P. aeruginosa, and further improvement in activities against both species is desirable. With this in mind, our research has been directed towards the development of new cephalosporins. E1077 {(-)-(6R,7R)-7-[2-(5-amino-1,2,4-thia-

diazol-3-yl)-2-(Z)-fluoromethoxyiminoacetoamido]-3-[(E)-3(RS) carbamoylmethylethylmethylammonio 1 propenyl] 8oxo-5-thia-1-azabicyclo(4,2,O)oct-2-ene-2-carboxylate} (Fig. 1) is a novel parenteral cephalosporin which has a broad antibacterial spectrum and potent activities against S. aureus and P. -

-

-

-

aeruginosa. In this paper, we compare

the in vitro activity of E1077 with the activities of cefpirome (22), ceftazidime (26), cefuzonam (6), cefoperazone (12), and cefotaxime (16). In addition, the susceptibility of the compound to hydrolysis by ,-lactamases and the affinity of 3-lactamases for the compound are reported. (This work was presented in part at the 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, Ga., 21 to 24 October 1990.) MATERIALS AND METHODS Antibiotics. E1077 and cefpirome were synthesized at Tsukuba Research Laboratories, Eisai Co. Ltd. The other antibacterial agents used were obtained commercially: ceftazidime (Glaxo Japan Co., Tokyo, Japan), cefuzonam (Lederle Japan Co., Tokyo, Japan), cefotaxime (Chugai *

Corresponding author. 589

590

WATANABE ET AL.

N.OCH2F FIG. 1. Chemical structure of E1077.

except for fastidious organisms. Test strains were grown for

18 h in fresh Mueller-Hinton broth (BBL). These overnight cultures were diluted into the same fresh medium, and 5 ,ul of each bacterial suspension, corresponding to about 104 CFU, was spotted with a multiple-inoculum replicator (Microplanter; Sakuma Seisakusho, Tokyo, Japan) onto agar plates that contained twofold serial dilutions of antibiotics. Bacterial suspensions for streptococci, H. influenzae, M. catarrhalis, N. gonorrhoeae, and obligate anaerobes were prepared from the corresponding fresh agar plates by dilution in liquid medium and applied to agar plates. The concentrations of the bacterial suspensions were determined by measuring turbidity and were verified by determining standard colony counts on antibiotic-free agar medium plates. For studies involving inoculum size, final inocula of 106, 105, 104, and 103 CFU were prepared and inoculated as described above. Agar plates were incubated for 18 to 24 h at 30°C for Pseudomonas species other than P. aeruginosa, Xanthomonas maltophilia, Flavobacterium species, and Alcaligenes species and at 37°C for other bacteria. For anaerobes, incubation was carried out anaerobically in GasPak jars (BBL), and for streptococci, H. influenzae, M. catarrhalis, and N. gonorrhoeae, incubation was carried out in an atmosphere of 5% CO2. The MIC was considered to be the lowest concentration that completely inhibited visible growth on agar plates. Selected strains were also tested by a microdilution method with cation-supplemented Mueller-Hinton broth. Final inocula of 5 x 105 CFU/ml were prepared by diluting overnight cultures into fresh broth medium. The MIC was determined by visual inspection for lack of turbidity after 24 h of incubation at 37°C. Samples (10 ,ul) from clear wells were transferred to antibiotic-free plates. The MBC, as defined by a 99.9% reduction in CFU relative to the inoculum, was determined by the method of Pearson et al. (18). For the determination of pH effects, the pH of Mueller-Hinton agar was adjusted with hydrochloric acid or sodium hydroxide to the desired level. The susceptibility breakpoints used for methicillin and ceftazidime were .6.25 ,ug/ml. Susceptibility to 13-lactamases. Various j-lactamases were prepared as described previously (8). ,B-Lactamase hydrolysis studies were performed spectrophotometrically on a Hitachi 220A spectrophotometer (20). The reaction mixture consisted of 3 ml of a substrate in 50 mM phosphate buffer (pH 7.0) and 50 ,ul of enzyme solution. The substrate concentration used was 100 ,uM. The enzyme reaction was carried out at 30°C. UV spectra were recorded for each compound in the presence and absence of a selected 1-lactamase. On the basis of the resulting difference spectrum, an appropriate wavelength was selected for the assay, and a molar extinction coefficient (Ac) was calculated from the differential absorbance change at that wavelength. The spectral parameters used in this study were as follows: E1077, 304 nm, Ac = 15.9 mM-1 cm-'; cefpirome, 283 nm, Ac = 7.13 mM-1 cm-'; ceftazidime, 260 nm, Ac = 9.59 mM-

