ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1979, P. 549-553
Vol. 16, No. 5
0066-4804/79/11-0549/00$02.00/0
Antibacterial Activity of Ceftizoxime (FK 749), a New Cephalosporin, Against Cephalosporin-Resistant Bacteria, and Its Stability to fi-Lactamase HITOSHI KOJO,' MINORU NISHIDA,I SACHIKO GOTO,2 AND SHOGO KUWAHARA2 Research Laboratories, Fujisawa Pharmaceutical Co., Ltd. Osaka,'and Department of Microbiology, Toho University, School of Medicine, Tokyo,2 Japan Received for publication 30 July 1979
Antibacterial activity of FK 749 against ampicillin-resistant clinical isolates of Escherichia coli was compared with those of other newly developed cephalosporins. FK 749 was the most active against strains possessing R-plasmids specifying ampicillin resistance and those whose resistance was chromosomally determined. The susceptibility of ampicillin-susceptible E. coli to FK 749 was not decreased by transduction of ampicillin resistance-specifying plasmids. However, most of the transconjugants acquired a high level of resistance to cefoperazone and cefamandole and a moderate level to cefotiam. FK 749 was highly stable to both penicillinase- and cephalosporinase-type ,8-lactamases, including R-plasmid-mediated f8-lactamase. Its level of resistance to ,B-lactamases was comparable to those of cefoxitin, cefmetazole, and cefotaxime, slightly superior to that of cefuroxime, and much superior to those of cefotiam, cefamandole, and cefoperazone.
Cephalosporin antibiotics are superior to other families of antibiotics in their low toxicity and significant antibacterial activity against a large number of gram-positive and -negative bacteria. However, the following two recent situations are of great concern for the antibacterial activity of cephalosporins against gram-negative bacteria. The first is the gradual increase of noncephalosporin-susceptible gram-negative species among clinical isolates, such as Serratia, Enterobacter, Citrobacter, Proteus, and Pseudomonas (3). The other is the elevation of the level of cephalosporin resistance mediated by plasmids specifying fl-lactamase, resulting in the increase of moderately cephalosporin-resistant strains in Enterobacteriaceae including Escherichia coli and Klebsiella pneumoniae (2, 5, 6). This report describes the antibacterial activity of FK 749, a cephalosporin derivative newly developed in our research laboratories (4), against the above-mentioned cephalosporin-resistant bacteria, especially against R-plasmidpossessing strains. This report also describes the stability of FK 749 to /3-lactamases. MATERIALS AND METHODS Bacterial strains. E. coli CSH2 (naF), 17 strains of E. coli CSH2 harboring R-plasmids specifying ampicillin resistance (Apr), and 12 chromosomally ampicillin-resistant clinical isolates of E. coli were kindly provided by T. Yokota of Juntendo University. Twelve clinical isolates of E. coli possessing R-plasmids determining ampicillin resistance were selected from the
culture collection of our laboratory. Ten fl-lactamresistant strains were selected among the clinical isolates of the following species and used for preparation of ,B-lactamases: Serratia marcescens, Enterobacter cloacae, E. coli, Proteus vulgaris, Pseudomonas aeruginosa, K. pneumoniae, Proteus mirabilis, and Staphylococcus aureus. Antibiotics. The antibiotics used in this study included ceftizoxime (FK 749; Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan), cefotiam (SCE 963; Takeda Chemical Industries, Ltd., Osaka, Japan), cefamandole (Eli Lilly & Co., Indianapolis, Ind.), cefuroxime (Glaxo Research Ltd., Greenford, England), cefotaxime (HR 756; Hoechst-Roussel, Frankfurt, West Germany), cefmetazole (Sankyo Co., Ltd., Tokyo, Japan), cefoxitin (Merck Institute for Therapeutic Research, Rahway, N.J.), cefoperazone (T-1551; Toyama Chemical Co., Toyama, Japan), cephaloridine (Eli Lilly), cefazolin (Fujisawa), penicillin G (Fujisawa), and ampicillin (Beecham Pharmaceuticals, Betchworth, England). Antibiotic susceptibility testing. The minimal inhibitory concentrations (MICs) of test antibiotics were determined by the agar dilution method with the use of a multipoint inoculator (1). The inoculum was undiluted overnight culture in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.). After incubation at 37°C for 20 h, the lowest concentration that inhibited macroscopic colonial growth was regarded as the MIC. Preparation of 8-lactamase. The cells were grown at 37°C in heart infusion broth to which penicillin G was added as an inducer. After overnight incubation, the cells were harvested by centrifugation, washed once, and suspended in 0.067 M potassium phosphate buffer (pH 7.0). The cell suspensions were sonicated at 20 kilocycles for 20 min. After cellular 549
550
KOJO ET AL.
