Microbiology © 1990 S. Karger A G . Basel 0009-3157/90/0364-0259 $ 2.75/0
Chemotherapy 1990;36:259-267
In vitro Activity of Cefepime, a New Parenteral Cephalosporin, against Recent European Blood Isolates and in Comparison with Piperacillin/Tazobactam K. Dornbusch, E. Mörtsell, E. Göransson Department of Clinical Microbiology, Karolinska Hospital, Stockholm, Sweden
Key Words. Cefepime ■Piperacillin/tazobactam • In vitro activity
Introduction Different chemical modifications of the parenteral cephalosporins have produced compounds with increased potency, in creased (3-Iactamase stability, broadened spectrum of activity, and more favourable pharmacokinetics. The newer cephalo
sporins have become established as safe and effective therapy of serious infections [10, 13, 15, 16]. Cefepime is a new paren teral aminothiazolemethoximino cephalo sporin which bears a quaternerized Nmethylpyrrolidine side chain at the C-3 position [2]. It has activity against Enterobacteriaceae, Pseudomonas aeruginosa,
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Abstract. Cefepime, a new parenteral cephalosporin with broad antibacterial spec trum and stability to the hydrolysis to many bacterial (3-lactamases, was tested against recent blood culture isolates (369 strains of gram-negative bacilli and 131 strains of sta phylococci) collected in 29 European laboratories by the microdilution method in Mueller-Hinton broth. Cefepime was very active against the gram-negative bacilli (MICso^O.016-0.064 mg/1; MIC 90 0.064-4 mg/1) and less active against Pseudomonas (MIC 50 4 mg/1; MIC9o> 16 mg/1) or Acinetobacter (M IC 50 and MIC 9o> 16 mg/1). The staphylococci were also inhibited (MIC 50 8 mg/1; MIC 90 16 mg/1). Cefepime was very active against bacteria producing different plasmid-encoded (3-lactamases (MIC 0.0160.5 mg/1). Piperacillin was not active against the latter strains (MIC from 2 to > 64 mg/ 1), but the presence of the (3-lactamase inhibitor tazobactam restored the activity of pip eracillin. The bactericidal activity of cefepime and piperacillin/tazobactam against (3-lactamase-producing strains was confirmed by the by the killing curve technique.
Dornbusch/Môrtsell/Gôransson
staphylococci, and other gram-positive bacteria [2, 11]. In this study the antibacte rial activity of cefepime has been tested against gram-negative bacilli and staphy lococci from blood specimens, isolated in different European laboratories, and also against bacteria with production of char acterized (3-lactamases. The bactericidal activity of the compound and its stability to (3-lactamases have also been studied and compared to that of piperacillin in combi nation with the (3-Iactamase inhibitor tazobactam ( YTR 830) [ 12].
Materials and Methods Bacteria Recent blood isolates comprising Escherichia coli (n = 58), Klebsiella sp. (n = 40), Enterobacter sp. (n = 62), Citrobacter sp. (n = 10), Serratia sp. (n = 35), Proteus mirahihs (n = 7), other Proteus sp. (n = 14), Morganella sp. (n= 17), Acinetobacter sp. (n = 38), Pseudomonas sp. (n = 88), Staphylococcus aureus (n = 71), and other staphylococci (n = 60), collected from 29 European laboratories as a part of a collab orative study [9], were tested. They were identified by routine laboratory methods [14], supplemented with API tests (Analytical Laboratories) when necessary. All bacteria but 23 strains of E. coli, 25 strains of S. aureus and 29 strains of other staphylococci were resistant to cefazoline (> 16 mg/1). Thirty-five per cent of the gram-negative bacilli and > 80% of the staphylococci were resistant to gentamicin (>4m g/ I). The great majority of the staphylococci (>90%) produced penicillinase as judged by the nitrocefin test (Glaxo). Strains of E. coli. Pseudomonas aerugi nosa, and Acinetobacter hydrophila, producing char acterized (3-lactamases as TEM 1 and 2; OX A 1,2, 3, 4, 5, 6, and 7; SHV 1 and 2; HMS 1; TLE l;OHIO I; ROB I; PSE 1, 2, 3, and 4; LCR 1; CARB 4, and AER I, were also tested. Antibiotics Cefepime as the sulphate (lot No. D87040; po tency 829 pg/mg) was obtained from Bristol Myers (Wallingford, Conn., USA), piperacillin monohy
drate (batch No. 7H19A; potency 970 pg/mg) and tazobactam (lot No. PC0801; potency 997 pg/mg) from Cyanamid (Gosport, Hants, UK). Stock solu tions were prepared in phosphate-buffered saline (pH 7.2-7.4). In all cases fresh stock solutions were used for the preparation of antibiotic microtiter plates which were frozen at -80 °C until use. Susceptibility Testing All strains were tested by determination of mini mum inhibitory concentration (MIC) by the microdi lution method. Serial twofold dilutions of antibiotic at concentrations ranging from 0.032 to 32 mg/1 were prepared in Mueller-Hinton broth (BBL; 50 pl/well) to which bacteria were added (105-106 CFU/ml; 50 pl/well). The MIC was determined by visual tur bidity after 20 h of incubation at 37°C and defined as the lowest concentration of antibiotic which inhib ited growth completely. MIC determinations were controlled by also including the reference strains E. coli ATCC25922, P. aeruginosa ATCC27853, and S. aureus ATCC25923 and ATCC292I3. With a few strains MIC was also determined by the macrobroth dilution method (final volume 1 ml/ tube; Mueller-Hinton broth) [8] and with the same bacterial inoculum as described above. The mini mum bactericidal concentration (MBC) was defined at 99.9% killing of inoculum and determined by spreading 0.1 ml of culture from each tube onto blood agar plates for viable counts. Killing Curves The test strain was grown to early log-phase (105—10ft CFU/ml; CFU = colony-forming units) when the antibiotic was added at a final concentra tion of 0.5-1 xMBC and cultivation was continued [5]. The strain was also cultivated in parallel in the absence of antibiotic as a control. Samples for viable counts and antibiotic assays by the agar well diffu sion method [4] were collected at time intervals. Bac tericidal activity was defined as a thousandfold de crease in viable counts of inoculum (99.9% killing). Preparation of /)- Lactamases Crude enzymes were prepared by sonication of washed bacterial cells while cooled in an ice bath as described in detail earlier [17] and characterized by isoelectric focusing in polyacrylamide gel (Ampholine PAG plate, pH 3.5-9.5; LKB, Stockholm, Sweden).
