In-vitro antibacterial activity of DQ-2556 and its stability to various beta-lactamases.

Journal of Antimicrobial Chemotherapy (1990) 26, 329-341

In-vitro antibacterial activity of DQ-2556 and its stability to various (Mactamases T. Fujunoto", M. Watanabe*, M. Inooe* and S. Mitsuhashr* 'Episome Institute, Fujimi-mwa, Seta-gun; ''Laboratory of Drug Resistance in Bacteria, Gunma University School of Medicine, Showa-machi, Maebashi, Gunma-ken, Japan DQ-2556, a new cephalosporin, showed a broad antibacterial spectrum over Gram-positive and -negative organisms. The activity of DQ-2556 against recent clinical isolates of Gram-positive cocci and Enterobacteriaceae was comparable with that of cefpirome, and superior to that of ceftazidime. DQ-2556 was almost as active as cefpirome against Pseudomonas aeruginosa, but was less active than ceftazidime. With the exception of Ps. aeruginosa, DQ-2556 was bactericidal against various organisms at either the MIC or twice the MIC. DQ-2556 bound preferentially to penicillin-binding proteins (PBPs) 2, 1 and 3 of Staphylococcus aureus. PBPs 3, 1A and IB of Escherichia coli and PBPs 1A, 3 and 4 of Ps. aeruginosa. DQ-2556 was stable to various penicillinases and cephalosporinases, but was unstable to oxyiminocephalosporinases. The A. values of DQ-2556 for the cephalosporinases of Citrobacter freundii and Enterobacter cloacae were only two- or three-fold higher than those of ceftazidime, indicating that DQ-2556 had a relatively high affinity for these enzymes compared with other recently developed cephalosporins. The MIC of DQ-2556 for Esch. coli increased four-fold in an OmpF-deficient mutant, indicating that the OmpF porin was one of the major routes for penetration of DQ-2556 into Esch. coli cells.

Introduction Qinical experience with the third generation cephalosporins during the past decade has highlighted the need for improvement of antibacterial activity of /Mactams against staphylococci, high-level cephalosporinase-producing strains of Citrobacter freundii and Enterobacter cloacae, Serraiia marcescens and Pseudomonas aeruginosa. The newer injectable cephalosporins under development, including cefpirome (Jones, Thornsberry & Barry, 1984) and DQ-2556 (Fujimoto et al., 1986), have advantages in some of these areas. Among the newer compounds, DQ-2556 is characterized by its well-balanced antibacterial activity against both Gram-positive and -negative organisms (Fujimoto et al., 1986) and by its favourable pharmacokinetic parameters (Nakashima et al., 1989). The present study compares the antibacterial activity of DQ-2556 against recent clinical isolates and /Mactamase-producing strains with that of ceftazidime (O'Callaghan et al., 1980), cefotaxime (Neu et al., 1979) and cefpirome. DQ-2556 was also compared with the reference cephalosporins in terms of its affinity for penicillin-binding proteins •Corretponding author Tenio Fujimoto, Episome Institute, 2220, Kogurc, Fujimi-mura, Seta-gun, Gunma-ken, 371-01, Japan. 0305-7453/90/090329+13 $02.00/0

329 © 1990 The British Society for Antimicrobial Chemotherapy

330

T. Fnpmoto et al

(PBPs), its stability and affinity for various /Mactamases, and its activity against outer membrane protein (OMP>deficient strains of Escherichia coli in the presence or absence of plasmids carrying genes encoding chromosomal /Mactamases. Materials and methods