debris was removed by centrifugation, the supernatant subjected to gel filtration on a Sephadex G100 column. The column was equilibrated with 0.067 M phosphate buffer (pH 7.0) and eluted with the same buffer. The enzyme fractions were pooled and stored at -200C. Assay of ,-lactamase activity. ,B-Lactamase activity was determined with a Hitachi 200-20 spectrophotometer equipped with a thermostatted cell holder. The enzyme was mixed in a 1-cm quartz cuvette with 150 jug of substrate and 200 itmol of phosphate buffer (pH 7.0) to make a final volume of 3.0 ml and was then incubated at 370C. The rate of hydrolysis of the ,Blactam ring was followed by the change in absorption at 240 nm for penicillin and at 260 nm for cephalosporins (8). The relative initial rate of hydrolysis was expressed as a percentage compared to the hydrolysis of cephaloridine for cephalosporinase and of penicillin G for penicillinase.
was
ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Antibacterial activity of FK 749 against ampicillin-resistant strains of E. colia Mean MIC (pg/mi) Antibiotic
R+ E. coli C+ E. coli FK 749 2.1 1.7 Cefotiam 5.6 9.4 Cefamandole 50 178 Cefuroxime 28 40 Cefotaxime 3.5 11 Cefmetazole 40 50 11 Cefoxitin 7.0 >800 >800 Cefoperazone 25 47 Cefazolin a Twelve each of R+ and C+ strains were used; R+, R-plasmid-harboring strains (clinical isolates) and C+, chromosomally resistant strains (clinical isolates). Incubation: heart infusion agar, 37°C, 18 to 20 h. Assay: 108 colony-forming units per ml; stamp method.
RESULTS
Antibacterial activity of FK 749 against ampicillin-resistant clinical isolates of E. coli Ampicillin-resistant clinical isolates of E. coli
were divided between those whose resistance was chromosomal and those whose resistance was mediated by an R-plasmid. The anti-
bacterial activity of FK 749 against those strains is shown in comparison with those of newly developed cephalosporin and cephamycin derivatives (Table 1). FK 749 was the most effective against both types ofresistant strains, with mean MICs of 2.1 ug/ml for R-plasmid-harboring strains and 1.7 pg/ml for chromosomally resistant strains. The mean MICs of FK 749 against the resistant strains were the same as those of the susceptible strains (4). Meanwhile, the other cephalosporin and cephamycin derivatives were 2 to 25 times less effective than FK 749 against R-plasmid-harboring strains and 5 to 100 times less effective against chromosomally resistant strains. Influence of R-plasmids encoding ampicillin resistance on the antibacterial activity of FK 749. The net influence of R-plasmids encoding ampicillin resistance on the antibacterial activities of cephalosporin derivatives was assessed by comparing the activity of cephalosporins against E. coli CSH2 to that against strains of E. coli CSH2 possessing R-plasmids. Strains possessing R-plasmids were constructed by conjugal transfer of R-plasmids from clinical isolates of E. coli (8 strains) and of K. pneumoniae (9 strains) to E. coli CSH2. Figure 1 shows the MICs of cephalosporins against E. coli CSH2 and the distribution of the MICs against 17 strains of E. coli CSH2 harboring Apr R-plasmids. Of the nine cephalosporins tested (FK 749, cefotiam, cefamandole, cefuroxime, cefotaxime, cefmetazole, cefoxitin, cefoper-
