Cephalosporin, Against Cephalosporin-Resistant Bacteria, and

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1979, P. 59-553 Vol. 16, No. 5 66-/79/11-59/$2./ Antibacterial Activity of Ceftizoxime (FK 79), 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 ofmedicine, Tokyo,2 Japan Received for publication 3 July 1979 Antibacterial activity of FK 79 against ampicillin-resistant clinical isolates of Escherichia coli was compared with those of other newly developed cephalosporins. FK 79 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 79 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 79 was highly stable to both penicillinase- and cephalosporinase-type,-lactamases, including R-plasmid-mediated f-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 79, a cephalosporin derivative newly developed in our research laboratories (), against the above-mentioned cephalosporin-resistant bacteria, especially against R-plasmidpossessing strains. This report also describes the stability of FK 79 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 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 79; 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 2 h, the lowest concentration 59 that inhibited macroscopic colonial growth was regarded as the MIC. Preparation of -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.67 M potassium phosphate buffer (ph 7.). The cell suspensions were sonicated at 2 kilocycles for 2 min. After cellular Downloaded from http://aac.asm.org/ on January 15, 219 by guest

55 KOJO ET AL. debris was removed by centrifugation, the supernatant was subjected to gel filtration on a Sephadex G1 column. The column was equilibrated with.67 M phosphate buffer (ph 7.) and eluted with the same buffer. The enzyme fractions were pooled and stored at -2C. Assay of,-lactamase activity.,b-lactamase activity was determined with a Hitachi 2-2 spectrophotometer equipped with a thermostatted cell holder. The enzyme was mixed in a 1-cm quartz cuvette with 15 jug of substrate and 2 itmol of phosphate buffer (ph 7.) to make a final volume of 3. ml and was then incubated at 37C. The rate of hydrolysis of the,blactam ring was followed by the change in absorption at 2 nm for penicillin and at 26 nm for cephalosporins (). 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. RESULTS Antibacterial activity of FK 79 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 antibacterial activity of FK 79 against those strains is shown in comparison with those of newly developed cephalosporin and cephamycin derivatives (Table 1). FK 79 was the most effective against both types of resistant 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 79 against the resistant strains were the same as those of the susceptible strains (). Meanwhile, the other cephalosporin and cephamycin derivatives were 2 to 25 times less effective than FK 79 against R-plasmid-harboring strains and 5 to 1 times less effective against chromosomally resistant strains. Influence of R-plasmids encoding ampicillin resistance on the antibacterial activity of FK 79. 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 ( 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 79, cefotiam, cefamandole, cefuroxime, cefotaxime, cefmetazole, cefoxitin, cefoper- ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Antibacterial activity of FK 79 against ampicillin-resistant strains of E. colia Mean MIC (pg/mi) Antibiotic R+ E. coli C+ E. coli FK 79 2.1 1.7 Cefotiam 5.6 9. Cefamandole 5 17 Cefuroxime 2 Cefotaxime 3.5 11 Cefmetazole 5 Cefoxitin 7. 11 Cefoperazone > > Cefazolin 25 7 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, 1 to 2 h. Assay: 1 colony-forming units per ml; stamp method. azone, and cefazolin), the antibacterial activity of cefoperazone was the most influenced by the R-plasmids, followed by that ofcefamandole and cefotiam. As shown in Fig. 1, 65% of the plasmidharboring strains of E. coli CSH2 were resistant to 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 1lg/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 79 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 79 is not reduced by R-plasmids specifying ampicillin resistance. Stability of FK 79 to f-lactamase. Stability of FK 79 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 f-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- Downloaded from http://aac.asm.org/ on January 15, 219 by guest

VOL. 16, 1979 CEFTIZOXIME (FK 79) AGAINST RESISTANT BACTERIA 551 16 I 9%-j FK79 Cefnetazole I I I I. -A. K I I I I.E I Cefotian I I I 9j i Ia Cefamandole I_777i K I I _Cefuroxcime a I I t- I I I 1 IX I I Cefotaxime _~ la II_ II "/ Cefoxitin Cefoperazane Cefazolin Aopicillin I I..,. I.2.7 3.13.5 5 2 >co.2.7 3.13.5 5 2 > EIC ( pg/nl ) FIG. 1. Influence of introduced ampicillin resistance plasmids on antibacterial activity of cephalosporins 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. Downloaded from http://aac.asm.org/ on January 15, 219 by guest drolysis of cephaloridine for cephalosporinase and of penicillin G for penicillinase. FK 79 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 cephalosporins. 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 79 to most classes of f-lactamases, but class Ic enzymes, which possesses a broad spectrum of