Test compounds DQ-2556 was provided by Daiichi Pharmaceutical Co. Ltd, Tokyo. Other compounds were obtained from the following sources: ceftazidime: Nihon Glaxo, Tokyo; cefotaxime and cefpirome: Hocchst Japan, Tokyo; methicillin and cefoxitin: Banyu Pharmaceuticals Co. Ltd, Tokyo; ampicillin and penicillin G: Meiji Seika Kaisha Ltd, Tokyo; and cephaloridine: Torii Yakuhin Co. Ltd, Tokyo. Compound solutions were prepared just prior to use. Bacterial strains Bacterial strains used were standard strains and recent clinical isolates. The clinical isolates were obtained from patients in geographically separate hospitals in Japan. All the isolates were stored in 50% glycerol at r-80°C until use. Determination of MICs MICs were determined by a routine two-fold agar dilution method with an inoculum of about KPcfu. The MIC was denned as the lowest concentration that prevented visible growth of bacteria after incubation at 37°C for 18-24 h. MICs for staphylococci were determined on sodium chloride-free Sensitivity Disk Agar-N (Nissui Seiyaku Co. Ltd, Tokyo) as the test medium, with an inoculum of about 10*cfu and an incubation time of 24 h at 37CC. Under these conditions, strains showing methicillin MICs of ^6-25mg/l or ^ 12-5 mg/1 were designated as methicillin-susceptible or -resistant strains, respectively. Determination of MBCs MBCs were determined by the method of Taylor et al. (1983), with an inoculum between 5x10* and 3xl0*cfu/ml of the logarithmic phase test organisms and a subculture volume of 10 /d. Media used were: Brain Heart Infusion Broth (Difco Laboratories, Detroit) supplemented with 5% Fildes Enrichment (Difco) for HaemophUus influenzae; Sensitivity Test Broth (STB; Nissui) supplemented with 0-4% potassium nitrate for Ps. aeruginosa; and STB for all other organisms. The MBC was defined as the lowest concentration that killed ^ 99-9% of the initial inoculum. Affinity for PBPs Affinities of DQ-2556 and the reference cephalosporins for PBPs were determined as described by Utsui & Yokota (1985) for Staphylococcus aureus, and as described by Spratt (1975) for Esch. coli and Ps. aeruginosa. The concentration of each compound that inhibited the subsequent binding of l4C-peniciUin G (Amersham Japan, Co. Ltd,

DQ-2556 in vitro

331

Tokyo) to each PBP by 50% (the IQa) was determined by screening the fluorograms with a densitometer (Densitoron PAN802; Jooko Co. Ltd, Tokyo). Stability and affinity for fi-lactamases /J-Lactamases were the standard preparations used in our institute. Each enzyme had > 95% purity and were stored at — 80 c C. These enzymes were classified as penicillinases, cephalosporinases or oxyiminocephalosporinases, according to the classification of Mitsuhashi & Inoue (1981). Hydrolysis was measured at a substrate concentration of 100 /iM with a spectrophotometric method at 30°C in 50 mM phosphate buffer (pH 70) as described previously (Minami, Inoue & Mitsuhashi, 1980). Hydrolysis of DQ-2556 was followed at a wavelength of 294 nm (AE = 21-72). Relative rates of hydrolysis were expressed as a percentage of penicillin G hydrolysis (for penicillinases) or cephaloridine hydrolysis (for cephalosporinases and oxyiminocephalosporinases). K^ and Vm values were estimated from Lineweaver-Burk plots. The inhibitory constant (tQ) of ceftazidime for penicillinase Type I was measured spectrophotometrically from a Dixon plot with cephaloridine as the substrate. Results Inhibitory activity against clinical isolates Table I summarizes the inhibitory activities of DQ-2556 and the reference /Mactams against 2217 recent clinical isolates. Bactericidal activity against clinical isolates As shown in Table II, DQ-2556 and the other cephalosporins tested had MBCs which were the same as, or twice, the MIC for Staph. aureus, Esch. coli, Klebsiella pneumoniae and H. influenzae. In contrast, the MBCs of DQ-2556 and cefpirome for Ps. aeniginosa were much higher than their MICs, while the MBCs of ceftazidime were four-fold greater than the MICs. The difference between the MICs and MBCs of DQ-2556 for this species was attributed to regrowth of organisms after an initial short phase of killing (data not shown). Affinity for PBPs The IC»s of DQ-2556, ceftazidime and cefpirome for the PBPs of Staph. aureus, Esch. coli and Ps. aeniginosa are summarized in Table III. DQ-2556 showed similar binding patterns to those of ceftazidime and cefpirome. The affinities for PBPs 2, 1 and 3 of Staph. aureus were highest with cefpirome, followed by DQ-2556 and ceftazidime, whereas the affinities for PBPs 3, 1A and IB of Esch. coli were highest with DQ-2556, followed by cefpirome and ceftazidime. These results correlated with the antibacterial activities of these compounds against the test strains. DQ-2556 had some affinity for PBP 4 of Staph. aureus, for which ceftazidime and cefpirome had no affinity, but the ICJO of DQ-2556 was > 10mg/l. As shown in Figure 1, DQ-2556 showed good affinity for PBPs 1 A, 3 and 4 of Ps. aeniginosa. The affinity of DQ-2556 for these PBPs was equal to or higher than the affinities of ceftazidime and cefpirome, while the activity of DQ-2556 against the strain was lower than that of ceftazidime (Table IH).