ANTIMICROB. AGENTS CHEMOTHER.

cm-1; cefotaxime, 264 nm, Ac = 7.25 mM-1 cm-1; cephalothin, 262 nm, Ae = 7.66 mM-1 cm-'; cephaloridine, 260 nm, Ac = 10.2 mM'1 cm-1; and penicillin G, 233 nm, Ae = 1.14 mM1 cm-. The Ki values were calculated from Dixon plots (4). 1-Lactamase induction. An overnight culture was diluted 20-fold into 10 ml of fresh Mueller-Hinton broth and incubated with shaking at 37°C. After 2.5 h of incubation (mid-log phase), inducers were added. Incubation was continued for 2 h, and the cells were harvested and washed twice with 50 mM phosphate buffer (pH 7.0). The cells were suspended in the same buffer and disrupted with a model 350 Sonifier (Branson Sonic Power Co., Danbury, Conn.) in ice-water. Cell debris and unbroken cells were removed by centrifugation, and the resulting supernatant fluid was used as a crude enzyme (13). ,-Lactamase activity was determined with 100 ,uM cephalothin as the substrate (21). The protein concentration was estimated by the method of Lowry et al. with bovine serum albumin as the standard (10). RESULTS Antibacterial activity. The in vitro antibacterial activities of E1077 against various clinical isolates were compared with the activities of cefpirome, ceftazidime, cefuzonam, cefoperazone, and cefotaxime (Table 1). E1077 showed a broad antibacterial spectrum against a wide range of bacteria covering gram-positive cocci, including staphylococci, Enterococcus faecalis, members of the family Enterobacteriaceae, non-glucose-fermenting rods, including P. aeruginosa, and anaerobes. Against methicillin-susceptible strains of S. aureus and Staphylococcus epidermidis, E1077 was active. The MIC which was required to inhibit 90% of the strains tested (MIC90) was 0.78 to 1.56 ,ug/ml, and E1077 was 4- to 16-fold more active than ceftazidime and cefotaxime and roughly comparable in activity to cefpirome and cefuzonam. However, methicillin-resistant strains of S. aureus and S. epidermidis were moderately to highly resistant to E1077; the MIC50s were 12.5 and 3.13 ,ug/ml, respectively, and the MIC90s were 100 and 12.5 ,ug/ml, respectively. It was at least twice as potent as the comparison compounds tested. Against streptococci, E1077 exhibited high inhibitory activity (MIC%, 100 ,ug/ml). However, E1077 hardly showed activity against other species of enterococci, such as Enterococcus avium and Enterococcus faecium, as did the comparison compounds. E1077 was highly active against most members of the family Enterobacteriaceae, except for Serratia marcescens and Proteus vulgaris strains, 90% of which were inhibited at an E1077 concentration of 0.78 ,ug/ml. The activity of E1077 against all species was higher than those of the other compounds tested and was about two- to fourfold higher than that of cefpirome, with the exception of P. vulgaris. Against P. vulgaris, E1077, with an MIC90 of 3.13 ,ug/ml, was 2- to 16-fold less active than the comparison compounds. However, E1077 was more active against Enterobacter cloacae, Enterobacter aerogenes, and Citrobacter

TABLE 1. Comparative in vitro activities of E1077 and other P-lactam antibiotics against clinical isolates Organism (no. of strains) and antibacterial agent

MIC (pg/ml) Range

50%

Cefpirome Ceftazidime Cefuzonam Cefotaxime Staphylococcus aureus, methicillin resistant (50) E1077 Cefpirome Ceftazidime Cefuzonam Cefotaxime

0.39-0.78 0.39-3.13 6.25-25 0.39-1.56 1.56-6.25

0.78 0.78 12.5 0.78 3.13

12.5 1.56-100 50 3.13->100 25->100 >100 1.56-> 100 100 12.5->100 >100

0.78 1.56 12.5 0.78 3.13

100 >100 >100 >100 >100

Staphylococcus epidermidis, methicillin susceptible (19) E1077

Cefpirome Ceftazidime Cefuzonam Cefotaxime

0.20-3.13 0.20-3.13 3.13-25 0.10-3.13 0.39-25

0.39 0.39 6.25 0.39 1.56

1.56 3.13 25 3.13 12.5

Staphylococcus epidermidis, methicillin resistant (28) E1077 Cefpirome

Ceftazidime Cefuzonam Cefotaxime Streptococcus pyogenes, group A (27) E1077 Cefpirome Ceftazidime Cefuzonam Cefotaxime

