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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 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, , , , 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 ; 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 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 ), 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 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 of inoculum size was maining 21 strainsstramswerew either negativenegaTheor re-only evaluated by testing 10 strains each of S. aureus, S. weakly positive for fl-lactamase production; epidermidis, P. aeruginosa, and each genus of Enter- their geometric mean MIC was 3.7 ,ug/ml. The obacteriaceae at a 102 dilution of an overnight broth production of ,6-lactamase, therefore, seemed to culture, as well as at the standard 10-4 dilution. Fifteen have no significant effect on MICs of Ro 13-9904 TABLE 1. Comparative in vitro activity ofsix cephalosporin drugs MIC (pg/mi) Organism (no.(no.of strainsofstrainstemtd)tested)Antibiotic~Range For % of isolates: 50% 90% S. aureus (50) Ro 13-9904 3.1-12.5 3.1 6.3 Cefotaxime 1.6-6.3 1.6 1.6 Moxalactam 3.1-25 6.3 6.3 Cefamandole 0.2-1.6 0.4 0.8 Cefoxitin 1.6-12.5 3.1 3.1 Cephalothin 0.2-0.8 0.4 0.4 S. epidermidis (50) Ro 13-9904 0.2->100 3.1 25 Cefotaxime 0.2->100 0.8 12.5 Moxalactam 6.3->100 12.5 100 Cefamandole 0.1-25 0.4 1.6 Cefoxitin 0.2-50 3.1 25 Cephalothin 0.1-50 0.2 0.8 Group D streptococci (50) Ro 13-9904 12.5->100 >100 >100 Cefotaxime 1.6->100 >100 >100 Moxalactam >100 >100 >100 Cefamandole 25->100 25 50 Cefoxitin 50->100 >100 >100 Cephalothin 12.5->100 25 50 S. pyogenes (15) Ro 13-9904 0.0125-0.025 0.0125 0.025 Cefotaxime 0.0125 0.0125 0.0125 Moxalactam 0.4-1.6 0.8 0.8 Cefamandole 0.05 0.05 0.05 Cefoxitin 0.4-0.8 0.8 0.8 Cephalothin 0.1 0.1 0.1 S. pneumoniae (15) Ro 13-9904 0.006-0.1 0.0125 0.05 Cefotaxime 0.006-0.1 0.0125 0.0125 Moxalactam 0.02-6.3 0.8 1.6 Cefamandole 0.006-0.8 0.05 0.2 Cefoxitin 0.2-25 0.8 1.6 Cephalothin 0.006-0.8 0.05 0.2 H. influenzae (15) Ro 13-9904 0.003-0.0125 0.003 0.0125 Cefotaxime 0.006-0.025 0.0125 0.025 Moxalactam 0.025-0.1 0.025 0.1 Cefamandole 0.02-3.1 0.4 1.6 Cefoxitin 1.6-12.5 3.1 12.5 Cephalothin 1.6-12.5 1.6 6.3 N. gonorrhoeae (15) Ro 13-9904 0.003-0.0125 0.003 0.0125 Cefotasime 0.003-0.05 0.003 0.05 Moxalactam 0.006-0.2 0.025 0.2 Cefamandole 0.2-3.1 0.2 3.1 Cefoxitin 0.2-1.6 0.8 1.6 Cephalothin 0.4-6.3 0.8 6.3 N. meningitidis (15) Ro 13-9904 0.003-0.006 0.003 0.003 Cefotaxime 0.003-0.0125 0.003 0.006 Moxalactam 0.003-0.025 0.006 0.0125 Cefamandole 0.006-0.4 0.05 0.1 Cefoxitin 0.025-0.4 0.4 0.4 Cephalothin 0.1-0.8 0.4 0.8 B. fragilis (20) Ro 13-9904 3.1->100 50 >100 Cefotaxime 3.1->100 50 >100 Moxalactam 0.4->100 1.6 100 Cefamandole 6.3->100 50 100 Cefoxitin 3.1-50 25 50 Cephalothin 12.5->100 50 >100 437 TABLE 1-Continued

MIC (pg/mi) (no. ofOrganismAtboistrain tested) Antbiotic For % of isolates: 50% 90% E. coli (50) Ro 13-9904 0.0125-0.4 0.05 0.1 Cefotaxime 0.0125-0.4 0.05 0.2 Moxalactam 0.05-0.4 0.1 0.2 Cefandole 0.2-50 0.8 12.5 Cefoxitin 1.6-50 3.1 6.3 Cephalothin 3.1->100 6.3 100 Klebsiella sp. (50) Ro 13-9904 0.0125-6.3 0.05 0.1 Cefotaxime 0.025-1.6 0.05 0.1 Moxalactam 0.05-0.4 0.1 0.2 Cefamandole 0.4->100 0.8 25 Cefoxtin 1.6->100 3.1 6.3 Cephalothin 1.6->100 3.1 50