azone, and cefazolin), the antibacterial activity of cefoperazone was the most influenced by the R-plasmids, followed by that of cefamandole and cefotiam. As shown in Fig. 1, 65% of the plasmidharboring strains of E. coli CSH2 were resistant to 400 pg and above of cefoperazone and cefamandole per ml, whereas the MICs of cefoperazone and cefamandole against plasmid-free E. coli CSH2 were 1.56 and 3.13 pLg/ml, respectively. The MICs of cefotiam against the plasmid-possessing strains ranged widely from 1.56 to 100lg/ml, whereas cefotiam inhibited the plasmid-free strains at 1.56 pg/ml. Meanwhile, the effect of the R-plasmids on the antibacterial activities of cefuroxime, cefotaxime, and cephamycins (cefoxitin and cefmetazole) was notably less, although their activities were reduced twoto fourfold in the presence of the R-plasmids. In contrast, the MICs of FK 749 against strains possessing the Apr R-plasmids were shown to coincide with its MIC against the plasmid-free strain. This result clearly indicates that the antibacterial activity of FK 749 is not reduced by R-plasmids specifying ampicillin resistance. Stability of FK 749 to f-lactamase. Stability of FK 749 to ,B-lactamase was compared with that of the nine ,B-lactam antibiotics cefotiam, cefamandole, cefuroxime, cefmetazole, cefoxitin, cefoperazone, cefazolin, cephaloridine, and penicillin G. According to the classification proposed by Richmond and Sykes (7), nine distinct types of f4-lactamases were selected from clinical isolates of gram-negative bacilli and used for assay, together with the enzyme from ampicillin-resistant S. aureus. Class I enzymes are cephalosporinases, whereas class IH, III, IV, and V, and S. aureus enzymes are termed penicillinases. The relative initial rates of hydrolysis of the cephalosporins were expressed in percentage of hy-
CEFTIZOXIME (FK 749) AGAINST RESISTANT BACTERIA
VOL. 16, 1979 16
Cefnetazole
FK749
I
12
8 4 0
9%-j
-A.
I I I I.
Cefotian
12
551
KI I I
_ ~
I
I
Cefoxitin
8 4
0
I
9j
I
I
.E I
Ia
i
Cefoperazane
Cefamandole
12
8 4
0
12
I_777i K
I
I
|||| l
_Cefuroxcime
Cefazolin
a
8
II_
4
0
a
I
I
t- I
I
1 IX
I
I
"/
II
I
Aopicillin
Cefotaxime
12 0
8 4
0
I
0.2 0.78 3.13 12.5
50
200
800>8co
0.2
I
.
.
, .
0.78 3.13 12.5
I 50
200
800>800
EIC ( pg/nl ) resistance plasmids on antibacterial activity of cephalosporins FIG. 1. Influence of introduced ampicillin against E. coli CSH2. Solid bars represent the MICs of the antibiotics against the plasmid-free strain of E. coli CSH2; open circles indicate the distribution ofMICs for the plasmid-possessing strains of E. coli CSH2.
drolysis of cephaloridine for cephalosporinase and of penicillin G for penicillinase. FK 749 was extremely stable against all types of,-lactamases, as were the two cephamycin derivatives, cefmetazole and cefoxitin (Table 2). On the other hand, cefotiam was hydrolyzed at significant rates by all of the class I cephalosporinases and was also appreciably susceptible to some of the penicilhinases, including R-plasmid-mediated enzyme (class III), as compared with other cepha-
losporins. Cefamandole and cefoperazone were hydrolyzed at substantial rates by most of the type I cephalosporinases, the former by classes Ia (1), Ib, and Ic and the latter by classes Ia (1), Ic, and Id. Both cefamandole and cefoperazone were markedly hydrolyzed by class III and IV peniclinases at rates similar to those for cephaloridine. Cefuroxime was as stable as FK 749 to most classes of f8-lactamases, but class Ic enzymes, which possesses a broad spectrum of
552
ANTIMICROB. AGENTS CHEMOTHER.
KOJO ET AL.