Streptococcus agalactiae, group B (25) E1077 Cefpirome Ceftazidime Cefuzonam Cefotaxime

Streptococcus pneumoniae (26) E1077 Cefpirome Ceftazidime Cefuzonam Cefotaxime

0.78-50 0.78-50 12.5-> 100 0.78->100 6.25->100

TABLE 1-Continued Organism (no. of strains) and antibacterial agent

12.5 3.13 3.13 25 25 50 100 3.13 12.5 >100

Cefpirome Ceftazidime Cefuzonam Cefotaxime Piperacillin Enterococcus faecium (34) E1077 Cefpirome Ceftazidime Cefuzonam Cefotaxime Piperacillin Neisseria gonorrhoeae (16) E1077 Cefpirome Ceftazidime Cefuzonam Cefotaxime

.0.006-0.025

.0.006

0.025 0.025 0.20 '0.006 0.025

0.025-0.20 0.05-0.20 0.39-1.56 0.012-0.10 0.025-0.10

0.05 0.05 0.39 0.025 0.05

0.10 0.10 0.78 0.05 0.10

0.05-0.20

.0.006-0.012

0.012 '0.006 0.10

.0.006

E1077 Cefpirome Ceftazidime Cefuzonam Cefotaxime

Escherichia coli (54) E1077

Cefpirome Ceftazidime

Cefpirome Ceftazidime Cefuzonam Cefotaxime

Piperacillin Enterococcus avium (27) E1077

90%

0.39-> 100 100 3.13->100 >100 50 0.78-> 100 0.78->100 100 25 3.13->100

> 100

1.56->100 6.25->100 >100 12.5->100 25->100 0.39->100

>100 >100 > 100 >100 >100 >100

.0.006-0.39 .0.006-0.78

>100 >100 >100 >100 >100 >100

>100 >100 >100 100

0.025-0.39

0.025 0.10 0.10 0.10 0.05

0.39 0.78 0.39 0.39 0.39

0.012-1.56 0.05-3.13 0.025-0.20 0.10-1.56 0.05-0.78

0.10 0.39 0.05 0.20 0.20

0.78 3.13 0.10 1.56 0.78

.0.006-0.05 0.012-0.20 0.025-1.56 0.025-0.78 0.012-0.78

0.025 0.05 0.10 0.10 0.05

0.025 0.10 0.20 0.10 0.10

0.025-0.20 0.025-0.39 0.05-0.78 0.05-0.78 0.025-0.39

0.025 0.05 0.20 0.10 0.05

0.10 0.20 0.78 0.20 0.39

0.012-0.10 0.025-0.20 0.05-0.78 0.025-0.20 0.025-0.20

0.025 0.05 0.10 0.05 0.05

0.05 0.20 0.78 0.20 0.20

0.025-0.78 0.025-3.13 0.20->100 0.10-50 0.10-100

0.05 0.10 0.78 0.39 0.39

0.39 1.56 100 50 25

0.012-12.5 0.10->100 0.025-> 100 0.025->100

0.05 0.10 0.39 0.20 0.39

0.78 6.25 100 100 100

0.025-3.13 0.025-6.25 0.05-25

0.025 0.05 0.20

0.05 0.10 12.5

.0.006-0.78 .0.006-0.39

Klebsiella pneumoniae (54) E1077

Cefpirome Ceftazidime Cefuzonam Cefotaxime

Klebsiella oxytoca (26) E1077 Cefpirome Ceftazidime Cefuzonam Cefotaxime

Citrobacterfreundii (34) E1077

0.012-0.05 0.012-0.05 0.10-0.78 '0.006-0.05

.0.006-0.10

0.025 0.025 0.20 0.012 0.025

0.025 0.05 0.39 0.012 0.025

Cefpirome Ceftazidime Cefuzonam Cefotaxime Enterobacter cloacae (66)

Enterococcus faecalis (74) E1077

50%

Moraxella catarrhalis (27)

Cefuzonam Cefotaxime

.0.006-0.025 .0.006-0.025

MIC (uig/ml) Range

90%

Staphylococcus aureus, methicillin susceptible (58) E1077

591

IN VITRO ACTIVITY OF E1077

VOL. 36, 1992

E1077 12.5 0.20-100 1.56 0.78->100 12.5 50 >100 6.25->100 >100 25 100 0.39-> 100 0.20->100 >100 >100 3.13 0.78-12.5 6.25

1.56->100

50

>100

Continued

Cefpirome Ceftazidime Cefuzonam Cefotaxime Enterobacter aerogenes (35) E1077 Cefpirome Ceftazidime

.0.006-3.13

Continued on following page

592

WATANABE ET AL.