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In vitro Activity of Cefepime
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Table 1. Antibacterial activity of cefepime against recent clinical blood isolates Organism E. coli Cef-s Cef-r Klebsiella sp. All cef-r Enterobacter sp. All cef-r Citrobacter sp. All cef-r Serratia sp. All cef-r P. mirabilis All cef-r Other Proteus sp. All cef-r Morganellasp. All cef-r Acinetobacter sp. Pseudomas sp. S. aureus Cef-s Cef-r Other staphylococci Cef-s Cef-r
n
MIC*,
MIG*)
Range, mg/1
23 35
¿10.016 0.064
0.064 0.125
¿0.016-1 ¿0.016-0.5
40
0.064
0.25
¿0.016-2
62
0.064
4
¿0.016-8
10
0.032
2
¿0.016-2
35
0.064
1
¿0.016-4
7
0.064
0.125
0.032-0.0125
14
0.064
0.064
0.032-0.5
17 38 88
0.064 16 4
1 > 16 > 16
¿0.016-4 2- > 16 1-16
25 46
4 >16
16 > 16
l- > 16 2-> 16
29 31
>
8 16
16 >16
0.5-> 16 4- > 16 0.032 4 2 2
E. coli ATCC 25922 P. aeruginosa ATCC 27853 S. aureus ATCC 25923 S. aureus ATCC 29213
Antibiotic Stability to Crude p-Lactamases To 1 ml of antibiotic solution (100 mg/1) 1 ml of crude enzyme was added, and the mixture was incu bated at 37 °C. As a control for spontaneous inactiva tion of antibiotic, 1 ml of phosphate-buffered saline was added to 1 ml of antibiotic solution and in
cubated at 37 °C. At time intervals (0, 1, 2, 4, 6, and 24 h) samples were assayed for antibiotic concentra tions by the agar well diffusion method [4] on DST agar (Oxoid) and with use of E. coli ATCC25922 as the indicator strain for cefepime and P. aeruginosa 10701 for piperacillin.
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Cef-s = cefazoline susceptible; Cef-r = cefazoline resistant.
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Table 2. In vitro activity of cefepime and pipera cillin plus tazobactam at the ratio of 8:1 against bac teria producing characterized p-lactamases Organism
MIC, mg/1 cefepime
piperacillin piperacillin + tazobactam
E. coli TEM 1 0.064 TEM 2 0.125 OXA 1 0.5 OXA2 0.032 0.032 OXA 3 OXA 4 0.125 0.064 OXA 5 OXA 7 0.064 SHV 1 0.064 SHV2 0.032 HMS 1 0.064 T L E 1 ¿0.016 OHIO 1 0.5 ROB 1 0.25
> 16 >64 32 8 16 16 8 64 >64 2 >64 >64 >64 >64
16 32 16 2 1 8 2 8 32 2 8 4 64 2
P. aeruginosa PSE 1 PSE 2 PSE 3 PSE 4 OXA 6 LCR 1 CARB4
>64 64 >64 >64 >64 32 >64
64 32 16 >64 64 16 >64
16
8
4 4 2 4 8 2 8
A . hydrophila 0.064 AER 1
Results Antibacterial Activity The antibacterial activity of cefepime against the European blood isolates was high and is shown in table 1. The majority of strains of Enterobacteriaceae were in hibited by *¿0.016-0.064 mg/1, whereas Pseudomonas (MIC50 4 mg/1) and Acine-
tobacter (M IC5o 16 mg/1) were less sus ceptible. Cefepime also showed activity against the staphylococci (M IC50 4-8 mg/1) except the cefazoline-resistant strains (MIC50 > 16 mg/1). The activity against the reference strains was also within standard ranges (table 1). Activity against Bacteria Producing Characterized fi-Lactamases Cefepime produced low MIC values against the strains of E. coli, P. aeruginosa, and A. hydrophila with production of P-lactamases located on different plasmids (table 2). For comparison, piperacillin, which is hydrolyzed by these enzymes, was less active (MIC 2->64 mg/1) against these strains, but in the presence of en zyme inhibitor tazobactam the activity of piperacillin was increased (table 2). Bactericidal Activity With all five strains tested, producing both chromosomal and plasmidal p-lactamases, the MBC values were the same or fourfold higher than each respective MIC except with the strain of P. aeruginosa (table 3). The combination piperacillin/ tazobactam was more bactericidal than piperacillin alone against these strains. As can be seen from figure 1 and 2, cefepime was rapidly bactericidal at 1 x MBC concentrations against E. coli B3 or Klebsiella pneumoniae B2919. Re growth was obtained with E. coli within 24 h, and the compound was slowly inacti vated. No regrowth was seen with K. pneu moniae, and no antibiotic inactivation could be observed. The compound was in hibitory during 6 h against Enterobacter cloacae B53 with production of inducible p-lactamase (fig. 3) and P. aeruginosa
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In vitro Activity of Cefepime
Table 3. Bactericidal activity of cefepime against clinical blood isolates producing characterized (3-lactamases Bacterium
Isoelectric point
Cefepime -----------MIC
MCB
Piperacillin -------------------MCB MIC
Piperacillin + tazobactam MIC
E. coli B3 K.pneumonia B2919 (K. 1) E. cloacae B53 P. aeruginosa PSE-2 S. aureus B15771
5.3; 10.8 6.6 7.9 6.1 -
0.25 0.125 0.032 4 2
0.25 0.125 0.5 >32 8
>64 16 16 64 -
>64 32 32 >64 >64
32 4 2 32 -
MBC 32 4 2 >64 32
MIC and MCB are expressed as milligrams per liter.