332

T. Fujimoto et aL

Table L Antibacterial activity of DQ-2556 and the other /f-lactams against 2217 clinical isolates Organism (no. of strains)

Compound

range

MIC (mg/1) 50% 1-56 12-5 313 0-78 1-56

90% 1-56 25 313 1-56 3.13

Methicillin-susccptible Staph. aureus (121)

DQ-2556 ceftazddime cefotaxime cefpirome mcthkallin

0-19-12-5 313->10O 0-39-100 0-19-25 0-78-6-25

Methicillin-resistant Staph. aureus (91)

DQ-2556 'ceftazidime cefotaxime cefpirome methicillin

1-56-100 12-5-> 100 6-25->100 1-56-100 125->100

Methicillin-susceptible coagulase-negative staphylococci (96)

DQ-2556 ceftazidime cefotaxime cefpirome methicillin

0-10-313 1-56-25 0-19-12-5 0-10-313 0-19-6-25

Methicillin-resistant coagulase-negative staphylococci (88)

DQ-2556 ceftazidime cefotaxime cefpirome methicillin

1-56-50 6-25-> 100 313->1OO 0-78-50 12-5->100

Streptococcus pneumoniae (17)

DQ-2556 ceftazidime cefotaxime cefpirome ampicillin

0006-0025 0-10-0-39 0-006-0O25 0006-0025 0025-0-10

0013 0-10 0O13 0013 0O25

0O25 0-19 0O25 0O25 005

Str. pyogenes (100)


100 25->100 0-78-> 100 l-56->100

Enterococcus faecium (33)

DQ-2556 ceftazidime cefotaxime cefpirome

100->100 >100 100->100 50->100

Esch. coli (146)


0O06-O-78 0025-12-5 0013-6-25 0013-0-39

25 >100 >100 50 >100 0-78 6-25 1-56 0-39 313 313 25 12-5 1-56 25

50 >100 >100 100 >100 1-56 25 6-25 1-56 6-25 12-5 100 >100 12-5 >100

12-5 >100 100 6-25

50 >100 >100 25

>100 >100 >100 >100

>100 >100 >100 >100

0O13 0-10 0O5 0O25

0025 0-19 0-10 005

DQ-2556 in ritro

333

TtWe L—Continued Orpftniffm

MIC (mg/1) 50%

(no. of strains)

Compound

range

K. pneumoniae (107)

DQ-2556 ceftazidime cefotaxime ccfpirome

0006-0.78 0025-313 0006-1-56 0013-1-56

0013 OlO 005 0025

0025 019 OlO 005

K. oxytoca (106)


< 0003-078 0013-039 O006-1-56 0006-078

0013 005 0025 0025

0025 OlO 005 005

Prot. mirabilis (103)