ANTIMICROB. AGENTS CHEMOTHER.

TABLE 1-Continued Organism (no. of strains) and antibacterial agent

Cefuzonam Cefotaxime Serratia marcescens (63) E1077

Cefpirome Ceftazidime Cefuzonam Cefotaxime Proteus mirabilis (40) E1077

Cefpirome Ceftazidime Cefuzonam Cefotaxime Proteus vulgaris (42) E1077

Cefpirome Ceftazidime Cefuzonam Cefotaxime Providencia rettgeri (39) E1077

Cefpirome Ceftazidime Cefuzonam Cefotaxime

TABLE 1-Continued

MIC (pug/ml) Range

50%

0.025-6.25 0.025-12.5

0.20 0.10

0.05-12.5 0.05-100 0.10->100 0. 10-> 100 0.10-> 100 0.025-0.10 0.05-0.10 0.05-0.10 0.05-0.20 0.012-0.05

0.025-25 0.025-12.5 0.025-0.78 0.05-1.56 0.012-6.25 100 0.78-> 100 12.5-> 100 1.56-> 100

0.025 0.05 0.10 0.10 0.10 0.05 0.05 0.20 0.012 0.025 3.13 12.5 3.13 50 12.5

0.20 0.20 12.5 3.13

6.25 0.10 0.10 0.20 0.05 0.025

antibacterial agent

E1077

Cefpirome

Ceftazidime Cefuzonam

Cefoperazone Xanthomonas maltophilia (39) E1077

Cefpirome

Ceftazidime Cefuzonam

Cefoperazone Acinetobacter calcoaceticus (50) E1077

Cefpirome

Ceftazidime Cefuzonam

Cefoperazone

MIC Range

6.25-> 100 6.25-> 100 0.78-> 100 6.25-> 100 12.5-> 100 6.25-> 100 12.5-> 100 3.13-> 100 1.56-> 100 6.25-> 100

0.39-25 0.78-100 1.56-25 1.56-> 100 25-> 100

(,ug/ml) 50%

90%

12.5 >100 25 100 3.13 12.5 12.5 100 50 >100 >100 >100 >100 >100 100

>100 >100 >100 >100 > 100

1.56 12.5 3.13 25 3.13 25 25 100 50 > 100

Alcaligenes spp. (36) E1077

Cefpirome

Ceftazidime Cefuzonam

Cefoperazone Flavobacterium spp. (38) E1077

Cefpirome Ceftazidime Cefuzonam

Cefoperazone Peptostreptococcus spp. (42) E1077

Cefpirome Ceftazidime Cefuzonam

Bacteroides fragilis (49) E1077

Cefpirome Ceftazidime Cefuzonam

0.20-> 100 1.56-> 100 0.20-25 0.78-> 100 0.39-25 0.39-> 100 0.78-> 100 3.13-> 100 6.25-> 100 3.13-> 100

0.025-1.56 0.05-6.25 0.10-25 0.012-3.13 0.78-> 100 6.25-> 100 3.13-> 100 0.78-100

6.25 50

3.13 12.5 3.13 25 25 100 25 50

0.39 1.56 3.13 0.20

25 > 100

12.5 100 12.5 100 100 > 100 >100 > 100 1.56 6.25 12.5 1.56

3.13 100 12.5 > 100 12.5 > 100 3.13 50

25 100 50

0.78-25 3.13 3.13-> 100 25 6.25 1.56-> 100 6.25-> 100 100 6.25-> 100 25

50

freundii. The MIC90s of E1077 for E. cloacae, E. aerogenes, and C. freundii were 0.78, 0.05, and 0.39 ,ug/ml, respectively, and the compound showed much higher activity (>64-fold) than the extended-spectrum cephalosporins, such as ceftazidime, cefuzonam, and cefotaxime, against these species. Against S. marcescens, E1077 was as active, with an MIC90

>100 100

of 3.13 ,ug/ml, as was ceftazidime but was two- to fourfold more active than cefpirome and cefuzonam. In addition,

0.39-25 1.56-100 0.78-25 6.25-> 100 6.25-> 100

25 50 12.5 >100 >100

3.13 12.5 6.25 50 25

> 100 > 100

12.5

Pseudomonas cepacia (35)