552 KOJO ET AL. ANTIMICROB. AGENTS CHEMOTHER. Antibiotic' TABLE 2. Stability of FK 79 to /-lactamase Relative rate of hydrolysis by each class of,b-lactamase^ Ia (1) Ia (2) lb Ic Id II III IV V S. aureus FK79 1.3 1.2 3..6.7..7.6. <1. Cefotiam 1.5 9.7 53 19 35 1.2.2.7 1. <1.3 Cefamandole 3.7 2.1 22 255 5.7 <.2 13.7 26.5 2. 9.3 Cefuroxine 1..2.9 163 1.9.6.2 1.5.5 <.6 Cefmetazole <.1. <.3 <1.1 <1.1 <.3.2 <1..3 <1.6 Cefoxitin <.1 2.5 1.5.6 <. <.1.3.3.3 <.5 Cefoperazone 65.2 3.1 6.3 176 3.7.2.9 1. 1.3 6.1 Cefazolin 9.5 11 9 392 16.1.5 15.2. 3. Cephaloridine 1 1 1 1 1. 1.3 32.6 6.7.5 PeniciUin G <.1 16. 13 5.7. 1 1 1 1 1 a Substrate concentration: 5 ug/ml. 'Relative initial velocity: cephaloridine = 1 for cephalosporinase, penicillin G = 1 for penicilhinase. Ultraviolet assay, 37 C,.67 M potassium phosphate buffer (ph 7.). Classes and sources of,b-lactamase were: Ia (1), S. marcescens 7; Ia (2), E. cloacae 91; Ib, E. coli 35 (C+); Ic, P. vulgaris 9; Id, P. aeruginosa 11 (C+); II, P. mirabilis 133; III, E. coli 1 (R+); IV, K. pneumoniae 13; V, P. aeruginosa 7 (R+); S. aureus, S. aureus 35. substrate profile, hydrolyzed this antibiotic rapidly. FK 79 was extremely stable to class I enzymes from the non-cephalosporin-susceptible gram-negative species such as Enterobacter spp., Citrobacter freundii (not shown), S. marcescens, and P. aeruginosa, as well as chromosomally resistant E. coli. FK 79 showed excellent antibacterial activity against these organisms. Furthermore, the stability of FK 79 to R-plasmid-coded,B-lactamase was shown to be excellent. This result explains the fact that the antibacterial activity of FK 79 was scarcely reduced in the presence of Apr R-plasmids. DISCUSSION R-plasmids specifying ampicillin resistance confer a high level of resistance to the penicillins on gram-negative bacterial species. However, there has been relatively little attention to this type of R-plasmids from the viewpoint of cephalosporin resistance, since only some of these plasmids could confer moderate levels of resistance to the cephalosporins. However, it is noteworthy that the frequency of isolation of strains possessing Apr R-plasmids conferring moderate levels of resistance to the cephalosporins has been increasing recently. The explanation for this phenomenon would seem to be that the level of production of plasmid-mediated,b-lactamases has been elevated, together with the fact that the Apr R-plasmids have spread extensively among the enteric bacteria. A series of newly developed cephalosporins were tested for their antibacterial activities against Apr R-plasmid-containing strains in this study. The results show that the antibacterial activities of cefoperazone, cefamandole, and cefotiam were markedly reduced in the presence of Apr R-plasmids, to the same level as the existing cephalosporins such as cephaloridine. Notably, the antibacterial activities of cefoperazone and cefamandole were reduced more than that of cefazolin. Meanwhile, the antibacterial activities of the methoxyimino cephalosporin derivatives, as well as the cephamycin derivatives, were much less influenced by the Apr R-plasmids. Particularly, the antibacterial activity of FK 79 was found to be completely free from the plasmids. Among the above-mentioned antibiotics, FK 79 was the most active against Apr R-plasmid-containing strains of E. coli. The exceptional stability of FK 79 to R-plasmid-mediated,B-lactamase is due to this property against the Apr R-plasmids. However, a discrepancy was observed with cefoxitin between its extreme stability to the plasmid-mediated enzyme and the slight decrease in its antibacterial activity against the plasmid-possessing strains. This might result from its comparatively poor ability to penetrate through the bacterial outer membrane (H. Kojo, Y. Shigi, and M. Nishida, unpublished data). The cephamycin derivatives showed excellent stability to,b-lactamases from non-cephalosporin-susceptible species such as Enterobacter spp., C. freundii, S. marcescens, and indole-positive Proteus spp., whose resistance to the cephalosporins was assumed to originate principally from their cephalosporinases. Against these species, however, the cephamycins were not as active as expected from their stability to the cephalosporinases. Meanwhile, FK 79 was similar to the cephamycins in its extreme stability to species-specific,b-lactamases, but it was quite different from the cephamycins in that it showed a significant antibacterial activity against the non-cephalosporin-susceptible species (). The discrepancy between the antibacterial activities Downloaded from http://aac.asm.org/ on January 15, 219 by guest

VOL. 16, 1979 CEFTIZOXIME (FK 79) AGAINST RESISTANT BACTERIA 553 of FK 79 and cephamycins against these species might be elucidated by comparative studies on their ability to penetrate through the outer membrane and their activities against the target enzymes of these species. LITERATURE CITED 1. Committee on MIC of Japan Society of Chemotherapy. 1975. Recommendation on the determination of minimal inhibitory concentration (MIC). Chemotherapy (Tokyo) 23:1. 2. Datta, N., and P. Kontomichalou. 1965. Penicillinase synthesis controlled by infectious R factors in Enterobacteriaceae. Nature (London) 2:239-21. 3. Goto, S. 1976. Gram-negative bacilli: its resistance and the superinfection. Jpn. J. Bacteriol. 31:32.. Kamimura, T., Y. Matumoto, N. Okada, Y. Mine, M. Nishida, S. Goto, and S. Kuwahara. 1979. Ceftizoxime (FK 79), a new parenteral cephalosporin: in vitro and in vivo antibacterial activities. Antimicrob. Agents Chemother. 16:5-5. 5. Matsumoto, F., M. Ohmori, and K. Shiba. 1976. The drug sensitivities of gram-negative bacilli isolated from urinary tract infections from 1972 with particular reference to cephalosporins and aminoglycosides. Chemotherapy (Tokyo) 2:131-135. 6. Ooka, T., H. Hashimoto, and S. Mitsuhashi. 197. Comparison of penicilhinase produced by R factors isolated from ampicillin-resistant gram-negative bacteria. Jpn. J. Microbiol. 1:3-. 7. Richmond, M. H., and R. B. Sykes. 1973. The fl-lactamase of gram-negative bacteria and their possible physiological role. Microb. Physiol. 9:31-5.. Samuni, A. 1975. A direct spectrophotometric assay and determination of Michaelis constants for the,b-lactamase reaction. Anal. Biochem. 63:17-26. Downloaded from http://aac.asm.org/ on January 15, 219 by guest