Time, h
Fig. 1. Growth of P-lactamase producing E. coli B3 (isoelectric point 5.3+10.8) in the presence of 0.25 mg/1 of cefepime (O; MBC 0.25 mg/1). In the di agram the antibiotic concentrations in the culture is given (mg/1). x = Control.
Fig. 2. Growth of ß-lactamase-producing K. pneu moniae B2919 (Kl) in the presence of 0.125 mg/1 of cefepime (O; MBC 0.125 mg/1). In the diagram the antibiotic concentration in the culture is given (mg/1). x = Control.
(fig. 4) at 0.5 x MBC and with slow anti biotic inactivation (31%). Cefepime was also slowly bactericidal against S. aureus B15771 (fig. 5), with no regrowth and with 29% inactivation of antibiotic. For comparison and by use of the
same strains piperacillin/tazobactam at 1 x MBC was rapidly bactericidal against E. coli B3 during at least 6 h. This was not the case with piperacillin alone which was inactivated by this strain (fig. 6). The combination at l x MBC was inhibitory
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Time, h
Dornbusch/M örtsell/Göransson
Fig. 3. Growth of E. cloacae B53 with inducible p-lactamase production (isoelectric point 7.9) in the presence of 0.25 mg/l of cefepime (O; MBC 0.5 mg/ 1). In the diagram the antibiotic concentration in the culture is given (mg/l). x = Control.
Fig. 5. Growth of penicillinase-producing S. au reus B15771 in the presence of 8 mg/l of cefepime (O; MBC 8 mg/l). In the diagram the antibiotic con centration in the culture is given (mg/l). x = Con trol.
Fig. 4. Growth of ß-lactamase-producing P. aeru ginosa (PSE-2) in the presence of 32 mg/l of cefe pime (O; MBC >32 mg/l). In the diagram the anti biotic concentration in the culture is given (mg/l). x = Control.
Fig. 6. Growth of E. coli B3 (isoelectric point 5.3+10.8) in the presence of 128 mg/l of piperacillin (O; MBC >64 mg/l) and piperacillin (32 mg/l) plus tazobactam (4 mg/l) (□; MBC combination at the ra tio of 8:1 32 mg/l). In the diagram the piperacillin concentration alone (PP) or in the combination (PP + YTR) is given, x = Control.
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In vitro Activity of Cefepime
Time, h Fig. 7. Growth of K. pneumoniae B2919 (K.1) in the presence of 16 mg/l of piperacillin (O; MBC > 16 mg/1) and piperacillin (4 mg/l) plus tazobactam (0.5 mg/l; □; MBC combination 4 mg/l). In the diagram the concentration of piperacillin alone (PP) or in the combination (PP + YTR) is given, x = Control.
265
Time, h Fig. 9. Hydrolytic activity of ß-lactamase prepa ration from E. coli B3, P. aeruginosa (PSE-2) (O), K. pneumoniae B2919 (■), and E. cloacae B53 ( • ) on 50 mg/l of cefepime. x = Control, 50 mg/l cefepime in phosphate-buffered saline.
Fig. 8. Growth of E. cloacae B53 in the presence of 32 mg/l of piperacillin (O; MBC 32 mg/l) and piperacillin (32 mg/l) plus tazobactam (4 mg/l) (□) or 2 mg/l plus 0.25 mg/l respectively (■; MBC of combination 2 mg/l). In the diagram the concentra tion of piperacillin (PP) alone or in the combination (PP + YTR) is given.
P-Lactamase Stability Crude (3-lactamase preparations from cultures of E. coli B3 and P. aeruginosa PSE-2 inactivated cefepime within 1 h, from K. pneumoniae Kl within 6 h, and from E. cloacae producing inducible (3-lactamase within 24 h (fig. 9). Spontane ous inactivation of the compound was 40% within 24 h. All enzymes inactivated
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against K. pneumoniae B2929 (fig. 7) and E. cloacae B53 (fig. 8) for up to 6 h and re growth was obtained within 24 h. A simi lar result was obtained with piperacillin alone, but then higher concentrations of piperacillin were needed. The combina tion was also bactericidal against the peni cillinase-producing strain of S. aureus B15771.
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266
Discussion As reported earlier, with ceftazidime, cefotaxime, and aztreonam [6, 9] and later with ceftibuten and piperacillin in combi nation with tazobactam at the ratio of 8:1 [7], also cefepime was very active against the European blood isolates (table 1), the majority of which were resistant to céfazo line and/or gentamicin. This is in agree ment with reports of other investigators [1, 3, 11] who have reported high activity of cefepime also against E. cloacae, Serratia marcescens, and M. morganii and against ceftazidime- and cefotaxime-resistant gram-negative bacilli. The compound showed similar activity as that of cefti buten, was twofold or more active than ceftazidime, and was manifold more ac tive than piperacillin/tazobactam against the gram-negative bacilli [7], Strains of Acinetobacter sp. were, however, resistant to cefepime. In contrast to ceftibuten, cefepime was also active against staphylo cocci, although not against the cefazolineresistant strains. Thus 6% of the gram negative bacilli were considered resistant (MIC > 16 mg/1), comprising only strains of Acinetobacter sp. (n = 10) and Pseu domonas sp. (n = 11). By comparing the results from an ear lier study with the same bacteria [7], 22% of the gram-negative bacilli were resistant to ceftibuten (MIC > 16 mg/1), 14% to cef tazidime, and 15% to piperacillin/tazo bactam (MIC > 64 mg/1).