0013-019 0025-039 0013-019 0025-039

005 005 0025 OlO

005 OlO 005 019

Prot. vulgaris (75)


0025-12-5 005-039 0025-25 005-12-5

019 OlO OlO 019

1-56 019 313 313

M. morganii (108)


0006-313 005-25 0013-12-5 0013-019

O025 OlO OlO 0025

019 313 1-56 OlO

Prov. rettgeri (50)


100 0025-100

O05 039 019 O05

6-25 100 50 313

Enterobacter cloacae (105)


0O13->100 005-100 0 0 2 5 - > 100 0 0 2 5 - > 100

005 078 039 OlO

25 50 100 6-25

Ser. marcescens (120)


O025->100 O10->100 O19->100 005->100

019 039 1-56 019

Ceftazidime-susceptible Ps. aeruginosa (101)


078-100 039-12-5 O78->100 039-100

6-25 1-56 12-5 6-25

90%

50 50 >100 50 25 6-25 100 12-5

T. Fujimoto et aL

334

Table L—Continued Organism (no. of strains) Ccftazidimc-rcsistant Ps. aeruginosa (26)

MIC (mg/1) Compound

range

50%

DQ-2556 ceftazidime ccfotaxime cefpirome

12-5- > 100 25-100 100->100 6-25-> 100

DQ-2556 ceftazidime ccfotaxime cefpirome

1-56-25 0-39-1-56 0-78-12-5 1-56-25


313->1OO 0-78-> 100 6-25->100 25->100


1-56-50 3-13-25 6-25-> 100 1-56-25

DQ-2556 ceftazidime cefotaxime cefpirome ampidllin

0006-010 04)25-0-19 < 0003-0025 0013-0-10 0-10->100

0025 0-10 0013 0025


sj 0-003-0-10 ^0003-0-19 < 0003-010 >S 0003-010 005-50

0013

Bran, catarrhalis (38)

DQ-2556 ceftazidime cefotaxime cefpirome ampidllin

0-025-0-78 O013-0-10 0O25-0-78 005-1-56 ^0003-12-5

Bact. fragilis (35)

DQ-2556 ceftazidime cefotaxime cefpirome cefoxitin

Ps. cepada (51)

X. maltophilia (50)

Adnetobacter (35)

calcoaceticus

H. influenzae (88)

N. gonorrhoeae (15)

Cl. perfringens (16)

Cl. diffidle (27)

DQ-2556 ceftazidime cefotaxime cefpirome cefoxitin DQ-2556 ceftazidime cefotaxime cefpirome cefoxitin

l-56->100 313->10O 0-39->100 313->1OO 3-13-50 0O06-O78 0O13-O-78 0006-1-56 0O06-O-78 0-19-0-78 12-5-50 25-100 2 5 - > 100 25-50 50->100

>100

90% >100

50

100

>100

>100

50

100

6-25 0-78

313 6-25

50 50 100 100 6-25 6-25

25 313

019 005 0O13 0025

039 019 005 O10 019 039

50 50 6-25

50 12-5 O013 0025 0013 0013

039

25 100 50 25 100

25 1-56 12-5 12-5

100 >100 >100 >100 12-5 12-5

50 25 005 010 0013

005 125 005 019 0O5 005 25 078 010 039 078 313 >100 >100

100 >100

25 0025

005 005 005 078

50 100 >100

50 >100

335

DQ-2556 in witro

Table IL Bactericidal activity of DQ-2556 and the other cephalosporins against clinical isolates MBC (mg/1) 90% 50%

MIC (mg/1) 90% 50%

Orffflnisni

(no. of strains)

Compound

Staph. aweus (20)

DQ-2556 ceftazidime cefpirome

1-56 12-5 0-78

1-56 12-5 078

1-56 25 1-56

1-56 25 1-56

EscKcoli (20)