Enterobacter sp. (36) Ro 13-9904 0.025-100 0.2 1.6 Cefotaxime 0.05-3.1 0.2 0.8 Mosalactam 0.05-6.3 0.1 0.4 Cefainandole 0.4->100 3.1 50 Cefoxitin 3.1->100 >100 >100 Cephalothin 12.5->100 >100 >100 Serratia sp. (23) Ro 13-9904 0.1-1.6 0.2 0.4 Cefotaxime 0.06-1.6 0.2 0.4 Moxalactam 0.1-0.8 0.2 0.4 Cefamandole 3.1->100 25 >100 Cefoxitin 6.3-100 12.5 100 Cephalothin 50->100 >100 >100 P. rettgeri (50) Ro 13-9904 0.003-1.6 0.0125 0.4 Cefotaxime 0.006-3.1 0.05 1.6 Moxalactam 0.05-1.6 0.1 0.4 Cefamandole 0.1-50 0.8 25 Cefoxtin 0.8-50 1.6 6.3 Cephalothin 3.1->100 100 >100 P. mirabilis (43) Ro 13-9904 0.003-0.025 0.006 0.006 Cefotaxime 0.0125-0.025 0.0125 0.025 Moxalactam 0.1-0.2 0.1 0.2 CefamandO1e 0.4-100 0.8 1.6 Cefoxitin 1.6-6.3 3.1 3.1 Cephalothin 1.6->100 3.1 6.3 Sabnonella sp. (34) Ro 13-9904 0.05-0.4 0.1 0.4 Cefotaxime 0.05-0.4 0.1 0.4 Moxalactam 0.1-0.4 0.1 0.2 Cefamandole 0.2-100 12.5 50 Cefoxitin 1.6-12.5 3.1 6.3 Cephalothin 1.6-100 25 50 Shigella sp. (10) Ro 13-9904 0.025-0.05 0.05 0.05 Cefotaxime 0.0125-0.05 0.025 0.05 Moxalactam 0.1-0.4 0.1 0.2 Cefamandole 0.2-12.5 0.4 0.8 Cefoxitin 1.6-6.3 3.1 3.1 Cephalothin 3.1-25 6.3 12.5 P. aerugmnosa (50) Ro 13-9904 1.6->100 25 50 Cefotaxime 0.8->100 12.5 100 Moxalactam 6.3-100 25 50 Cefamandole >100 >100 >100 Cefoxitin >100 >100 >100 Cephalothin >100 >100 >100

438 VOL. 19, 1981 COMPARATIVE STUDIES OF Ro 13-9904 439 against S. aureus. When S. epidermidis was negative strains. MICs of the f8-lactamase-neg- tested, however, the geometric mean MIC for ative strains were not significantly affected by the 26 ,8-lactamase-positive strains was 8 ,ug/ml, increased inoculum size. When the ,8-lactamase- as compared with 2 jig/ml for the 24 strains positive strains were tested, the geometric mean which were weakly positive or negative. MICs ofthe antibiotics increased as follows, with Ro 13-9904 and cefotaxime were the most the 2 log10 inoculum size increase: cefamandole, active agents against strains of S. pyogenes. Cef- 105 times, cephalothin, 22 times; Ro 13-9904, 5 amandole and cephalothin were intermediate in times; cefoxitin, 2.4 times; moxalactam, 1.3 activity, whereas cefoxitin and moxalactam were times; and cefotaxime, 0.9 times. the least active. There was little difference The effects of altering the media and the pH among the activities of Ro 13-9904, cefotaxime, on the activities of the antibiotics were also cephalothin, and cefamandole against S. pneu- evaluated. There were no significant differences moniae. Again, cefoxitin and moxalactam were between results obtained in Mueller-Hinton the least active, with concentrations of 1.6 ,g/ broth and those obtained in Trypticase soy ml required to inhibit 90% of the strains. None broth. The effects ofpH in the range of6 through of the antibiotics tested demonstrated signifi- 8 were for the most part slight and genus depen- cant activity against group D streptococci. dent. The most significant pH effect was ob- The strains of Haemophilus tested included served when strains of S. aureus were tested. As six ,B-lactamase-producing strains with ampicil- the pH was raised from 6 to 8, MICs increased. lin MICs ranging from 6.3 to 100 ,ug/ml. The Although this effect was apparent for each anti- third-generation cephalosporins, particularly Ro biotic, the size of the increase varied; the geo- 13-9904, demonstrated marked activity against