The bactericidal activity of cefepime was confirmed by the low MBC values (table 3) and the killing curve results (fig. 1-5) against strains producing different (3-lactamases. For comparison, the pres ence of tazobactam restored the bacteri cidal activity of piperacillin (fig. 6-8). Therefore, the excellent activity of cefe pime, as of other newer (3-lactam antibiot ics (tables 2, 3), might be explained by its stability to many (3-lactamases followed by rapid access to the penicillin-binding pro teins (tables 2 and 3). However, an inter esting observation was that crude enzyme preparations from tenfold concentrated cultures inactivated cefepime and pipera cillin/tazobactam at different degrees (fig-9). In conclusion, the narrow range of MIC values of cefepime together with its stability to many (3-lactamases gives it a predictable degree of in vitro activity against a given and wide spectrum of bac teria. It is also suggested that cefepime is active against bacteria with derepressed constitutive (3-lactamase production, as also observed by other investigators [18, 19]. It was shown that the activity of cefe pime against strains with chromosomally encoded high-level production of (3-lactamases was only little reduced because of its low affinity for these enzymes.
References 1 Bodey, G. P.; Ho, D. H.: Blanc, B. de: In vitro studies with BMY-28142, a new broad-spectrum cephalosporin. Antimicrob. Agents Chemother. 27: 265-268(1985). 2 Cefepime. Investigator Brochure, Bristol-Myers Company, Pharmaceutical Research and Devel opment Division (1987).
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piperacillin within 1 h, while when com bined with tazobactam, the inactivation was slower, except with the enzymes from E. coli B3 and P. aeruginosa PSE-2.
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12 Jacobs, M. R.; Aronoff, S.C.; Johenning, S.; Schlaes, D. M.; Yamabe, S.: Comparative activi ties of the beta-lactamase inhibitors YTR-830, clavulanate and sulbactam combined with ampicillin and broad spectrum penicillins against de fined beta-lactamase producing aerobic gram negative bacilli. Antimicrob. Agents Chemother. 29: 980-985(1986). 13 Lambert, H.; Phillips, I.; Wise, R.J.: Ceftazidime in clinical practice. J. antimicrob. Chemother. 12: suppl. A (1983). 14 Manual of clinical microbiology; 4th ed. Editors: Lennette, E.H., Balows, A., Hausler, W.J., Jr., Shadomy, H.J. (American Society for Clinical Microbiology, Washington 1986). 15 Masuyoshi, S.; Arai, S.; Miyamoto, M.; Mitsuhashi, S.: In vitro antimicrobial activity of cefo taxime, a new cephalosporin. Antimicrob. Agents Chemother. 18: 1-8(1980). 16 Callaghan, C.H.: Description and classification of new cephalosporins and their relationship with the established compounds. J. antimicrob. Che mother. 5: 635-671 (1979). 17 Olsson-Liljequist, B.; Dornbusch, K.; Nord, C. E.: Characterization of three different beta-lac tamases from the Bacteroides fragilis group. An timicrob. Agents Chemother. 18: 220-225(1980). 18 Phelps, D.J.; Carlton, D. D.; Farrel, C.A.; Kess ler, R.E.: Affinity of cephalosporins for (5-lactamases as a factor in antibacterial efficacy. Anti microb. Agents Chemother. 29: 845-848 ( 1986). 19 Sanders, C.C.; Sanders, W. E., Jr.: Type I p-lactamases of gram-negative bacteria: interactions with p-lactam antibiotics. J. Infect. Dis. 154: 792-800(1986).
K. Dornbusch Department of Clinical Microbiology Karolinska Hospital S—104 01 Stockholm (Sweden)
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3 Conrad, D.A.; Scribner, R.K.; Weber, A. H.; Marks, M.I.: In vitro activity of BMY-28142 against pediatric pathogens including isolates from cystic fibrosis sputum. Antimicrob. Agents Chemother. 28: 58-61 (1985). 4 Dornbusch, K.: Assay of gentamicin concentra tions in serum specimens by microbiological and radioenzymatic methods. Scand. J. infect. Dis. 9: 227-229(1977). 5 Dornbusch, K.; Frolander, F.; Cacciapuoti, A. F.; Naples, L.; Hare, R.S.; Miller, G.M.: In vitro ac tivity of Sch 34343 against gram-negative bacteria producing characterized beta-lactamases. J. antimicrob. Chemother. 15: suppl. C, pp. 85-88 (1985). 6 Dornbusch, K.; Bengtsson, S.; Brorson, J.E.; Fritz, H.; Henning, C.; Kronvall, G.; Larsson, P.; Malmborg, A.S.; Thore, M.; Tarnvik, A.; Walder M.: Susceptibility to beta-lactam antibiotics and gentamicin of gram-negative bacilli isolated from hospitalized patients. A Swedish multicenter study. Scand. J. infect. Dis. 20: 641-644(1988). 7 Dornbusch, K.; Kronvall, G.; Goransson, E.; Mortsell, E.: Comparative in vitro activity of the penem FCE 22101 against recent European blood culture isolates. J. antimicrob. Chemother. 23: suppl. C, pp. 43-52(1989). 8 Ericsson, H. M.; Sherris, J.C.: Antibiotic sensitiv ity testing: report of an international collabora tive study. Acta pathol. microbiol. scand., B, Mi crobiol. Immunol., suppl. 217 (1971). 9 European Study Group on Antibiotic Resistance: Susceptibility to beta-lactam antibiotics in septi cemia isolates from 29 European laboratories. Eur. J.clin. Microbiol. 6: 515-520(1987). 10 Geddes, A.M.: Studies of beta-lactam antibiotics in systemic infections. Evaluation of new betalactam antibiotics. Rev. infect. Dis. 8: suppl. 3, pp. 333-348(1986). 11 Giamarellou, H.; Sahin, A.; Chryssouli, Z.: Com parative in vitro evaluation of BMY-28142, a new broad-spectrum cephalosporin, versus other beta-lactams against multiresistant gram-nega tive isolates. Drugs exp. clin. Res. 13: 149-153 (1987).