0-10 0-39 0-10

0-19 0-78 0-19

0-10 0-39 0-10

0-19 0-78 039

K. pneumoniae (20)


0-10 0-19 0-10

0-19 0-39 0-39

0-10 0-39 0-10

019 039 039

Ps. aeruginosa (20)


12-5 313 6-25

25 6-25 12-5

>100 6-25 >100

H. inftuenzae (20)

DQ-2556 ceftazidime

0O5 0-19

005 0-19

0O25 0-10

>100 25 >100 005 019

Table HL Comparative affinities of DQ-2556, crftnzidimr and cefpirome for penicillin-binding proteins (PBPs) Organism Staph. aweus 209P JC-1

EscKcoli K12C600

Ps. aeruginosa NCTC10490

PBPs PBP I PBP2 PBP 3 PBP 4

DQ-2556

1A IB 2 3 4 5/6

PBP PBP PBP PBP PBP PBP

1A IB 2 3 4 5

cefpirome

(1-56)

(12-5)

(078)

045 031 5-2 >10

034 046 9-1 >10

018 034 2-4 >10

(0013) PBP PBP PBP PBP PBP PBP

IC S0 (mg/IT ceftazidime

1-6 1-7 9-6 0018

2-8 >100 (1-56) 0064

5-8 >100

O10 032 >100

(O10)

1-2 2-7 68 0096 >100 >100 (039)

012 10 >100 0081

1-8 >100

(0025)

6-5 2-6 9-7 O097

16 >100 (1-56) 0068

6-6 >100

026 023 >100

"MICs of each compound for the three organisms tested are shown in parentheses.

336

T. Fnjirooto et aL PBPs

1A IB 2 3 4 5

a

b

c

d

e

f

g

h

i

j

k

l

Figure 1. Binding of DQ-2556 to the PBPs of Ps. aentginosa NCTC 10490. Concentration of DQ-2556 (mg/1) used in each track was as follows: a and 1, 0; b, 0O03; c, 001; d, 003; e, 0-1; f, 0-3; g, 10; h, 3-0; i, 10; j , 30; k, 100.

Antibacterial activity against fi-lactamase-producing strains

As shown in Table IV, DQ-2556 had potent activity against penicillinase-producing organisms such as Esch. coli, Staph. aureus and K. pneumoniae. Among the strains of Esch. coli that produced plasmid-mediated pcnicillinases, only the strain that produced penicillinase Type II was less susceptible to DQ-2556 than the host. DQ-2556 had potent activity against strains of Esch. coli, Providencia rettgeri and Morganella morganii that produced cephalosporinases. DQ-2556 also had moderately high activity against cephalosporinase-producing strains of Enterobacter cloacae and Ser. marcescens, but not against Ps. aeruginosa or strains of Citro.fretmdii that produced high levels of cephalosporinase. Among the oxyiminocephalosporinaseproducing strains, Xanthomonas maltophilia GN12873 was resistant to all the compounds tested. Against the other oxyiminocephalosporinase-producing organisms, the activity of DQ-2556 tended to be moderate or somewhat less than the other cephalosporins tested. Overall, the activity of DQ-2556 against these organisms was similar to that of cefpirome, except in the case of strains such as Citro.freundii GN7391 (Tajima et al., 1980) and Enterobacter cloacae GN7471 (Minami et al., 1980) that produced high levels of cephalosporinase; indeed DQ-2556 was four- to eight-fold less active against Citro.freundii GN7391 and Enterobacter cloacae GN7471 than cefpirome. DQ-2556 was more active than ceftazidime against penicillinase- and cephalosporinase-

DQ-2556 ia wtiro

337

Table IV. Antibacterial activity of DQ-2556 and other cephalosporins against /Mactamaseproducing strains Type of /Mactamase"

Organism Esck Esck Esck Esck Esck

coli coli coli coli coli

ML4901 ML4901 ML4901 ML4901 ML4901

(Rms212) (Rms213) (Rtel6> (Rmsl49)

