Comparative in Vitro Studies of Ro 13-9904, a New Cephalosporin Derivative T
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1981, p. 435-442 Vol. 19, No. 3 0066-4804/81/030435-08$02.00/0 Comparative In Vitro Studies of Ro 13-9904, a New Cephalosporin Derivative T. C. EICKHOFF* AND J. EHRET Department ofMedicine, Denver General Hospital, and Division ofInfectious Diseases, Department of Medicine, University of Colorado School ofMedicine, Denver, Colorado 80262 The in vitro activity of Ro 13-9904, a new cephalosporin derivative, was compared with the activities of cephalothin, cefamandole, cefoxitin, cefotaxime, and moxalactam against 591 clinical isolates of gram-negative and gram-positive organisms. The spectra of activity and potency of Ro 13-9904 and cefotaxime were quite similar; they were the most active agents against Enterobacteriaceae, Streptococcus pyogenes, Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis. Moxalactam was only slightly less active against these organisms. Ro 13-9904, cefotaxime, and moxalactam were approximately equal in activity against Pseudomonas aeruginosa; concentrations of 50 to 100 ,Ig/ml inhibited over 90% of the strains tested. Cefamandole and cephalothin were the most active drugs tested against staphylococci. Moxalactam demonstrated the highest intrinsic activity against Bacteroides fragilis; a concentration of 1.6 yg/ ml inhibited over 50% of the strains. All six of the antibiotics were essentially inactive against group D streptococci. The action of all of the antibiotics was bactericidal, with minimal bactericidal concentrations generally being no more than twofold greater than miniimal inhibitory concentrations. The only exception to this was found when large inocula of Staphylococcus aureus were tested. Increased inoculum size generally sharply reduced the activity of Ro 13-9904, cefotaxime, and moxalactam against Enterobacteriaceae and P. aeruginosa. The chemotherapy of serious bacterial infec- The purpose of this study was to compare the tions, particularly those which are hospital ac- in vitro activity of Ro 13-9904 with the activities quired, is a continuing clinical challenge. Orga- of other drugs representing the three major gen- nisms such as Serratia, Acinetobacter, Pseu- erations of cephalosporin development. The first domonas cepacia, and others once thought to generation was represented by cephalothin, the cause disease only rarely, if at all, are now rec- second was represented by cefamandole and ce- ognized as common pathogens. The need for foxitin (a cephamycin antibiotic), and the third antimicrobial agents with broad spectra of activ- was represented by Ro 13-9904, cefotaxime, and ity and resistance to the /3-lactamases of gram- moxalactam. negative organisms associated with nosocomial infection has motivated research resulting in the MATERLALS AND METHODS development of a great number ofcephalosporin derivatives. Since the discovery of the fungus Antibiotics. Standard reference powders were kindly provided as follows: Ro 13-9904 by Hoffinann- Cephalosporium acremonium in Sardinian sew- La Roche, Inc.; cefotaxime by Hoechst-Roussel Phar- age in 1945 (1), the cephalosporins have evolved maceutical, Inc.; cefoxitin sodium by Merck Sharp & into a complex group of antibiotics with signifi- Dohme; and cefamandole lithium, sodium cephalo- cant differences in potency and antibacterial thin, and moxalactam by Eli Lilly & Co. spectrum. Ro 13-9904 (Fig. 1) is a new product Bacteria. A total of 591 strains of bacteria were of the chemical manipulation of side chains tested, with the following distribution: 50 Escherichia joined to the 7-amino-cephalosporanic acid nu- coli, 50 Klebsiella, 36 Enterobacter, 23 Serratia, 50 cleus. It has been reported to be highly active Proteus rettgeri, 43 Proteus mirabilis, 34 Salmonella, against common gram-negative organisms and 10 Shigella, 50 P. aeruginosa, 50 Staphylococcus au- to inactivation reus, 50 Staphylococcus epidermidis, 50 group D relatively resistant by cephalo- streptococci, 15 Streptococcus pyogenes, 15 Strepto- sporinases; preliminary data also suggest that coccus pneumoniae, 15 Haemophilus influenzae, 15 the expanded spectrum of activity includes Neisseria gonorrhoeae, 15 Neisseria meningitidis, Pseudomonas aeruginosa, Serratia marcesens, and 20 Bacteroides fragilis strains. These organisms and most strains ofEnterobacter (compound Ro were isolated from clinical sources from 1977 to 1979 13-9904 preliminary data, Hoffmann-La Roche, and identified in the Clinical Microbiology Laboratory, Inc., 1979). University Hospital, University of Colorado Health 435 436 EICKHOFF AND EHRET ANTIMICROB. AGENTS CHEMOTHER. NH2 strains each ofH. influenzae and N. gonorrhoeae were S N tested, using inocula of 105 and 107 colony-forming H H CHEON units per