ANTIMICROBIAL AGzNTs A CazmoT AY, Apr. 1976, p. 690-695 Copyright C 1976 American Society for Microbiology

Vol. 9, No. 4 Printed in U.SA.

Antibacterial Activity of Cefuroxime, a New Cephalosporin Antibiotic, Compared with That of Cephaloridine, Cephalothin, and Cefamandole SUSANNAH EYKYN, CLARE JENKINS, ANNA KING, AND IAN PHILLIPS* Department of Microbiology, St. Thomas's Hospital Medical School, London, SE1, England

Received for publication 18 November 1975

The in vitro activity of cefuroxime, a new cephalosporin derivative, was compared with that of cephaloridine, cephalothin, and cefamandole against strains of gram-positive and gram-negative bacteria recently isolated from clinical sources. Cefuroxime showed very similar activity to cefamandole against Staphylococcus aureus, Haemophilus influenzae, and most members of the Enterobacteriaceae. It was more active than cefamandole against gonococci, pneumococci, and most streptococci. Increasing the inoculum size appeared to have less effect on the minimum inhibitory concentrations of cefuroxime for gram-negative bacilli than has been found with the other cephalosporin derivatives, and minimum bactericidal concentrations of cefuroxime were only marginally greater than minimum inhibitory concentrations.

Cefuroxime, 6R,7R-3-carbamoyloxy-methyl7-(2z)-2-methoximino (fur-2-yl) acetamido-ceph3-em-4-carboxylate, is a new semisynthetic parenteral cephalosporin with a broad antibacterial spectrum. In this study, its in vitro antibacterial activity was compared with that of cephaloridine, cephalothin, and cefamandole (Fig. 1).

faciens (9); Acinetobacter anitratus (7); A. lwoffii (10); Serratia marcescens (16); Pseudomonas aeruginosa (30); P. acidovorans (15); P. cepacia (9); P. fluorescens (7); P. maltophilia (12); P. stutzeri (7); P.

thomasii (8); P. pseudoalcaligenes (12); and Bacteroides fragilis (100). Inocula for susceptibility testing were prepared by overnight culture in nutrient broth (Southern Group Laboratories) supplemented with 10% sapMATERIALS AND METHODS onin-lysed horse blood for investigation ofN. gonorThe organisms investigated were isolated from rhoeae and B. fragilis. For testing H. influenzae, the specimens received in the clinical laboratory of St. strain was grown overnight on chocolate blood agar Thomas' Hospital over the past 3 years, except for (Oxoid blood agar base no. 2, CM 271, plus 10% four ampicillin-resistant strains of Haemophilus in- heated-defibrinated horse blood) and then susfluenzae that were provided by Glaxo Research Lab- pended in nutrient broth. The broth was diluted to oratories. Beta-lactamase production was demon- give an inoculum of approximately 103 colony-formstrated for these four strains using both the iodine ing units (CFU) when used with a multiple inoculaplate technique (R. B. Sykes, personal communica- tor. For S. aureus, an inoculum of approximately 107 tion) and the chromogenic cephalosporin substrate CFU was also prepared, and for H. influenzae, inocula of approximately 105 and 107 were also prepared. as described by O'Callaghan et al. (3). The following bacteria were examined: Staphylo- A limited number of investigations were carried out in a liquid medium; for these, the nutrient broth was coccus aureus (12 penicillin-susceptible, 28 penicillin-resistant and methicillin-susceptible, and 40 diluted so that 0.02 ml contained approximately 104, methicillin-resistant strains); coagulase-negative 106, and 108 CFU. Minimum inhibitory concentrations (MICs) were staphylococci (39); enterococci (40); alpha-hemolytic and nonhemolytic streptococci (Streptococcus bovis, determined on solid medium (Oxoid DST agar CM 4; S. sanguis, 4; S. mitior, 4; S. mutans, 3; S. milleri, 261) containing suitable doubling dilutions of cefu2; S. salivarius, 1; unclassified, 2); S. pneumoniae roxime, cephaloridine, cephalothin, or cefamandole. (19); beta-hemolytic streptococci (Lancefield groups For N. gonorrhoeae and B. fragilis, 10% lysed blood A 16, B 9, C 2, F 1, G 12); Neisseria gonorrhoeae was added; for H. influenzae, Oxoid Columbia agar (100); H. influenzae (4 beta-lactamase-producing base (CM 331) was used with 2% V factor (Southern strains, 20 non-beta-lactamase-producing strains); Group Laboratories) and 1% X factor (Southern Escherichia coli (44); Citrobacter koseri (30); C. Group Laboratories) added. The MIC was defined as freundii (40); Klebsiella aerogenes (50); K. ozaenae the lowest concentration of antibiotic at which no (19); Proteus mirabilis (29); P. vulgaris (20); P. mor- visible growth appeared. Minimum bactericidal conganii (20); P. rettgeri (18); Enterobacter cloacae (29); centrations (MBCs) were determined in 1.5% gluE. aerogenes (20); Providencia stuartii (27); P. alcali- cose indicator broth (Oxoid nutrient broth no. 2, CM 690