Chemotherapy 1990;36:259-267
In vitro Activity of Cefepime, a New Parenteral Cephalosporin, against Recent European Blood Isolates and in Comparison with Piperacillin/Tazobactam K. Dornbusch, E. Mörtsell, E. Göransson Department of Clinical Microbiology, Karolinska Hospital, Stockholm, Sweden
Key Words. Cefepime ■Piperacillin/tazobactam • In vitro activity
Introduction Different chemical modifications of the parenteral cephalosporins have produced compounds with increased potency, in creased (3-Iactamase stability, broadened spectrum of activity, and more favourable pharmacokinetics. The newer cephalo
sporins have become established as safe and effective therapy of serious infections [10, 13, 15, 16]. Cefepime is a new paren teral aminothiazolemethoximino cephalo sporin which bears a quaternerized Nmethylpyrrolidine side chain at the C-3 position [2]. It has activity against Enterobacteriaceae, Pseudomonas aeruginosa,
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Abstract. Cefepime, a new parenteral cephalosporin with broad antibacterial spec trum and stability to the hydrolysis to many bacterial (3-lactamases, was tested against recent blood culture isolates (369 strains of gram-negative bacilli and 131 strains of sta phylococci) collected in 29 European laboratories by the microdilution method in Mueller-Hinton broth. Cefepime was very active against the gram-negative bacilli (MICso^O.016-0.064 mg/1; MIC 90 0.064-4 mg/1) and less active against Pseudomonas (MIC 50 4 mg/1; MIC9o> 16 mg/1) or Acinetobacter (M IC 50 and MIC 9o> 16 mg/1). The staphylococci were also inhibited (MIC 50 8 mg/1; MIC 90 16 mg/1). Cefepime was very active against bacteria producing different plasmid-encoded (3-lactamases (MIC 0.0160.5 mg/1). Piperacillin was not active against the latter strains (MIC from 2 to > 64 mg/ 1), but the presence of the (3-lactamase inhibitor tazobactam restored the activity of pip eracillin. The bactericidal activity of cefepime and piperacillin/tazobactam against (3-lactamase-producing strains was confirmed by the by the killing curve technique.
Dornbusch/Môrtsell/Gôransson
staphylococci, and other gram-positive bacteria [2, 11]. In this study the antibacte rial activity of cefepime has been tested against gram-negative bacilli and staphy lococci from blood specimens, isolated in different European laboratories, and also against bacteria with production of char acterized (3-lactamases. The bactericidal activity of the compound and its stability to (3-lactamases have also been studied and compared to that of piperacillin in combi nation with the (3-Iactamase inhibitor tazobactam ( YTR 830) [ 12].
Materials and Methods Bacteria Recent blood isolates comprising Escherichia coli (n = 58), Klebsiella sp. (n = 40), Enterobacter sp. (n = 62), Citrobacter sp. (n = 10), Serratia sp. (n = 35), Proteus mirahihs (n = 7), other Proteus sp. (n = 14), Morganella sp. (n= 17), Acinetobacter sp. (n = 38), Pseudomonas sp. (n = 88), Staphylococcus aureus (n = 71), and other staphylococci (n = 60), collected from 29 European laboratories as a part of a collab orative study [9], were tested. They were identified by routine laboratory methods [14], supplemented with API tests (Analytical Laboratories) when necessary. All bacteria but 23 strains of E. coli, 25 strains of S. aureus and 29 strains of other staphylococci were resistant to cefazoline (> 16 mg/1). Thirty-five per cent of the gram-negative bacilli and > 80% of the staphylococci were resistant to gentamicin (>4m g/ I). The great majority of the staphylococci (>90%) produced penicillinase as judged by the nitrocefin test (Glaxo). Strains of E. coli. Pseudomonas aerugi nosa, and Acinetobacter hydrophila, producing char acterized (3-lactamases as TEM 1 and 2; OX A 1,2, 3, 4, 5, 6, and 7; SHV 1 and 2; HMS 1; TLE l;OHIO I; ROB I; PSE 1, 2, 3, and 4; LCR 1; CARB 4, and AER I, were also tested. Antibiotics Cefepime as the sulphate (lot No. D87040; po tency 829 pg/mg) was obtained from Bristol Myers (Wallingford, Conn., USA), piperacillin monohy
drate (batch No. 7H19A; potency 970 pg/mg) and tazobactam (lot No. PC0801; potency 997 pg/mg) from Cyanamid (Gosport, Hants, UK). Stock solu tions were prepared in phosphate-buffered saline (pH 7.2-7.4). In all cases fresh stock solutions were used for the preparation of antibiotic microtiter plates which were frozen at -80 °C until use. Susceptibility Testing All strains were tested by determination of mini mum inhibitory concentration (MIC) by the microdi lution method. Serial twofold dilutions of antibiotic at concentrations ranging from 0.032 to 32 mg/1 were prepared in Mueller-Hinton broth (BBL; 50 pl/well) to which bacteria were added (105-106 CFU/ml; 50 pl/well). The MIC was determined by visual tur bidity after 20 h of incubation at 37°C and defined as the lowest concentration of antibiotic which inhib ited growth completely. MIC determinations were controlled by also including the reference strains E. coli ATCC25922, P. aeruginosa ATCC27853, and S. aureus ATCC25923 and ATCC292I3. With a few strains MIC was also determined by the macrobroth dilution method (final volume 1 ml/ tube; Mueller-Hinton broth) [8] and with the same bacterial inoculum as described above. The mini mum bactericidal concentration (MBC) was defined at 99.9% killing of inoculum and determined by spreading 0.1 ml of culture from each tube onto blood agar plates for viable counts. Killing Curves The test strain was grown to early log-phase (105—10ft CFU/ml; CFU = colony-forming units) when the antibiotic was added at a final concentra tion of 0.5-1 xMBC and cultivation was continued [5]. The strain was also cultivated in parallel in the absence of antibiotic as a control. Samples for viable counts and antibiotic assays by the agar well diffu sion method [4] were collected at time intervals. Bac tericidal activity was defined as a thousandfold de crease in viable counts of inoculum (99.9% killing). Preparation of /)- Lactamases Crude enzymes were prepared by sonication of washed bacterial cells while cooled in an ice bath as described in detail earlier [17] and characterized by isoelectric focusing in polyacrylamide gel (Ampholine PAG plate, pH 3.5-9.5; LKB, Stockholm, Sweden).