ml prepared by taking organisms grown C-CO-NHHC3-N-NK Na overnight on GC agar, suspending them in Mueller- N 0-N - Ss- }t Hinton broth, and diluting the culture 10-2 and 10-4 in OCH3 CH2 N O Mueller-Hinton broth. production. The production of fi- COQONaS® lactamasef-Lactamasewas assessed with the chromogenic cepha- C18H,8O7N8S3Na2 MW 598.5 losporin, nitrocefm (Glaxo Laboratories) (16). Media effect. The effect of media composition on FIG. 1. Chemical structure ofRo 13-9904. the activity of Ro 13-9904 was evaluated by comparing the MICs of 50 strains tested in Trypticase soy broth with those obtained in Mueller-Hinton broth. Ten strains each of S. aureus, S. epidermidis, E. coli, and P. aeruginosa were studied. Renepter.e. Klebsiella,pH effect. The same strains (see above) were used Susceptibilityetesting: brothMICs. The minimal to study the effect of altering the pH of the media. ihbitory concentrations (MICs) of the drugs againstThpHvleoMulrHionbthwsajtd S. aureus, S. epidermidis, group D streptococci, P. to va ues of 6,7, and 8 with either 1 N sodium hydrox- aeruginosa, and the various genera of Enterobacteri- ide or 1 N hydrochloric acid. Hydrogen ion concentra- aceae were determined by a microtiter broth dilution tion was determined on a Corning model 12 research technique. Serial twofold dilutions of freshly prepared pH meter. antibiotics were made in Mueller-Hinton broth (Difco); group D streptococci were tested in Trypti- case soy broth (BBL Microbiology Systems [BBL]). RESULTS Overnight broth ciltures of the organismwere diluted The comparative in vitro activities on Ro 13- 10-4, and 0.05 ml was added to 0.05 ml of the diluted 9904 and the other five antibiotics studied are antibiotic in microtiter plates (Dynatech Laborato- summarized in Table 1. The spectrum of activity ries). The final inoculum contained approximately 106 and potency of Ro 13-9904 was almost identical colony-forming units per ml. After overnight incuba- to that of cefotaxime; they were the most active tion at 35°C in ambient air, MIC was determined as ts exceptagain st the lowest concentration of antibiotic in which there agentstested,tested, except against staphylococci,aciveen- was no visible growth. The miniimal bactericidal con- terococci, B. fragilis, and P. aeruginosa. Against centrations (MBCs) were determined by using an ad- Enterobacteriaceae, Ro 13-9904 and cefotaxime aptation of the Steers et al. replicator (22) to subcul- were most active, followed by moxalactam, cef- ture approximately 0.003 ml from each well of the amandole, cefoxitin, and cephalothin, in that microtiter plates. All subcultures were incubated over- order. Cefoxitin was more active than cefaman- night at 35°C in ambient air, MBC was determined as dole when tested against Serratia. the lowest concentration of antibiotic yielding no The susceptibility pattern of P. aeruginosa growth or only one colony. Agar dilution method. The agar dilution tech- demonstrated a sharp dlstinction between nique, employing the inocula-replicating method of the third-generation cephalosporins and older Steers et al. (22), was used to determine the MICs for agents. The geometric mean MICs of Ro 13- S. pneumoniae, S. pyogenes, N. meningitidis, N. gon- 9904, cefotaxime, and moxalactam clustered orrhoeae, H. influenzae, and B. fragilis. Strains of around 21 ,ug/ml; a concentration of 12.5 ,ug of Streptococcaceae were tested on Mueller-Hinton agar Ro 13-9904 or moxalactam per ml inhibited 48% supplemented with 5% defibrinated sheep blood; Neis- of the strains, and the same concentration of seriaceae and Haemophilus were tested for suscepti- cefotaxime inhibited 52% of the strains. MICs of bility on GC medium base (Difco) supplemented with cefamandole, cefoxitin, and cephalothin were 1% IsoVitaleX (BBL). The susceptibility of strains ofIg/ml.uniformly higher than 100 Bacteroides was tested on brain heart infusion agar than (BBL) supplemented with 1% hemin-vitamin K (Scott Against staphylococci,hlocci cefamandolecephland ceph- Laboratories, Inc.). The replicator delivered 0.003 ml alothin were significantly more active than the of inoculum containing approximately 106 colony- other drugs. Cefotaxime, Ro 13-9904, and cefox- forming units per ml. S. pneumoniae and S. pyogenes itin were intermediate in activity, and moxalac- test plates were incubated overnight at 35°C in am- tam was the least active. bient air; N. meningitidis, N. gonorrhoeae, and H. Of the S. aureus strains tested, 29 were f- influenzae test plates were incubated in a 10% C02 lactamase positive by the nitrocefin chromo- atmosphere at 350C for 24 h; B. fragilis test plates genic test; the geometric mean MIC of Ro 13- were incubated in a GasPak jar (BBL) at 350C for 24 994 for these strains was 3.3 The re- h. 9904 as 3.3 jig/ml. Inoculum effect. The effect