CEFUROXIME ANTIBACTERIAL ACTIVITY

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-17IL2.

-

67) containing doubling dilutions of antibiotic in a volume of 2 ml. After overnight incubation a standard loopful of nutrient broth (containing approximately 0.006 ml) was transferred to antibiotic-free blood agar (Oxoid blood agar base no. 2, CM 271, plus 4% defibrinated horse blood). The MBC was defined as the lowest concentration that yielded no growth on subculture. All cultures were incubated aerobically at 37 C for 18 h, except those of B. fragilis, which were incubated in a GasPak jar (BBL).

C-NH

S

CH

I

_

RESULTS Cumulative percentages of strains of S. aureus susceptible to the four cephalosporins are

CHrO/ C CH3

COOH

OCH-C-

CH-SIIN

OH

-

N

I

COOH

CH3

C

-CONH

o ICN

%CH2

O

CO

NH2

COOH

CH3

691

FIG. 1. Chemical structures of (top to bottom) cephaloridine, cephalothin, cefamandole, and cefuroxime.

shown in Table 1. Results for penicillin-susceptible, penicillin-resistant and methicillin-susceptible and methicillin-resistant strains are given separately. Table 2 lists results for alphahemolytic and nonhemolytic streptococci, betahemolytic streptococci, and pneumococci, and Table 3 gives results for enterococci, coagulasenegative staphylococci, and H. influenzae. The effect of increasing inoculum size on the MICs of ampicillin and cefuroxime for both beta-lactamase-producing and non-beta-lactamase-producing strains of H. influenzae is shown in Table 4. The results for N. gonorrhoeae and B. fragilis are shown in Table 5. The results of susceptibility testing of the majority of gram-negative bacilli are given in Table 6, in three groups, using the same arbitrary divisions as were used in our previous in vitro assessment of cefamandole (1). The first group includes organisms usually susceptible to

TABLE 1. Activity of cephaloridine, cephalothin, cefamandole, and cefuroxime against Staphylococcus .. . . Penicillin susceptible

aureusa ~~~Penicillin resistant, methicillin sus-

. Methicillin resistant

ceptible

MIC Cepha- Cepha- Cefa- Cefur- Cepha- Cepha- Cefa- Cefur- Cepha- Cepha- Cefa- Cefur(,ug/ml) loridine lothin mandole oxime loridine lothin mandole oxime loridine lothin mandole oxime l o3b

1 07 1 03 1 07 103 107 103 107 103 107 103 107 1 03 107 103 107 103 107

103

107 103 107 103 107

(12) (12) (12) (12) (12) (12) (12) (12) (28) (28) (28) (28) (28) (28) (28) (28) (40) (40) (40) (40) (23) (23) (40) (40) 0.01 0.03 25 25 0.06 100 92 17 8 0.12 92 75 25