Continued

against Providencia rettgeri and Morganella morganii, E1077 and cefpirome were more active than ceftazidime and cefuzonam. Another antibacterial feature of E1077 is its potency against P. aeruginosa. The M'C50 and the MIC90 of E1077 for P. aeruginosa were 3.13 and 25 ,ug/ml, respectively, and its activity was two- to fourfold higher than those of ceftazidime and cefpirome. Cefuzonam was the least active of the compounds tested. E1077 also showed moderate activities against Pseudomonas fluorescens (MIC90, 12.5 ,ug/ml) and

IN VITRO ACTIVITY OF E1077

VOL. 36, 1992

593

TABLE 2. Effect of inoculum size on the activity of E1077' Geometric

Organism'

S. aureus, MS S. aureus, MR E. faecalis E. coli E. cloacae, CS E. cloacae, CR P. aeruginosa, CS P. aeruginosa, CR

mean

MIC, in

1

104

0.52 (0.39-0.78) 4.74 (1.56-25) 2.06 (0.78-25) 0.016 (C0.006-0.05) 0.037 (0.012-0.10) 0.39 (0.10-1.56) 0.59 (0.39-0.78) 14.4 (6.25-50)

0.59 (0.39-0.78) 7.18 (1.56-25) 4.12 (1.56-25) 0.021 (0.012-0.05) 0.043 (0.025-0.10) 0.52 (0.10-1.56) 0.90 (0.78-1.56) 16.5 (6.25-50)

CFU: pig/ml (range), at the following 105

0.59 (0.39-0.78) 12.5 (3.13-50) 4.74 (1.56-50) 0.021 (C0.006-0.05) 0.043 (0.025-0.10) 0.90 (0.20-3.13) 1.36 (0.78-3.13) 28.7 (12.5-100)

106

0.78 (0.78-0.78) 25 (3.13-50) 9.74 (3.13-100) 0.043 (0.025-0.10) 0.13 (0.05-0.39) 1.18 (0.20-3.13) 2.72 (1.56-6.25) 57.4 (25-100)

a Determined on Mueller-Hinton agar. b Five isolates of each species were tested. MS, methicillin susceptible; MR, methicillin resistant; CS, ceftazidime susceptible; CR, ceftazidime resistant.

Pseudomonas putida (MIC90, 25 p,g/ml), as did ceftazidime. However, E1077 was less active against Pseudomonas cepacia than was ceftazidime, yet the activity of the former was almost equal to those of cefpirome and cefuzonam. Against X. maltophilia, all compounds tested were inactive (MICg, > 100 p,g/ml). Against Acinetobacter calcoaceticus, E1077 was at least twice as active, with an MIC90 of 12.5 ,ug/ml, as the comparison compounds. E1077, even with an MIC90 of 25 ,g/ml, was fourfold more active against Alcaligenes species than was cefpirome, yet was two times less active than were ceftazidime and cefoperazone. E1077 was inactive against Flavobacterium species, as were the comparison compounds. E1077 showed high activities against N. gonorrhoeae and M. catarrhalis, with MIC90s of 0.39 and 0.78 ,ug/ml, respectively; however, the activity of E1077 against M. catarrhalis was at least two times lower than that of ceftazidime. Cefotaxime was two- to fourfold more active than E1077 against H. influenzae, but the latter, with an MIC90 of 0.10 ,ug/ml, showed activity as high as those of the other compounds. Included in these studies were ampicillinresistant strains of N. gonorrhoeae (3 of 16), M. catarrhalis (11 of 27), and H. influenzae (5 of 25). For the anaerobes, E1077 was as active as cefuzonam against Peptostreptococcus species, with an MIC90 of 1.56 ,ug/ml, and its activity was four- to eightfold higher than those of cefpirome and ceftazidime. The M'C50 and the MIC90 of E1077 for Bacteroides fragilis were 3.13 and 100 p,g/ml, respectively, and its activity was equal to or two times lower than that of cefuzonam but four- to eightfold higher than those of the other comparison compounds. About 20% of the strains tested were highly resistant to all compounds tested. The MBCs of E1077 in Mueller-Hinton broth were identical to or at most two times as high as the MICs for six strains each of S. aureus, E. faecalis, Escherichia coli, Kiebsiella pneumoniae, E. cloacae, C. freundii, S. marcescens, Proteus mirabilis, P. vulgaris, P. aeruginosa, and A. calcoaceticus. The comparison compounds also showed bactericidal effects (MBCs/MICs) similar to those of E1077. Effect of growth conditions on in vitro activity. The effects of the medium composition, the medium pH, the presence of horse serum, and inoculum size on the activity of E1077 were determined with five strains each of S. aureus, E. faecalis, E. coli, E. cloacae, and P. aeruginosa, including 13-lactamase-producing strains. The type of medium did not make a significant difference ( 100