metric mean MICs increased as follows: Ro 13- these organisms. MICs of Ro 13-9904, cefotax- 9904, 14-fold; cefotaxime, 4.7-fold; cefamandole, ime, and moxalactam were not appreciably in- 3.1-fold; and moxalactam, 2.4-fold. MICs for En- creased by ,8-lactamase production. terobacteriaceae were not significantly affected Two 13-lactamase-positive strains were in- by pH alteration. P. aeruginosa demonstrated cluded among the N. gonorrhoeae strains tested. a slight trend toward decreased MICs as the pH MICs of these strains were at the upper extreme increased. The most marked example of a de- of the range for each drug. The third-generation crease was found with Ro 13-9904; the geometric cephalosporins demonstrated excellent activity mean MIC for 10 strains of P. aeruginosa was against N. gonorrhoeae strains, whether they 30.8 jig/ml at pH 6 and 13.4 ,ig/ml at pH 8. were /?-lactamase positive or not. Activity against N. meningitidis was similarly excellent. The susceptibility pattern of B. fragilis was DISCUSSION unique in that moxalactam and cefoxitin were This study demonstrates the wide variations the most active of the cephalosporins. Ro 13- in spectrum and potency that now exist among 9904, cefotaxime, and cefamandole were inter- the cephalosporin antibiotics. As this and nu- mediate in activity, and cephalothin was the merous other studies (3, 9-11, 19, 24) have es- least active. High-level resistance was not nec- tablished, the first generation of cephalosporin essarily associated with f)-lactamase production, antibiotics, represented by cephalothin, have as measured by the chromogenic cephalosporin only limited activity against gram-negative ba- test. Nine strains exhibited resistance to all of cilli because of their susceptibility to attack by the cephalosporins, with moxalactam MICs of numerous ,B-lactamases. Their activity against 6.3 ,ug/ml or higher; six of these strains produced gram-positive cocci, with the exceptions of en- ,8-lactamase weakly or not at all. The remaining terococci and -resistant staphylo- three strains were strong ,8-lactamase producers. cocci, is generally excellent. Cephalothin and There was wide variation in inoculum effect cefamandole are the most active cephalosporins (Table 2). S. aureus was unique in that an in- against staphylococci and are known to resist creased inoculum size resulted in a sharp in- destruction by staphylococcal ,8-lactamase (4, 6). crease in the MBC/MIC ratio for most of the The second-generation cephalosporins, repre- cephalosporins tested. In general, the third-gen- sented by cefamandole and cefoxitin, expanded eration cephalosporins and cefamandole were the cephalosporin spectrum to include more most affected by increased inoculum size when gram-negative bacilli, as shown in this and other Enterobacteriaceae were tested. As has been studies (3, 9, 11, 12). This is due to their varying previously established (3, 17, 4, 12), cefoxitin was degrees of ability to resist hydrolysis by gram- affected very little by inoculum size. The 15 negative ,8-lactamases. Cefamandole, in this and strains of H. influenzae tested were composed other studies (3, 13), has been shown to have of 8 ,B-lactamase-positive and 7 f8-lactamase- good in vitro activity against gram-positive cocci 440 EICKHOFF AND EHRET ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Effect of inoculum size on geometric mean MICs and MBCs (g/ml) ofsix cephalosporin drugsa Geometric mean with inoculum size (colony-fonning units per ml)