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In vitro Activity of Cefepime
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Table 1. Antibacterial activity of cefepime against recent clinical blood isolates Organism E. coli Cef-s Cef-r Klebsiella sp. All cef-r Enterobacter sp. All cef-r Citrobacter sp. All cef-r Serratia sp. All cef-r P. mirabilis All cef-r Other Proteus sp. All cef-r Morganellasp. All cef-r Acinetobacter sp. Pseudomas sp. S. aureus Cef-s Cef-r Other staphylococci Cef-s Cef-r
n
MIC*,
MIG*)
Range, mg/1
23 35
¿10.016 0.064
0.064 0.125
¿0.016-1 ¿0.016-0.5
40
0.064
0.25
¿0.016-2
62
0.064
4
¿0.016-8
10
0.032
2
¿0.016-2
35
0.064
1
¿0.016-4
7
0.064
0.125
0.032-0.0125
14
0.064
0.064
0.032-0.5
17 38 88
0.064 16 4
1 > 16 > 16
¿0.016-4 2- > 16 1-16
25 46
4 >16
16 > 16
l- > 16 2-> 16
29 31
>
8 16
16 >16
0.5-> 16 4- > 16 0.032 4 2 2
E. coli ATCC 25922 P. aeruginosa ATCC 27853 S. aureus ATCC 25923 S. aureus ATCC 29213
Antibiotic Stability to Crude p-Lactamases To 1 ml of antibiotic solution (100 mg/1) 1 ml of crude enzyme was added, and the mixture was incu bated at 37 °C. As a control for spontaneous inactiva tion of antibiotic, 1 ml of phosphate-buffered saline was added to 1 ml of antibiotic solution and in
cubated at 37 °C. At time intervals (0, 1, 2, 4, 6, and 24 h) samples were assayed for antibiotic concentra tions by the agar well diffusion method [4] on DST agar (Oxoid) and with use of E. coli ATCC25922 as the indicator strain for cefepime and P. aeruginosa 10701 for piperacillin.
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Cef-s = cefazoline susceptible; Cef-r = cefazoline resistant.
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Table 2. In vitro activity of cefepime and pipera cillin plus tazobactam at the ratio of 8:1 against bac teria producing characterized p-lactamases Organism
MIC, mg/1 cefepime
piperacillin piperacillin + tazobactam
E. coli TEM 1 0.064 TEM 2 0.125 OXA 1 0.5 OXA2 0.032 0.032 OXA 3 OXA 4 0.125 0.064 OXA 5 OXA 7 0.064 SHV 1 0.064 SHV2 0.032 HMS 1 0.064 T L E 1 ¿0.016 OHIO 1 0.5 ROB 1 0.25
> 16 >64 32 8 16 16 8 64 >64 2 >64 >64 >64 >64
16 32 16 2 1 8 2 8 32 2 8 4 64 2
P. aeruginosa PSE 1 PSE 2 PSE 3 PSE 4 OXA 6 LCR 1 CARB4
>64 64 >64 >64 >64 32 >64
64 32 16 >64 64 16 >64
16
8
4 4 2 4 8 2 8
A . hydrophila 0.064 AER 1
Results Antibacterial Activity The antibacterial activity of cefepime against the European blood isolates was high and is shown in table 1. The majority of strains of Enterobacteriaceae were in hibited by *¿0.016-0.064 mg/1, whereas Pseudomonas (MIC50 4 mg/1) and Acine-
tobacter (M IC5o 16 mg/1) were less sus ceptible. Cefepime also showed activity against the staphylococci (M IC50 4-8 mg/1) except the cefazoline-resistant strains (MIC50 > 16 mg/1). The activity against the reference strains was also within standard ranges (table 1). Activity against Bacteria Producing Characterized fi-Lactamases Cefepime produced low MIC values against the strains of E. coli, P. aeruginosa, and A. hydrophila with production of P-lactamases located on different plasmids (table 2). For comparison, piperacillin, which is hydrolyzed by these enzymes, was less active (MIC 2->64 mg/1) against these strains, but in the presence of en zyme inhibitor tazobactam the activity of piperacillin was increased (table 2). Bactericidal Activity With all five strains tested, producing both chromosomal and plasmidal p-lactamases, the MBC values were the same or fourfold higher than each respective MIC except with the strain of P. aeruginosa (table 3). The combination piperacillin/ tazobactam was more bactericidal than piperacillin alone against these strains. As can be seen from figure 1 and 2, cefepime was rapidly bactericidal at 1 x MBC concentrations against E. coli B3 or Klebsiella pneumoniae B2919. Re growth was obtained with E. coli within 24 h, and the compound was slowly inacti vated. No regrowth was seen with K. pneu moniae, and no antibiotic inactivation could be observed. The compound was in hibitory during 6 h against Enterobacter cloacae B53 with production of inducible p-lactamase (fig. 3) and P. aeruginosa
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262
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In vitro Activity of Cefepime
Table 3. Bactericidal activity of cefepime against clinical blood isolates producing characterized (3-lactamases Bacterium
Isoelectric point
Cefepime -----------MIC
MCB
Piperacillin -------------------MCB MIC
Piperacillin + tazobactam MIC
E. coli B3 K.pneumonia B2919 (K. 1) E. cloacae B53 P. aeruginosa PSE-2 S. aureus B15771
5.3; 10.8 6.6 7.9 6.1 -
0.25 0.125 0.032 4 2
0.25 0.125 0.5 >32 8
>64 16 16 64 -
>64 32 32 >64 >64
32 4 2 32 -
MBC 32 4 2 >64 32
MIC and MCB are expressed as milligrams per liter.