21 7 4

5

3

75 36 50 20 10 13 100 100 100 50 50 100 57 96 54 7 38 13 38 100 83 58 50 64 100 89 86 7 14 11 53 13 63 100 100 100 75 100 96 36 68 68 73 15 95 82 96 93 100 100 98 18 95 93 100 96 100 43 100

0.25 0.5 1.0 2.0 4.0 8.0 16

326'°

100

0 1 C

100

3 15 25 45 83 98

5 10 17 17 9 25 25 65 17 33 30 100 17 48 43 74 93 68 80 100 ~~~ ~ 100 93 90 ~~~~~~~~~~~100

C X100 100

Results are expressed as cumulative percentages of strains susceptible to the amount of antibiotic shown in the MIC column. a

b

Inoculum expressed as CFU. Numbers in parentheses are numbers of strains tested.

692

ANnmicRoB. AGZNTS. CHZMOTHZ]t.

EYKYN ET AL.

TABLz

2. Activity of cephaloridine, cephalothin, cefamandole, and cefuroxime against streptococci and pneumococcia

Alpha-hemolytic and nonhemolytic Beta-hemolytic streptococci (40 S. pneumoniae (19 strains) strains) (20 strains) streptococci MIC (g/ Cephal- Cephal- Cefa- Cefu- Cephal- Cephal- Cefa- Cefu- Cephal- Cephal- Cefa- Cefuoridine othin mandole roxime oridine othin mandole roxime oridine othin mandole roxime 78 20 40.003 53 16 8 15 95 25 0.006 100 5 32 50 18 95 35 5 40 0.01 53 42 100 80 93 3 98 55 5 50 0.03 100 89 95 95 65 100 70 20 20 75 0.06 100 98 100 95 75 35 55 85 0.12 100 100 85 70 85 90 0.25

100 95 95 100 a Results are expressed as cumulative percentages of strains susceptible to the amount of antibiotic shown in the MIC column. The inoculum was approximately 103 CFU in all cases. 0.5 1.0 2.0

100

90

90 100

TABLz 3. Activity of cephaloridine, cephalothin, cefamandole, and cefuroxime against coagulase-negative staphylococci, enterococci, and H. influenzaea Coagulase-negative staphylococci

Enterococci (40 strains)

H.

influenza

(24 strains)

(39 strains)b MIC (Ag/ ml) Cephal- Cephal- Cefa- Cefu- Cephal- Cephal- Cefa- Cefu- Cephal- Cephal- Cefa- Cefuoridine othin mandole roxime oridine othin mandole roxime oridine othin mandole roxime 3 44 0.01 5 59 0.03 8 3 8 85 0.06 8 8 10 31 67 0.12 87 38 63 8 4 64 28 77 90 0.25 100 96 17 13 54 77 90 87 0.5 100 88 50 92 79 92 90 1.0 100 75 3 85 95 95 2.0 95 100 3 87 97 95 95 4.0 5 5 90 97 95 95 8.0 8 5 20 90 49 100 95 100 16 8 85 98 100 100 100 32 100 18 100 64 100 >128 a Results are expressed as cumulative percentages of strains susceptible to the amount of antibiotic shown in the MIC column. The inoculum was approximately 103 CFU in all cases. b No. of strains tested. c Four beta-lactamase-producing strains and 20 non-beta-lactamase-producing strains.

cephalosporin C, the second includes those in- ble and widely used for over 10 years and with

trinsically resistant, and the third includes various nonfermenting bacteria. The effect of increasing inoculum size on the MIC of cefuroxime for selected gram-negative bacilli is shown in Table 7. The MBC for these organisms and its relation to their MIC is given in Table 8.