Organism Antibiotic 105 10 MIC/ (no. of strains) AniotcMC (no.MIC MBC MICMBC/MIC MBC/ 10 mC S.cMrCR17MBC MIC MBC S. aureus (10) Ro 13-9904 4.1 5.1 1.2 4.4 75.8 17.2 1.1 Cefotaxime 1.8 1.8 1.0 2 28.8 14.8 1.1 Moxalactam 5.1 5.9 1.2 5.1 33.1 6.5 1.0 Cefamandole 0.61 0.61 1.0 1.7 5.5 3.2 2.8 Cefoxitin 2.9 2.9 1.0 4.4 12.5 2.8 1.5 Cephalothin 0.33 0.35 1.1 0.7 7.3 10.4 2.3 S. epidernidis (10) Ro 13-9904 3.6 4.8 1.3 5.9 8.3 1.4 1.6 Cefotaxime 1.2 1.6 1.3 2.2 3.2 1.5 1.8 Moxalactam 13.5 16.6 1.2 20.4 28.8 1.4 1.5 Cefamandole 0.37 0.43 1.2 0.85 0.98 1.2 2.3 Cefoxitin 3.6 4.5 1.3 5.5 6.7 1.2 1.5 Cephalothin 0.26 0.3 1.2 0.37 0.46 1.2 1.4 E. coli (10) Ro 13-9904 0.06 0.07 1.2 0.33 0.53 1.6 5.5 Cefotaxime 0.065 0.1 1.5 0.53 0.65 1.2 8.2 Moxalactam 0.14 0.15 1.1 0.74 2 2.7 5.3 Cefamandole 3.6 4.4 1.2 70.8 70.8 1 19.7 Cefoxitin 3.9 3.9 1 5.1 11.7 2.3 1.3 Cephalothin 17.7 53.6 3.0 >100 >100 - - Klebsiella sp. (10) Ro 13-9904 0.07 0.07 1 0.21 0.23 1.1 3 Cefotaxime 0.05 0.05 1 0.21 0.21 1 4.2 Moxalactam 0.14 0.15 1.1 0.57 0.61 1.1 4.1 Cefamandole 1.4 2.2 1.6 8.3 8.9 1.1 5.9 Cefoxitin 2.4 2.4 1 5.8 5.8 1 2.4 Cephalothin 4.8 5.5 1.2 20.3 26.8 1.3 4.2 Enterobacter sp (10) Ro 13-9904 0.12 0.19 1.6 1.7 2.6 1.5 14.2 Cefotaxime 0.1 0.15 1.5 2.4 5.9 1.5 24 Moxalactam 0.17 0.2 1.2 1.1 2.2 2 6.5 Cefamandole 2.8 3.6 1.3 >100 >100 - - Cefoxitin 87 >100 - >100 >100 - - Cephalothin 87 >100 - >100 >100 - - Serratia sp. (10) Ro 13-9904 0.13 0.15 1.2 15.5 28.8 1.9 119.2 Cefotaxime 0.14 0.26 1.9 23.4 46.7 2 167 Moxalactam 0.2 0.35 1.8 10.9 18.6 1.7 54.5 Cefamandole 19.0 21.8 1.2 >100 >100 - Cefoxitin 16.5 20.3 1.2 37.9 87.1 2.3 2.3 Cephalothin >100 >100 >100 >100 - - P. mirabilis (10) Ro 13-9904 0.004 0.007 1.8 0.49 0.8 1.6 122.5 Cefotaxime 0.023 0.028 1.2 1.7 2.2 1.3 73.9 Moxalactam 0.1 0.1 1 5.4 5.4 1 54 Cefamandole 1.3 1.4 1.1 4.4 4.4 1 3.4 Cefoxitin 3.1 3.1 1 8.3 8.3 1 2.7 Cephalothin 5.1 5.1 1 8.87 8.87 1 1.7 P. rettgeri (10) Ro 13-9904 0.013 0.013 1 1.94 4.46 2.3 149.2 Cefotaxime 0.05 0.07 1.4 2.23 2.23 1 44.6 Moxalactam 0.1 0.15 1.5 6.3 6.3 1 63 Cefamandole 0.75 0.92 1.2 25.7 46.8 1.8 34.3 Cefoxitin 1.59 2.1 1.3 8.85 9.5 1.1 5.6 Cephalothin 28.7 38 1.3 >100 >100 - VOL. 19, 1981 COMPARATIVE STUDIES OF Ro 13-9904 441 TABLE 2-Continued Geometric mean with inoculum size (colony-forming units per ml): Organism Antibiotic 10 10 MIC/ (no. of strains) MBC/ MICMBC MBC/ 13CMIC MBC MI MBC MIC Salmonella sp. (10) Ro 13-9904 0.11 0.11 1 0.2 0.3 1.5 1.8 Cefotaxime 0.12 0.12 1 0.26 0.35 1.4 2.2 Moxalactam 0.15 0.15 1 0.99 0.99 1 6.6 Cefamandole 2.75 3.6 1.3 11.8 16.6 1.4 4.3 Cefoxitin 3.2 3.6 1.1 5.8 5.8 1 1.8 Cephalothin 8.9 8.9 1 35.4 35.4 1 4 Shigella sp. (10) Ro 13-9904 0.04 0.04 1 0.08 0.08 1 2 Cefotaxime 0.02 0.02 1 0.05 0.05 1 2.5 Moxalactam 0.14 0.14 1 0.17 0.17 1 1.2 Cefamandole 0.53 0.56 1.1 1.1 1.1 1 2.1 Cefoxitin 2.7 2.9 1.1 3.1 3.1 1 1.2 Cephalothin 6.3 6.3 1 25 28.7 1.2 4 P. aeruginosa (10) Ro 13-9904 12.5 23.3 1.9 >100 >100 Cefotaxime 11.7 16.5 1.4 >100 >100 Moxalactam 19.0 21.8 1.2 >100 >100 Cefamandole >100 >100 - >100 >100 Cefoxitin >100 >100 - >100 >100 Cephalothin >100 >100 - >100 >100 H. influenzae (15) Ro 13-9904 0.003 - - 0.008 - - 2.7 Cefotaxime 0.011 - - 0.011 - - 1 Moxalactam 0.038 - - 0.046 - - 1.2 Cefamandole 0.55 - - 9.6 - - 17.5 Cefoxitin 3.4 - - 7.6 - - 2.2 Cephalothin 2.5 - - 26.3 - - 10.5