Time, h
Fig. 1. Growth of P-lactamase producing E. coli B3 (isoelectric point 5.3+10.8) in the presence of 0.25 mg/1 of cefepime (O; MBC 0.25 mg/1). In the di agram the antibiotic concentrations in the culture is given (mg/1). x = Control.
Fig. 2. Growth of ß-lactamase-producing K. pneu moniae B2919 (Kl) in the presence of 0.125 mg/1 of cefepime (O; MBC 0.125 mg/1). In the diagram the antibiotic concentration in the culture is given (mg/1). x = Control.
(fig. 4) at 0.5 x MBC and with slow anti biotic inactivation (31%). Cefepime was also slowly bactericidal against S. aureus B15771 (fig. 5), with no regrowth and with 29% inactivation of antibiotic. For comparison and by use of the
same strains piperacillin/tazobactam at 1 x MBC was rapidly bactericidal against E. coli B3 during at least 6 h. This was not the case with piperacillin alone which was inactivated by this strain (fig. 6). The combination at l x MBC was inhibitory
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Time, h
Dornbusch/M örtsell/Göransson
Fig. 3. Growth of E. cloacae B53 with inducible p-lactamase production (isoelectric point 7.9) in the presence of 0.25 mg/l of cefepime (O; MBC 0.5 mg/ 1). In the diagram the antibiotic concentration in the culture is given (mg/l). x = Control.
Fig. 5. Growth of penicillinase-producing S. au reus B15771 in the presence of 8 mg/l of cefepime (O; MBC 8 mg/l). In the diagram the antibiotic con centration in the culture is given (mg/l). x = Con trol.
Fig. 4. Growth of ß-lactamase-producing P. aeru ginosa (PSE-2) in the presence of 32 mg/l of cefe pime (O; MBC >32 mg/l). In the diagram the anti biotic concentration in the culture is given (mg/l). x = Control.
Fig. 6. Growth of E. coli B3 (isoelectric point 5.3+10.8) in the presence of 128 mg/l of piperacillin (O; MBC >64 mg/l) and piperacillin (32 mg/l) plus tazobactam (4 mg/l) (□; MBC combination at the ra tio of 8:1 32 mg/l). In the diagram the piperacillin concentration alone (PP) or in the combination (PP + YTR) is given, x = Control.
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In vitro Activity of Cefepime
Time, h Fig. 7. Growth of K. pneumoniae B2919 (K.1) in the presence of 16 mg/l of piperacillin (O; MBC > 16 mg/1) and piperacillin (4 mg/l) plus tazobactam (0.5 mg/l; □; MBC combination 4 mg/l). In the diagram the concentration of piperacillin alone (PP) or in the combination (PP + YTR) is given, x = Control.
265
Time, h Fig. 9. Hydrolytic activity of ß-lactamase prepa ration from E. coli B3, P. aeruginosa (PSE-2) (O), K. pneumoniae B2919 (■), and E. cloacae B53 ( • ) on 50 mg/l of cefepime. x = Control, 50 mg/l cefepime in phosphate-buffered saline.
Fig. 8. Growth of E. cloacae B53 in the presence of 32 mg/l of piperacillin (O; MBC 32 mg/l) and piperacillin (32 mg/l) plus tazobactam (4 mg/l) (□) or 2 mg/l plus 0.25 mg/l respectively (■; MBC of combination 2 mg/l). In the diagram the concentra tion of piperacillin (PP) alone or in the combination (PP + YTR) is given.
P-Lactamase Stability Crude (3-lactamase preparations from cultures of E. coli B3 and P. aeruginosa PSE-2 inactivated cefepime within 1 h, from K. pneumoniae Kl within 6 h, and from E. cloacae producing inducible (3-lactamase within 24 h (fig. 9). Spontane ous inactivation of the compound was 40% within 24 h. All enzymes inactivated
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against K. pneumoniae B2929 (fig. 7) and E. cloacae B53 (fig. 8) for up to 6 h and re growth was obtained within 24 h. A simi lar result was obtained with piperacillin alone, but then higher concentrations of piperacillin were needed. The combina tion was also bactericidal against the peni cillinase-producing strain of S. aureus B15771.
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266
Discussion As reported earlier, with ceftazidime, cefotaxime, and aztreonam [6, 9] and later with ceftibuten and piperacillin in combi nation with tazobactam at the ratio of 8:1 [7], also cefepime was very active against the European blood isolates (table 1), the majority of which were resistant to céfazo line and/or gentamicin. This is in agree ment with reports of other investigators [1, 3, 11] who have reported high activity of cefepime also against E. cloacae, Serratia marcescens, and M. morganii and against ceftazidime- and cefotaxime-resistant gram-negative bacilli. The compound showed similar activity as that of cefti buten, was twofold or more active than ceftazidime, and was manifold more ac tive than piperacillin/tazobactam against the gram-negative bacilli [7], Strains of Acinetobacter sp. were, however, resistant to cefepime. In contrast to ceftibuten, cefepime was also active against staphylo cocci, although not against the cefazolineresistant strains. Thus 6% of the gram negative bacilli were considered resistant (MIC > 16 mg/1), comprising only strains of Acinetobacter sp. (n = 10) and Pseu domonas sp. (n = 11). By comparing the results from an ear lier study with the same bacteria [7], 22% of the gram-negative bacilli were resistant to ceftibuten (MIC > 16 mg/1), 14% to cef tazidime, and 15% to piperacillin/tazo bactam (MIC > 64 mg/1).