DISCUSSION In this study the antibacterial activity of the new parenteral cephalosporin, cefuroxime, was compared with that of cephaloridine and cephalothin, which have been commercially availa-

that of cefamandole, a newer analogue considerably more active against gram-negative bacilli than cephaloridine and cephalothin (1). All strains of S. aureus examined, including those resistant to methicillin, were less suceptible to cefuroxime than to cephaloridine or cephalothin. The activity of cefuroxime against staphylococci was very similar to that of cefamandole. No increase in the MIC of cefuroxime was noted with the larger inoculum of 107 CFU of penicillinase-producing staphylococci, which would suggest that this cephalosporin is stable to staphylococcal beta-lactamase. Among the

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CEFUROXIME ANTIBACTERIAL ACTIVITY

other gram-positive cocci investigated, cefuroxime was as active as cephaloridine against alpha-hemolytic, nonhemolytic, and beta-hemolytic streptococci and was: the most active of the four derivatives against pneumococci. The 39 strains of coagulase-negative staphylococci tested were most susceptible to cephaloridine and least susceptible to cefuroxime, but it is difficult to draw firm conclusions from susceptibility tests on such a heterogeneous collection of organisms. None of the cephalosporins showed useful activity against the enterococci, but cefuroxime was again least active. Cefuroxime was particularly active against TABLE 4. Activity of ampicillin and cefuroxime against 4 beta-lactamase-producing strains" and 20 non-beta-lactamase-producing strains of H. influenzae No. of strains with MIC shown with: MIC (llg/ Cefuroxime Ampicillin

10k'

105

107 5 (2) 20 (4) (1) 2 (1)

103

107 1 (1) 20 (4) 10'

>16 8.0 4.0 1 2.0 (1) 1 1.0 1 (2) 1 3 (1) 3 (1) 4 12 (3) 12 (2) 0.5 0.25 7 4 6 (1) 3 0.12 7 3 2 0.06 i2 a No. of beta-lactamase-producing strains are shown in parentheses. bInoculum size, CFU.

693

the large number of strains of gonococci tested. These included both penicillin-susceptible organisms and those showing increased resistance to penicillin with MICs ofpenicillin of 1.0 ,ug or more per ml. This finding has been reported in much greater detail by I. Phillips, A. King, C. Warren, and B. Watts (J. Antimicrob. Chemother., in press). Cefuroxime and cefamandole were highly active against H. influenzae including those strains producing beta-lactamase, with MICs of 0.5 ,ug or less per ml. Similar activity was reported for cefamandole by Kattan et al. (2). Cephaloridine was the least active of the four cephalosporins examined, but the MICs of this drug and those of cephalothin in this study are rather lower than those reported in our previous study and lower than those reported by Kattan et al. (2). However, susceptibility testing of H. influenzae to ampicillin and cephalosporins presents technical difficulties. It clearly emerges from Table 4 that for ampicillin the size of inoculum markedly affects the MIC; using the lower inoculum (our standard practice for most organisms), there was for many strains only a small or, in some cases, no difference in MIC for beta-lactamase producers and non-beta-lactamase producers. With cefuroxime, for most strains only

an

inoculum of 107

CFU produced a marked increase in MIC. Against the Enterobacteriaceae usually susceptible to cephalosporin C (group 1 in Table 6), which includedE. coli, C. koseri, K. aerogenes, K. ozaenae, and P. mirabilis, cefuroxime and cefamandole were considerably more active than either cephaloridine or cephalothin; 80 to

TABLE 5. Activity of cephaloridine, cephalothin, cefamandole, and cefuroxime against 100 strains of N. gonorrhoeae and B. fragilisa N. gonorrhoeae B. filis MIC (Qg/ml) Cephal- Cephal-. CefaMIC (glml) Cephal- Cephal- CefaCefuCefuoridine

othin

mandole roxime

ordine

othin

mandole roxime

0.003 1 0.5 0.007 15 1.0 0.015 14 63 2.0 0.03 36 73 4.0 1 6 0.06 46 78 8.0 3 3 7 11 0.12 12 49 86 16 26 61 15 38 0.25 35 52 92 32 82 91 65 58 0.5 56 66 64 95 93 89 93 70 1.0 1 66 81 99 >64 100 100 100 100 2.0 42 89 95 100 4.0 68 96 97 8.0 99 100 97 16.0 100 100 a Results are expressed as cumulative percentages of strains susceptible to the amount of antibiotic in the MIC column. The inoculum was approximately 103 CFU in all cases.