N. gonorrhoeae (15) Ro 13-9904 0.002 - - 0.004 - - 2 Cefotaxime 0.004 - - 0.01 - - 2.8 Moxalactam 0.025 - - 0.044 - - 1.8 Cefamandole 0.33 - - 0.55 - - 1.7 Cefoxitin 0.49 - - 0.66 - - 1.4 Cephalothin 0.87 - - 1.2 - - 1.4 -, Value not calculated. and many strains of E. coli, Klebsiella, Proteus, multiply resistant strains of Enterobacter, Ser- Serratia, Enterobacter, and Haemophilus; how- ratia, and indole-positive Proteus. This has also ever, it is susceptible to substantial hydrolysis been demonstrated by others for cefotaxime (2, by the Richmond type IIIa and IVc enzymes 14, 26) and moxalactam (7, 15, 25). Ro 13-9904 (18). Thus, many gram-negative organisms bear- and cefotaxime were approximately equal in ac- ing R factors remained resistant. Cefamandole tivity, with moxalactam being only slightly less is an example of the problem that, in the syn- active. Marked improvement in activity was also thesis of cephalosporin antibiotics, there seems evident against H. influenzae and N. gon- to be a correlation between potency and suscep- orrhoeae, including f8-lactamase-producing tibility to 8t-lactamases. Cefoxitin exemplifies strains. Perhaps the most significant advance, the converse; i.e., cephalosporins able to resist however, was the approximately equal antipseu- ,8-lactamases tend to have less intrinsic antibac- domonal activity evidenced by all three antibi- terial activity, particularly against gram-positive otics. At a concentration of 12.5 ,ug/ml, approx- cocci. imately 50% of the P. aeruginosa strains tested Ro 13-9904, with the other third-generation were inhibited. cephalosporins cefotaxime and moxalactam, was The marked effect of inoculum size when cer- shown in this study to have an impressive spec- tain species are tested is disturbing. Strains of S. trum of antibacterial activity. Ro 13-9904 ex- aureus have been reported to show disparities hibited significantly improved in vitro activity between MIC and MBC values for cephalothin against Enterobacteriaceae, including many (20) and cefotaxime (21), suggesting the phe- 442 EICKHOFF AND EHRET ANTIMICROB. AGENTS CHEMOTHER. nomenon of antibiotic tolerance (20, 23). The 9. Hamilton-Miller, J. M. T., D. W. Kerry, and W. Brum- tests in this study carried out with an inoculum fitt. 1974. An in vitro comparison of cefoxitin, a semi- synthetic cephamycin, with cephalothin. J. Antibiot. of 107 colony-forming units of S. aureus per ml 27:42-48. confirmed these results and extended them to 10. Klein, J. O., T. C. Eickhoff, J. G. Tilles, and M. Ro 13-9904. In all other instances, however, re- Finland. 1964. Cephalothin: activity in vitro, absorp- gardless of organism or inoculum size, the action tion and excretion in normal subjects and clinical ob- servations in 40 patients. Am. J. Med. Sci. 56:640-656. of Ro 13-9904 and the comparison drugs was 11. Moellering, R. C., Jr., M. Dray, and L. J. Kunz. 1974. bactericidal, with MBCs being generally no more Susceptibility of clinical isolates of bacteria to cefoxitin than twofold higher than MICs. Of particular and cephalothin. Antimicrob. Agents Chemother. 6: concern in this study and other studies (2, 14, 320-323. 