TABLE 2. Stability of FK 749 to /-lactamase Relative rate of hydrolysis by each class of ,B-lactamase^ Antibiotic' Ia (1)
FK749 Cefotiam Cefamandole
Ia (2)
lb
1.3 1.2 3.0 18.5 49.7 53 2.1 22 83.7 1.0 0.2 0.9 Cefuroxine
Vol. 16, No. 5
0066-4804/79/11-0549/00$02.00/0
Antibacterial Activity of Ceftizoxime (FK 749), a New Cephalosporin, Against Cephalosporin-Resistant Bacteria, and Its Stability to fi-Lactamase HITOSHI KOJO,' MINORU NISHIDA,I SACHIKO GOTO,2 AND SHOGO KUWAHARA2 Research Laboratories, Fujisawa Pharmaceutical Co., Ltd. Osaka,'and Department of Microbiology, Toho University, School of Medicine, Tokyo,2 Japan Received for publication 30 July 1979
Antibacterial activity of FK 749 against ampicillin-resistant clinical isolates of Escherichia coli was compared with those of other newly developed cephalosporins. FK 749 was the most active against strains possessing R-plasmids specifying ampicillin resistance and those whose resistance was chromosomally determined. The susceptibility of ampicillin-susceptible E. coli to FK 749 was not decreased by transduction of ampicillin resistance-specifying plasmids. However, most of the transconjugants acquired a high level of resistance to cefoperazone and cefamandole and a moderate level to cefotiam. FK 749 was highly stable to both penicillinase- and cephalosporinase-type ,8-lactamases, including R-plasmid-mediated f8-lactamase. Its level of resistance to ,B-lactamases was comparable to those of cefoxitin, cefmetazole, and cefotaxime, slightly superior to that of cefuroxime, and much superior to those of cefotiam, cefamandole, and cefoperazone.
Cephalosporin antibiotics are superior to other families of antibiotics in their low toxicity and significant antibacterial activity against a large number of gram-positive and -negative bacteria. However, the following two recent situations are of great concern for the antibacterial activity of cephalosporins against gram-negative bacteria. The first is the gradual increase of noncephalosporin-susceptible gram-negative species among clinical isolates, such as Serratia, Enterobacter, Citrobacter, Proteus, and Pseudomonas (3). The other is the elevation of the level of cephalosporin resistance mediated by plasmids specifying fl-lactamase, resulting in the increase of moderately cephalosporin-resistant strains in Enterobacteriaceae including Escherichia coli and Klebsiella pneumoniae (2, 5, 6). This report describes the antibacterial activity of FK 749, a cephalosporin derivative newly developed in our research laboratories (4), against the above-mentioned cephalosporin-resistant bacteria, especially against R-plasmidpossessing strains. This report also describes the stability of FK 749 to /3-lactamases. MATERIALS AND METHODS Bacterial strains. E. coli CSH2 (naF), 17 strains of E. coli CSH2 harboring R-plasmids specifying ampicillin resistance (Apr), and 12 chromosomally ampicillin-resistant clinical isolates of E. coli were kindly provided by T. Yokota of Juntendo University. Twelve clinical isolates of E. coli possessing R-plasmids determining ampicillin resistance were selected from the
culture collection of our laboratory. Ten fl-lactamresistant strains were selected among the clinical isolates of the following species and used for preparation of ,B-lactamases: Serratia marcescens, Enterobacter cloacae, E. coli, Proteus vulgaris, Pseudomonas aeruginosa, K. pneumoniae, Proteus mirabilis, and Staphylococcus aureus. Antibiotics. The antibiotics used in this study included ceftizoxime (FK 749; Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan), cefotiam (SCE 963; Takeda Chemical Industries, Ltd., Osaka, Japan), cefamandole (Eli Lilly & Co., Indianapolis, Ind.), cefuroxime (Glaxo Research Ltd., Greenford, England), cefotaxime (HR 756; Hoechst-Roussel, Frankfurt, West Germany), cefmetazole (Sankyo Co., Ltd., Tokyo, Japan), cefoxitin (Merck Institute for Therapeutic Research, Rahway, N.J.), cefoperazone (T-1551; Toyama Chemical Co., Toyama, Japan), cephaloridine (Eli Lilly), cefazolin (Fujisawa), penicillin G (Fujisawa), and ampicillin (Beecham Pharmaceuticals, Betchworth, England). Antibiotic susceptibility testing. The minimal inhibitory concentrations (MICs) of test antibiotics were determined by the agar dilution method with the use of a multipoint inoculator (1). The inoculum was undiluted overnight culture in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.). After incubation at 37°C for 20 h, the lowest concentration that inhibited macroscopic colonial growth was regarded as the MIC. Preparation of 8-lactamase. The cells were grown at 37°C in heart infusion broth to which penicillin G was added as an inducer. After overnight incubation, the cells were harvested by centrifugation, washed once, and suspended in 0.067 M potassium phosphate buffer (pH 7.0). The cell suspensions were sonicated at 20 kilocycles for 20 min. After cellular 549
550
KOJO ET AL.