Cefuzonam

6.25-> 100

P. aeruginosa (33) E1077

Cefpirome Ceftazidime Cefuzonam a

3.13-100 25-> 100 12.5-> 100 50-> 100

50%

90%

0.10 1.56 50 12.5

0.78 3.13 100 50

0.78 3.13 50 50

3.13 12.5 > 100 100

25 50 50 > 100

Ceftazidime-resistant strains from Table 1 are included.

E1077

0.05 0.025

0.05 0.05 0.012 0.20 0.05 0.78 0.05 1.56 0.05 0.012 0.025 25 1.56 0.78 3.13 3.13 >100

Cefpirome 0.10 0.39 0.10 0.20 0.025 0.78 0.05 3.13 0.10 3.13 0.10 0.025

0.20 100 6.25 3.13 1.56 6.25 100

Ceftazidime

Cefotaxime

0.39 0.10 0.39 25 0.20 100 0.39 >100 0.78

0.10 0.10 0.05 3.13 0.05 50 0.39 100 0.39 25 3.13 0.025 0.39 >100 25 1.56 0.39 50 100

3.13 6.25 0.05 3.13 50 3.13 0.39

0.05 3.13 50

derepressed; I, inducible; ND, not detected.

anaerobic gram-positive and gram-negative bacteria. One of the antibacterial features of E1077 is its potency against gram-positive cocci, such as staphylococci and E. faecalis. It is noteworthy that the activity of E1077 was high against E. faecalis, which was resistant or poorly susceptible to most of the previously used cephalosporins, including cefpirome. High activity against gram-positive cocci seems to be conferred on E1077 by the introduction of the propenylammonium moiety at the 3-position of the cephem nucleus (7). However, the precise role which the propenylammonium moiety plays in the mode of action of E1077 is unknown. In addition, against methicillin-resistant strains of S. aureus, the compound was relatively active compared with the comparison compounds. However, the MICs of E1077 were much higher (.8-fold) for methicillin-resistant strains than for methicillin-susceptible strains and were

Organism (no. of strains)a and antibacterial agent

MIC (,g/ml) of:

50 100 100 > 100

affected by the inoculum size, medium pH, culture temperature, and concentration of sodium chloride in the medium, as were those of the other 1-lactams. On the basis of this result, methicillin-resistant S. aureus is suggested to be intrinsically resistant to E1077 as well. A second important advantage is the activity of E1077 against P. aeruginosa. E1077 showed two- to fourfold higher activity than cefpirome and ceftazidime. Activity against P. aeruginosa seems to be enhanced by the aminothiadiazolyl group in the 7,-side chain, as observed in the cephalosporin E1040 (25). A third advantage of E1077 is that it is very active against most members of the family Enterobacteriaceae. Especially for strains derepressed for chromosomal f-lactamase, such as strains of Enterobacter and Citrobacter species, which are highly resistant to the cephalosporins with an extended spectrum, including ceftazidime and cefuzonam, the MIC90s of E1077 were 2 orders of magnitude lower than those of ceftazidime. Although the overproduction of chromosomal ,-lactamnase may not be the sole mechanism of resistance to these cephalosporins (11, 15, 21), the high activity of E1077 appears to result in part from the low affinity of the 3-lactamases for E1077, coupled with the high resistance of the compound to enzymatic hydrolysis, as reported for cefpirome (9) and cefepime (19). In addition, dipolar ionic compounds such as E1077 penetrate through porin channels of the outer membrane much more rapidly than do compounds having a net negative charge (17), and this good penetrability of E1077 may participate in part in its good antibacterial activity. However, cross-resistance between E1077 and ceftazidime was observed with ceftazidime-resistant strains of P. aeruginosa, although resistance in P. aeruginosa is suggested to be predominantly due to the overproduction of chromosomal ,B-lactamase (1) and the affinity of the P. aeruginosa ,-lactamase for E1077 was 1 order of magnitude lower than that for ceftazidime, as were those of the group 1 1-lactamases of other species. The resistance to E1077 and ceftazidime observed in P. aeruginosa strains may have been due to the overall poorer penetration of 1-lactams in

596

ANTIMICROB. AGENTS CHEMOTHER.

WATANABE ET AL.