PCaseType I PCaseType II PCaseTypem PCaseTypelV

MIC(i DQ-2556 0025 0025

010 0025 O025

CAZ

CTX

019 019 019 039 010

010 010 019 010 005

005 005 019 005 005 005 039

Stapk aureus MS 15009 Stapk aureus MS15OO9 (pI258) PCaseType V

O10 078

078

039

6-25

1-56

K. pneumoniae GN69 Esck coli GN14929 Esck coli GN5482 Citro. freundii GN346 Citro. freundii GN7391 Enterobacter cloacae GN5797 Enterobacter cloacae GN7471 PTOV. rettgeri GN5284 M. morganii GN5407 Ser. marcescens GN14931 Set. marcescens GN1O857 Ps. aeruginosa GN 10362 Ps. aeruginosa GN1O367

PCaseType I CSase CSase CSase CSase CSase CSase CSase CSase CSase CSase CSase CSase

0013 0025

019 019 078 50

K. oxytoca GN10650 Prot. vulgaris GN76 Prot. vulgaris GN7919 Ps.cepacia GN11164 X. makophilia GN12873

CXase CXase CXase CXase CXase

005 6-25

50 O10 078 0025 O025 0025

O025

010 039 25 100

>100

078 313 010 010 019

1-56 6-25 0013

078

1-56

005 039 25

6-25

078 313

50

25 078 0025

25 6-25

100

CPR

12-5

078

0025

005 313 019 50

0025

25 1 56 >100

0025

005 0025

078 12-5

010 010 0025 0025 0025 1-56 6-25

25 078 010 50 6-25 >100

•PCajc, Penicillinase; CSase, cephalosporinase; CXase, oxyiminocephalosporinase. *CAZ, Ceftazidime; CTX, cefotaxime; CPR, cefpirome.

producing organisms, with the exception of Ps. aeruginosa. DQ-2556 was less active than ceftazidime against oxyiminocephalosporinase-producing organisms such as K. oxytoca, Proteus vulgaris and Ps. cepacia. Stability and affinity for various fi-lactamases As shown in Table V, DQ-2556 was stable to various penicillinases and cephalosporinases, except penicillinase Type II, but was not stable to oxyiminocephalosporinases. The rates of DQ-2556 hydrolysis were almost equal to those of cefpirome and were generally higher than those of ceftazidime. Table VI shows the K^, Vma and V^K^ values of DQ-2556 and ceftazidime for representative /Mactamases. DQ-2556 and ceftazidime both had a low affinity for penicillinase Type I (A^, values > 100/XM). DQ-2556 had a somewhat lower affinity than ceftazidime for the cephalosporinases of Citro. freundii, Enterobacter cloacae and Ps. aeruginosa, and the oxyiminocephalosporinase of Prot. vulgaris, with two- to threefold higher K^ values than ceftazidime. However, the ten- to 60-fold higher

338

T. Fnpmoto et aL Table V. Stability of DQ-25S6 and the other cephalosporins to various /Mactamases

Origin

Relative rate of hydrolysis DQ-2556 CAZ CTX CPR

Type of /f-lactamase

Esch. coli ML4901 (Rms212) Each, coli ML4901 (Rms213) Esch. coli ML4901 (Rtel6) Esch. coli ML4901 (Rmsl49) Stapk aureus MS 15009 (pI258)

PCase Type I PCase Type II PCaseTypem PCase Type IV PCase Type V

Esch. coli GN5482 Citro. freundii GN7391 Enterobacter cloacae GN7471 M. morgana GN5407 Prov. rettgeri GN4430 Ser. marcescens GN10857 Ps. aeruginosa GN10362

CSase CSase CSase CSase CSase CSase CSase

K. oxytoca GN10650 Prot. vulgaris GN7919 Ps. cepacia GNU 164 X. maltophilia GN12873 X. maltophilia GN12873

CXase CXase CXase CXase (L-l) CXase (L-2)

007 17 017 004 006

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