694

ANTIMICROB. AGZNTS. CHRMOTHER.

EYKYN ET AL.

TABLE 6. Activity of cephaloridine, cephalothin, cefamandole, and cefuroxime against gram-negative bacillia Group 3 Group 2 Group 1b MIC (,zg/ Cephal- Cephal- Cefa- Cefu- Cephal- Cephal- Cefa- Cefu- Cephal- Cephal- Cefa- Cefuoridine othin mandole roxime oridine othin mandole roxime oridine othin mandole roxime ml) (172)" (172) (139) (172) (199) (199) (176) (199) (126) (126) (119) (126)

1 1 1 0.06 4 1 1 5 0.12 1 7 12 2 1 0.25 9 9 2 20 7 3 0.5 11 2 16 5 23 1.0 4 8 53 2 2 3 26 42 7 38 66 5 2.0 22 27 4 1 3 19 51 46 8 78 87 8 4.0 57 52 1 10 31 62 5 12 60 87 95 15 60 73 8.0 74 8 32 39 67 13 91 25 95 19 16.0 81 85 24 14 44 52 34 75 83 26 88 93 99 32.0 87 99 37 42 82 86 28 21 56 55 93 95 64.0 93 27 60 58 90 90 30 52 94 99 99 46 97 128.0 100 100 100 100 100 100 > 128 100 100 100 100 100 100 a Results are expressed as cumulative percentages of strains susceptible to the amount of antibiotic shown in the MIC column. The inoculum was approximately 103 CFU in all cases. A Group 1 consists ofE. coli, C. koseri, K. aerogenes, K. ozaenae, and P. mirabilis; group 2, C. freund ii, E. aerogenes, E. cloacae, S. marcescens, P. vulgaris, P. morganii, P. rettgeri, and Providencia spp; group 3, Acinetobacter spp., P. aeruginosa, and Pseudomonas s8p. c Number of strains tested.

TABLE 7. Effect of increasing inocula of strains of gram-negative bacillia on the MIC of cefuroxime MIC No. of strains

103 0.25 0.5 1.0 2.0 4.0

(g.g/ml) with inoculum (CFU) of 104 1.0 1.0 1-2 2-8 16-32 8-64

106

10'

1.0 1.0 64 4.0 1 1-4 4-16 3 11 2-32 8-128 6 16-64 32-64 16-64 3 8.0 16-128 a E. coli (5), C. freundii (3), C. koseri (2), K. aerogenes (4), K. ozaenae (1), P. mirabilis (2), P. rettgeri (2), P. morganii (2), E. aerogenes (1), E. cloacae (1), and P. stuartii (2). 1

90% of strains were inhibited by 4 gg or less of cefuroxime or cefamandole per ml, whereas only about 50% were as susceptible to cephaloridine and cephalothin. Cefuroxime was also as active as cefamandole against the Enterobacteriaceae intrinsically resistant to cephalosporin C (group 2 in Table 6). This group included C. freundii, Enterobacter spp., S. marcescens, indole-positive Proteus spp., and Providencia spp. Overall, 60% of this group were inhibited by 8 ug or less of both cefuroxime and cefamandole per ml, but only 12 to 15% were as susceptible to cephaloridine and cephalothin. Individual members of group 2, however, showed marked differences in susceptibility both to cefuroxime and to the other derivatives: for instance, 85% of the strains of P. vulgaris had a

cefuroxime MIC of 16 ,ug or more per ml, as did over 50% of the P. morganii and the P. stuartii,