debris was removed by centrifugation, the supernatant subjected to gel filtration on a Sephadex G100 column. The column was equilibrated with 0.067 M phosphate buffer (pH 7.0) and eluted with the same buffer. The enzyme fractions were pooled and stored at -200C. Assay of ,-lactamase activity. ,B-Lactamase activity was determined with a Hitachi 200-20 spectrophotometer equipped with a thermostatted cell holder. The enzyme was mixed in a 1-cm quartz cuvette with 150 jug of substrate and 200 itmol of phosphate buffer (pH 7.0) to make a final volume of 3.0 ml and was then incubated at 370C. The rate of hydrolysis of the ,Blactam ring was followed by the change in absorption at 240 nm for penicillin and at 260 nm for cephalosporins (8). The relative initial rate of hydrolysis was expressed as a percentage compared to the hydrolysis of cephaloridine for cephalosporinase and of penicillin G for penicillinase.
was
ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Antibacterial activity of FK 749 against ampicillin-resistant strains of E. colia Mean MIC (pg/mi) Antibiotic
R+ E. coli C+ E. coli FK 749 2.1 1.7 Cefotiam 5.6 9.4 Cefamandole 50 178 Cefuroxime 28 40 Cefotaxime 3.5 11 Cefmetazole 40 50 11 Cefoxitin 7.0 >800 >800 Cefoperazone 25 47 Cefazolin a Twelve each of R+ and C+ strains were used; R+, R-plasmid-harboring strains (clinical isolates) and C+, chromosomally resistant strains (clinical isolates). Incubation: heart infusion agar, 37°C, 18 to 20 h. Assay: 108 colony-forming units per ml; stamp method.
RESULTS
Antibacterial activity of FK 749 against ampicillin-resistant clinical isolates of E. coli Ampicillin-resistant clinical isolates of E. coli
were divided between those whose resistance was chromosomal and those whose resistance was mediated by an R-plasmid. The anti-
bacterial activity of FK 749 against those strains is shown in comparison with those of newly developed cephalosporin and cephamycin derivatives (Table 1). FK 749 was the most effective against both types ofresistant strains, with mean MICs of 2.1 ug/ml for R-plasmid-harboring strains and 1.7 pg/ml for chromosomally resistant strains. The mean MICs of FK 749 against the resistant strains were the same as those of the susceptible strains (4). Meanwhile, the other cephalosporin and cephamycin derivatives were 2 to 25 times less effective than FK 749 against R-plasmid-harboring strains and 5 to 100 times less effective against chromosomally resistant strains. Influence of R-plasmids encoding ampicillin resistance on the antibacterial activity of FK 749. The net influence of R-plasmids encoding ampicillin resistance on the antibacterial activities of cephalosporin derivatives was assessed by comparing the activity of cephalosporins against E. coli CSH2 to that against strains of E. coli CSH2 possessing R-plasmids. Strains possessing R-plasmids were constructed by conjugal transfer of R-plasmids from clinical isolates of E. coli (8 strains) and of K. pneumoniae (9 strains) to E. coli CSH2. Figure 1 shows the MICs of cephalosporins against E. coli CSH2 and the distribution of the MICs against 17 strains of E. coli CSH2 harboring Apr R-plasmids. Of the nine cephalosporins tested (FK 749, cefotiam, cefamandole, cefuroxime, cefotaxime, cefmetazole, cefoxitin, cefoper-