TABLE 7. Induction of P-lactamases in strains of C. freundii, E. cloacae, and P. aeruginosa by E1077 and other P-lactams 13-Lactamase sp act (nmol/min/mg of protein) at the following inducer concn (pg/ml)b: MIC (pug/ml) Inducer' Organism 0

C. freundii E12075/WT

E. cloacae E10045/WT

P. aeruginosa E03441/WT

6.25

25

100

0.39

1.56

15 18 21 507

16 18 17 1,640

16 16 18 2,970

36 26 17

262 91 15

718 396 26

53 88 43 2,340

61 72 54 5,100

204 313 55 10,400

3,800 3,490 88

12,800 9,360 469

8,980 11,000 1,530

339

1 2 1 203

4 4 2 264

59 91 13 511

168 304 153 635

339 522 179 509

0.10

13

None E1077 CPR CAZ IPM

0.05 0.10 0.78 0.39 45

None E1077 CPR CAZ IPM

0.05 0.39 0.39

None E1077 CPR CAZ IPM

1.56 6.25 3.13 3.13

0.05

1

a CPR, cefpirome; CAZ, ceftazidime; IPM, imipenem. b Determined with cephalothin as the substrate.

this species. The penetration of 1-lactams, in general, is about 100- to 500-fold lower in P. aeruginosa than in members of the family Enterobacteriaceae (17). The overproduction of P-lactamase coupled with the more limited penetration of the compound may have resulted in the cross-resistance seen between E1077 and ceftazidime in P. aeruginosa strains but not in other gram-negative bacteria. E1077 MICs for members of the family Enterobactenaceae increased slightly with ,-lactamase overproduction, but the MICs remained in the susceptible range, presumably because permeability was less restricted. Furthermore, E1077 was more active against anaerobes than were ceftazidime and cefpirome. Although E1077 showed potent activity against a wide range of the commonly isolated bacteria in this study, including E. faecalis, it should be kept in mind that E1077 exhibited poor activity against other enterococcal species, TABLE 8. Inhibitor constants of E1077 and other cephalosporins for the hydrolysis of cephalothin by group 1 P-lactamases' Enzyme producer

C. freundii E12075/SKR32

Inhibitor

E1077

Cefpirome Ceftazidime Cefotaxime E. cloacae E10045/SKR5

E1077

Cefpirome Ceftazidime Cefotaxime P. aeruginosa E03441/SKR2

E1077

Cefpirome Ceftazidime Cefotaxime

Ki (pM)b 5.52 35.6 0.73 0.0034 141 128 8.20 0.034

58.1 134 6.85 0.118

aThe Kms of enzymes produced by C freundii E12075/SKR32, E. cloacae E10045/SKR5, and P. aeruginosa E03441/SKR5 for cephalothin were 16.5, 34.2, and 68.5 ,uM, respectively. b Ki values were determined from Dixon plots with cephalothin as the substrate.

i.e., E. avium and E. faecium, as did the comparison compounds. The high activity against staphylococci, E. faecalis, and P. aeruginosa and the low cross-resistance with extendedspectrum cephalosporins clearly distinguish E1077 from previously used extended-spectrum compounds and indicate that this compound could be a useful agent for the treatment of serious infections. Further studies on in vivo activity and pharmacokinetic and toxicological behaviors are therefore warranted. REFERENCES 1. Bayer, A. S., J. Peters, T. R. Parr, Jr., L. Chan, and R. E. W. Hancock. 1987. Role of P-lactamase in in vivo development of ceftazidime resistance in experimental Pseudomonas aeruginosa endocarditis. Antimicrob. Agents Chemother. 31:253-258. 2. Bush, K. 1989. Characterization of P-lactamases. Antimicrob. Agents Chemother. 33:259-263. 3. Chambers, H. F., and A. Tomasz. 1987. Effect of NaCl and nafcillin on penicillin-binding protein 2a and heterogeneous expression of methicillin resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 31:1982-1988. 4. Dixon, M. 1953. The determination of enzyme inhibitor constants. Biochem. J. 55:170-171. 5. Hartman, B. J., and A. Tomasz. 1986. Expression of methicillin resistance in heterogenous strains of Staphylococcus aureus. Antimicrob. Agents Chemother. 29:85-92. 6. Hikida, M., M. Inoue, and S. Mitsuhashi. 1986. In vitro antibacterial activity of L-105, a new cephalosporin. J. Antimicrob. Chemother. 18:585-591. 7. Kamachi, H., M. Oka, Y. Narita, S. linuma, S. Aburaki, H. Yamashita, K. Tomatsu, J. Okumura, and T. Naito. 1990. Synthesis of a new series of cephalosporins having a 3-substituted-ammonio-1-propenyl group as the C-3 side chain. J. Antibiot. 43:533-543. 8. Katsu, K., K. Kitoh, M. Inoue, and S. Mitsuhashi. 1982. In vitro antibacterial activity of E-0702, a new semisynthetic cephalosporin. Antimicrob. Agents Chemother. 22:181-185. 9. Kobayashi, S., S. Arai, S. Hayashi, and K. Fujimoto. 1986. p-Lactamase stability of cefpirome (HR810), a new cephalosporin with a broad antimicrobial spectrum. Antimicrob. Agents Chemother. 30:713-718. 10. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J.