but 90% of the strains of P. rettgeri were inhibited by 8 ,ug or less per ml. Previous experience has shown that increasing the inoculum size may markedly affect the MIC with cephalosporins but that the magnitude of this increase is variable (1). Cefuroxime is no exception, as can be seen in Table 7, but the increase in MIC obtained with the larger inocula was rather smaller than that reported for cefamandole (1). Similarly, the ratio of MBC to MIC of cefuroxime for these same strains of gram-negative bacilli was considerably smaller than that found with cefamandole. Although these findings are of interest, it is impossible to assess their importance in clinical medicine. The nonfermenting bacteria in group 3 (Table 6) were in general resistant to the cephalosporins tested, but cefuroxime showed useful activity against eight of the nine strains of P. cepacia examined and against P. thomasii, which is also susceptible to cephalexin (1). The cephalosporins investigated were inactive against the majority of strains of B. fragilis, the only anaerobic organism examined. Sutter and Finegold (4), in their extensive study of the antimicrobial susceptibility of anaerobic bacteria, found B. fragilis to be rather more resistant than we did to both cephaloridine and cephalothin. It is worth noting, though, that their inoculum was 105 to 106 organisms, and they incubated the plates for 48 h compared with our inoculum of 103 and incubation time of 24 h.

695

CEFUROXIME ANTIBACTERIAL ACTIVITY

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TABLE 8. MIC and MBC of cefuroxime for 25 gram-negative bacillia MIC

(/glMl)

1.0-2.0 2.0-4.0 4.0-8.0 8.0-16 16-32 32-64 128 Organisms as listed in Table 7. 1.0 2.0 4.0 8.0 16.0 32.0 64.0

a

Inoculum 104 CFU No. ofMBC (g/m) Ratio of MBC to MIC strains

4 3 8 3 4 2 1

1-2 1-2 1-2 1-2 1-2 1-2 2

They did not investigate cefuroxime or cefamandole. From this study, we conclude that cefuroxime has a very broad antibacterial spectrum: it was in general the most active ofthe four derivatives tested. Its main advantages would seem to be superior activity against gonococci, Haemophilus spp., and the Enterobacteriaceae. In vitro assessment of any new antibiotic, however, is of limited value without appropriate pharmacokinetic and toxicological data, but should these data prove satisfactory, cefuroxime should certainly merit clinical trial. ACKNOWLEDGMENTS We thank Glaxo Research Ltd, Greenford, Middlesex England, for financial support and for the cefuroxime used

MIC

(/Ag/ml)

1.0 2.0 4.0 8.0 16.0 32.0 64.0

No. of strains

3 1 5 4 7 3 2

Inoculum 106 CFU Ratio of MBC to MIC MBC (,tg/m1)

1-2 4 8-32 8-16

16->128 64-128 128->128

1-2 2 2-8 1-2 1->8 2-4 2->2

in this trial, and Eli Lilly & Co. for supplies of cefamandole.

LITERATURE CITED 1. Eykyn, S., C. Jenkins, A. King, and I. Phillips. 1973. Antibacterial activity of cefamandole, a new cephalosporin antibiotic, compared with that of cephaloridine, cephalothin, and cephalexin. Antimicrob. Agents Chemother. 3:657-661. 2. Kattan, S., P. Cavanagh, and J. D. Williams. 1975. Relationship between ,-lactamase production by

Haemophilwu influenzae and sensitivities to penicillin

and cephalosporins. J. Antimicrob. Chemother. 1:7984. 3. O'Callaghan, C. H., A. Morris, S. A. Kirby, and A. H. Shingler. 1972. Novel method for detection of S-lactamase by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1:283-288. 4. Sutter, V. L., and S. M. Finegold. 1975. Susceptibility of anaerobic bacteria to carbenicillin, cefoxitin and related drugs. J. Infect. Dis. 131:417422.

Antibacterial activity of cefuroxime, a new cephalosporin antibiotic, compared with that of cephaloridine, cephalothin, and cefamandole.

The in vitro activity of cefuroxime, a new cephalosporin derivative, was compared with that of cephaloridine, cephalothin, and cefamandole against str...
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