azone, and cefazolin), the antibacterial activity of cefoperazone was the most influenced by the R-plasmids, followed by that of cefamandole and cefotiam. As shown in Fig. 1, 65% of the plasmidharboring strains of E. coli CSH2 were resistant to 400 pg and above of cefoperazone and cefamandole per ml, whereas the MICs of cefoperazone and cefamandole against plasmid-free E. coli CSH2 were 1.56 and 3.13 pLg/ml, respectively. The MICs of cefotiam against the plasmid-possessing strains ranged widely from 1.56 to 100lg/ml, whereas cefotiam inhibited the plasmid-free strains at 1.56 pg/ml. Meanwhile, the effect of the R-plasmids on the antibacterial activities of cefuroxime, cefotaxime, and cephamycins (cefoxitin and cefmetazole) was notably less, although their activities were reduced twoto fourfold in the presence of the R-plasmids. In contrast, the MICs of FK 749 against strains possessing the Apr R-plasmids were shown to coincide with its MIC against the plasmid-free strain. This result clearly indicates that the antibacterial activity of FK 749 is not reduced by R-plasmids specifying ampicillin resistance. Stability of FK 749 to f-lactamase. Stability of FK 749 to ,B-lactamase was compared with that of the nine ,B-lactam antibiotics cefotiam, cefamandole, cefuroxime, cefmetazole, cefoxitin, cefoperazone, cefazolin, cephaloridine, and penicillin G. According to the classification proposed by Richmond and Sykes (7), nine distinct types of f4-lactamases were selected from clinical isolates of gram-negative bacilli and used for assay, together with the enzyme from ampicillin-resistant S. aureus. Class I enzymes are cephalosporinases, whereas class IH, III, IV, and V, and S. aureus enzymes are termed penicillinases. The relative initial rates of hydrolysis of the cephalosporins were expressed in percentage of hy-
CEFTIZOXIME (FK 749) AGAINST RESISTANT BACTERIA
VOL. 16, 1979 16
Cefnetazole
FK749
I
12
8 4 0
9%-j
-A.
I I I I.
Cefotian
12
551
KI I I
_ ~
I
I
Cefoxitin
8 4
0
I
9j
I
I
.E I
Ia
i
Cefoperazane
Cefamandole
12
8 4
0
12
I_777i K
I
I
|||| l
_Cefuroxcime
Cefazolin
a
8
II_
4
0
a
I
I
t- I
I
1 IX
I
I
"/
II
I
Aopicillin
Cefotaxime
12 0
8 4
0
I
0.2 0.78 3.13 12.5
50
200
800>8co
0.2
I
.
.
, .
0.78 3.13 12.5
I 50
200
800>800
EIC ( pg/nl ) resistance plasmids on antibacterial activity of cephalosporins FIG. 1. Influence of introduced ampicillin against E. coli CSH2. Solid bars represent the MICs of the antibiotics against the plasmid-free strain of E. coli CSH2; open circles indicate the distribution ofMICs for the plasmid-possessing strains of E. coli CSH2.
drolysis of cephaloridine for cephalosporinase and of penicillin G for penicillinase. FK 749 was extremely stable against all types of,-lactamases, as were the two cephamycin derivatives, cefmetazole and cefoxitin (Table 2). On the other hand, cefotiam was hydrolyzed at significant rates by all of the class I cephalosporinases and was also appreciably susceptible to some of the penicilhinases, including R-plasmid-mediated enzyme (class III), as compared with other cepha-
losporins. Cefamandole and cefoperazone were hydrolyzed at substantial rates by most of the type I cephalosporinases, the former by classes Ia (1), Ib, and Ic and the latter by classes Ia (1), Ic, and Id. Both cefamandole and cefoperazone were markedly hydrolyzed by class III and IV peniclinases at rates similar to those for cephaloridine. Cefuroxime was as stable as FK 749 to most classes of f8-lactamases, but class Ic enzymes, which possesses a broad spectrum of
552
ANTIMICROB. AGENTS CHEMOTHER.
KOJO ET AL.
TABLE 2. Stability of FK 749 to /-lactamase Relative rate of hydrolysis by each class of ,B-lactamase^ Antibiotic' Ia (1)
FK749 Cefotiam Cefamandole
Ia (2)
lb
1.3 1.2 3.0 18.5 49.7 53 2.1 22 83.7 1.0 0.2 0.9 Cefuroxine