The bactericidal activity of cefepime was confirmed by the low MBC values (table 3) and the killing curve results (fig. 1-5) against strains producing different (3-lactamases. For comparison, the pres ence of tazobactam restored the bacteri cidal activity of piperacillin (fig. 6-8). Therefore, the excellent activity of cefe pime, as of other newer (3-lactam antibiot ics (tables 2, 3), might be explained by its stability to many (3-lactamases followed by rapid access to the penicillin-binding pro teins (tables 2 and 3). However, an inter esting observation was that crude enzyme preparations from tenfold concentrated cultures inactivated cefepime and pipera cillin/tazobactam at different degrees (fig-9). In conclusion, the narrow range of MIC values of cefepime together with its stability to many (3-lactamases gives it a predictable degree of in vitro activity against a given and wide spectrum of bac teria. It is also suggested that cefepime is active against bacteria with derepressed constitutive (3-lactamase production, as also observed by other investigators [18, 19]. It was shown that the activity of cefe pime against strains with chromosomally encoded high-level production of (3-lactamases was only little reduced because of its low affinity for these enzymes.
References 1 Bodey, G. P.; Ho, D. H.: Blanc, B. de: In vitro studies with BMY-28142, a new broad-spectrum cephalosporin. Antimicrob. Agents Chemother. 27: 265-268(1985). 2 Cefepime. Investigator Brochure, Bristol-Myers Company, Pharmaceutical Research and Devel opment Division (1987).
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piperacillin within 1 h, while when com bined with tazobactam, the inactivation was slower, except with the enzymes from E. coli B3 and P. aeruginosa PSE-2.
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12 Jacobs, M. R.; Aronoff, S.C.; Johenning, S.; Schlaes, D. M.; Yamabe, S.: Comparative activi ties of the beta-lactamase inhibitors YTR-830, clavulanate and sulbactam combined with ampicillin and broad spectrum penicillins against de fined beta-lactamase producing aerobic gram negative bacilli. Antimicrob. Agents Chemother. 29: 980-985(1986). 13 Lambert, H.; Phillips, I.; Wise, R.J.: Ceftazidime in clinical practice. J. antimicrob. Chemother. 12: suppl. A (1983). 14 Manual of clinical microbiology; 4th ed. Editors: Lennette, E.H., Balows, A., Hausler, W.J., Jr., Shadomy, H.J. (American Society for Clinical Microbiology, Washington 1986). 15 Masuyoshi, S.; Arai, S.; Miyamoto, M.; Mitsuhashi, S.: In vitro antimicrobial activity of cefo taxime, a new cephalosporin. Antimicrob. Agents Chemother. 18: 1-8(1980). 16 Callaghan, C.H.: Description and classification of new cephalosporins and their relationship with the established compounds. J. antimicrob. Che mother. 5: 635-671 (1979). 17 Olsson-Liljequist, B.; Dornbusch, K.; Nord, C. E.: Characterization of three different beta-lac tamases from the Bacteroides fragilis group. An timicrob. Agents Chemother. 18: 220-225(1980). 18 Phelps, D.J.; Carlton, D. D.; Farrel, C.A.; Kess ler, R.E.: Affinity of cephalosporins for (5-lactamases as a factor in antibacterial efficacy. Anti microb. Agents Chemother. 29: 845-848 ( 1986). 19 Sanders, C.C.; Sanders, W. E., Jr.: Type I p-lactamases of gram-negative bacteria: interactions with p-lactam antibiotics. J. Infect. Dis. 154: 792-800(1986).
K. Dornbusch Department of Clinical Microbiology Karolinska Hospital S—104 01 Stockholm (Sweden)
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3 Conrad, D.A.; Scribner, R.K.; Weber, A. H.; Marks, M.I.: In vitro activity of BMY-28142 against pediatric pathogens including isolates from cystic fibrosis sputum. Antimicrob. Agents Chemother. 28: 58-61 (1985). 4 Dornbusch, K.: Assay of gentamicin concentra tions in serum specimens by microbiological and radioenzymatic methods. Scand. J. infect. Dis. 9: 227-229(1977). 5 Dornbusch, K.; Frolander, F.; Cacciapuoti, A. F.; Naples, L.; Hare, R.S.; Miller, G.M.: In vitro ac tivity of Sch 34343 against gram-negative bacteria producing characterized beta-lactamases. J. antimicrob. Chemother. 15: suppl. C, pp. 85-88 (1985). 6 Dornbusch, K.; Bengtsson, S.; Brorson, J.E.; Fritz, H.; Henning, C.; Kronvall, G.; Larsson, P.; Malmborg, A.S.; Thore, M.; Tarnvik, A.; Walder M.: Susceptibility to beta-lactam antibiotics and gentamicin of gram-negative bacilli isolated from hospitalized patients. A Swedish multicenter study. Scand. J. infect. Dis. 20: 641-644(1988). 7 Dornbusch, K.; Kronvall, G.; Goransson, E.; Mortsell, E.: Comparative in vitro activity of the penem FCE 22101 against recent European blood culture isolates. J. antimicrob. Chemother. 23: suppl. C, pp. 43-52(1989). 8 Ericsson, H. M.; Sherris, J.C.: Antibiotic sensitiv ity testing: report of an international collabora tive study. Acta pathol. microbiol. scand., B, Mi crobiol. Immunol., suppl. 217 (1971). 9 European Study Group on Antibiotic Resistance: Susceptibility to beta-lactam antibiotics in septi cemia isolates from 29 European laboratories. Eur. J.clin. Microbiol. 6: 515-520(1987). 10 Geddes, A.M.: Studies of beta-lactam antibiotics in systemic infections. Evaluation of new betalactam antibiotics. Rev. infect. Dis. 8: suppl. 3, pp. 333-348(1986). 11 Giamarellou, H.; Sahin, A.; Chryssouli, Z.: Com parative in vitro evaluation of BMY-28142, a new broad-spectrum cephalosporin, versus other beta-lactams against multiresistant gram-nega tive isolates. Drugs exp. clin. Res. 13: 149-153 (1987).