12. Neu, H. C. 1974. Cefoxitin, a semisynthetic cephamycin 15) is the marked elevation of MICs ofthe third- antibiotic: antibacterial spectrum and resistance to hy- generation cephalosporins in response to an in- drolysis by gram-negative beta-lactamases. Antimicrob. crease in the size of Serratia, Enterobacter, Agents Chemother. 6:170-176. Proteus, and Pseudomonas inocula. Several in- 13. Neu, H. C. 1974. Cefamandole, a cephalosporin antibiotic with an unusually wide spectrum of activity. Antimi- vestigators have noted the marked effect of in- crob. Agents Chemother. 6:177-182. oculum size on MICs of cefamandole against 14. Neu, H. C. 1979. HR 756, a new cephalosporin active some gram-negative organisms, such as Entero- against gram-positive and gram-negative aerobic and bacter (5, 8). The full clinical significance of anaerobic bacteria. Antimicrob. Agents Chemother. 13: 273-281. these inoculum effects remains to be assessed, 15. Neu, H. C., N. Aswapokee, K. P. Fu, and P. Aswa- but it is a priori a cause for considerable concern. pokee. 1979. Antibacterial activity ofa new 1-oxa ceph- There are yet many strains of bacteria which alosporin compared with that of other ,B-lactam com- remain resistant to cephalosporin antibiotics, pounds. Antimicrob. Agents Chemother. 16:141-149. to 16. O'Callaghan, C. H., A. Morris, S. M. Kirby, and A. H. presumably due penetration barriers protect- Shingler. 1972. Novel method for detection of ,B-lac- ing the sites of synthesis in the tamases by using a chromogenic cephalosporin sub- inner bacterial membrane. Overall, however, the strate. Antimicrob. Agents Chemother. 1:283-288. third-generation antibiotics Ro 13-9904, cefotax- 17. Perea, E. J., J. Aznar, M. C. Garcia-Iglesias, and M. and moxalactam V. Borobio. 1978. Cefoxitin sodium activity against ime, offer substantial in vitro anaerobes: effect of the inoculum size, pH variation and advances over first- and second-generation ceph- different culture media. J. Antimicrob. Chemother. alosporin derivatives. Studies evaluating their 4(Suppl. B):55-60. clinical potential are obviously important and 18. Richmond, M. D., and S. Wotton. 1976. Comparative may provide additional study of seven cephalosporins: susceptibility to beta- insight into the signifi- lactamases and ability to penetrate the surface layers cance of some of the in vitro data reported of Escherichia coli. Antimicrob. Agents Chemother. herein. 10:219-222. 19. Sabath, L D., and M. Finland. 1967. Resistance of ACKNOWLEDGMENT and cephalosporins to beta-lactamases from gram-negative bacilli: some correlations with antibac- This study was supported by a grant from Hoffmann-La terial activity. Ann. N.Y. Acad. Sci. 145:237-245. Roche, Inc. 20. Sabath, L. D., N. Wheeler, M. Laverdiere, D. Bla- zevic, and B. J. Wilkinson. 1977. A new type of resistance of Staphylococcus aureus. Lancet LITERATURE CITED i:443-447. 1. Abraham, E. P. 1962. The cephalosporins. Pharmacol. 21. Sosna, J. P., P. R. Murray, and G. Medoff. 1978. Rev. 14:473-500. Comparison of the in vitro activities of HR756 with 2. Counts, G. W., and M. Turek. 1979. Antibacterial activ- cephalothin, cefoxitin, and cefamandole. Antimicrob. ity of a new parenteral cepholosporin-HR 756: com- Agents Chemother. 14:876-879. parison with cefamandole and ceforanide. Antimicrob. 22. Steers, E., E. L. Foltz, and B. S. Graves. 1959. 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