VOL. 36, 1992

Biol. Chem. 193:265-275. 11. Marchou, B., F. Bellindo, R. Charnas, C. Lucain, and J.-C. Pechere. 1987. Contribution of ,B-lactamase hydrolysis and outer membrane permeability to ceftriaxone resistance in Enterobacter cloacae. Antimicrob. Agents Chemother. 31:1589-1595. 12. Matsubara, N., S. Minami, T. Muraoka, I. Saikawa, and S. Mitsuhashi. 1979. In vitro antibacterial activity of cefoperazone (T-1551), a new semisynthetic cephalosporin. Antimicrob. Agents Chemother. 16:731-735. 13. Minami, S., A. Yotsuji, M. Inoue, and S. Mitsuhashi. 1980. Induction of P-lactamase by various P-lactam antibiotics in Enterobacter cloacae. Antimicrob. Agents Chemother. 18:382385. 14. Mitsuhashi, S., and M. Inoue. 1981. Mechanisms of resistance to P-lactam antibiotics, p. 41-56. In S. Mitsuhashi (ed.), Betalactam antibiotics. Springer-Verlag, New York. 15. Nayler, J. H. C. 1987. Resistance to 1-lactams in gram-negative bacteria:' relative contributions of ,3-lactamase and permeability limitations. J. Antimicrob. Chemother. 19:713-732. 16. Neu, H. C., N. Aswapokee, P. Aswapokee, and K. P. Fu. 1979. HR756, a new cephalosporin active against gram-positive and gram-negative aerobic and anaerobic bacteria. Antimicrob. Agents Chemother. 15:273-281. 17. Nikaido, H. 1985. Role of permeability barriers in resistance to P-lactam antibiotics. Pharmacol. Ther. 27:197-231. 18. Pearson, R. D., R. T. Steigbigel, H. T. Davis, and S. W. Chapman. 1980. Methods for reliable determination of minimal lethal concentrations. Antimicrob. Agents Chemother. 18:699708.

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19. Phelps, D. J., D. D. Carlton, C. A. Farrell, and R. E. Kessler. 1986. Affinity of cephalosporins for ,B-lactamases as a factor in antibacterial efficacy. Antimicrob. Agents Chemother. 29:845848. 20. Samuni, A. 1975. A direct spectrophotometric assay and determination of Michaelis constants for P-lactamase reaction. Anal. Biochem. 63:17-26. 21. Sanders, C. C. 1987. Chromosomal cephalosporinases responsible for multiple resistance to newer P-lactam antibiotics. Annu. Rev. Microbiol. 41:573-593. 22. Seibert, G., M. Limbert, I. Winkler, and T. Dick. 1983. Antibacterial activity in vitro and ,B-lactamase stability of the new cephalosporin HR810 in comparison with five other cephalosporins and two aminoglycosides. Infection 11:275-279. 23. Ubukata, K., N. Yamashita, and M. Konno. 1985. Occurrence of a ,B-lactam-inducible penicillin-binding protein in methicillinresistant staphylococci. Antimicrob. Agents Chemother. 27: 851-857. 24. Utsui, Y., and T. Yokota. 1985. Role of an altered penicillinbinding protein in methicillin- and cephem-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 28:397-403. 25. Watanabe, N., 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. 26. Wise, R., J. H. Andrews, and K. A. Bedford. 1980. Comparison of in vitro activity of GR20263, a novel cephalosporin derivative, with activities of other beta-lactam compounds. Antimicrob. Agents Chemother. 17:884-889.

In vitro evaluation of E1077, a new cephalosporin with a broad antibacterial spectrum.

E1077 is a novel parenteral cephalosporin with a wide spectrum of potent antibacterial activity against aerobic and anaerobic gram-positive and gram-n...
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