ANTIMICROBIAL Azw's AD CHEMOTHURAPY, Mar. 1976, P. 511-519
Copyright C 1976 American Society for Microbiology
Cefuroxime,
a
Vol. 9, No. 3 Printed in U.SA.
New Cephalosporin Antibiotic: Activity In Vitro
CYNTHIA H. O'CALLAGHAN,* R. B. SYKES, A. GRIFFITHS, AND J. E. THORNTON Glaxo Research Ltd., Greenford, Middlesex, England
Received for publication 25 September 1975
Cefuroxime is a new broad-spectrum cephalosporin antibiotic with increased stability to f8-lactamases. This stability, although no absolute in all cases, has the effect of widening the antibacterial spectrum of the compound so that many organisms resistant to the established cephalosporins are susceptible to cefuroxime. It is active against gram-positive organisms, including penicillinaseproducing staphylococci, but it is less active against methicillin-resistant strains. In addition to its high activity against non-,8-lactamase-producing gram-negative bacteria, cefuroxime effectively inhibits the growth of many 1Blactamase-producing strains, including Enterobacter, Klebsiella, and indole-positive Proteus spp. It is highly active against Neisseria gonorrhoeae, Neisseria meningitidis, and also Haemophilus influenzae, including ampicillin-resistant strains. Cefuroxime is rapidly bactericidal and induces the formation and subsequent lysis of filamentous forms over a small concentration range. All the cephalosporins in current clinical use have some limitations in their spectrum of antibacterial activity. None is very active against Neisseria or Haemophilus infZuenzae, whereas poor activity against many gramnegative bacilli can often be attributed to destruction by 4-lactamases. Other deficiencies encountered among this group of antibiotics include metabolic instability and high binding to serum proteins. Cefuroxime, 6R,7R-3-carbamoyloxymethyl-7-(2Z)-2-methoxyimino (fur2-yl) acetamido-ceph-3-em-4-carboxylate, is a new semisynthetic cephalosporin analogue that overcomes some of these disadvantages. Its structure is given in Fig. 1. MATERIALS AND METHODS Antibiotics. Cefuroxime sodium was prepared in our own laboratories as a creamy white crystalline solid with a molecular weight of 446.4; it is approximately 20% soluble in water. Cephaloridine, cephalothin, and cephalexin were commercially available materials made by Glaxo Laboratories Ltd.; cefazolin (Totacef, Bristol Laboratories) and ampicillin were purchased. Organisms. Most of the organisms tested were recent clinical isolates from Westminster Hospital (London), London Hospital, Papworth Hospital (Cambridge), and Southmead Hospital (Bristol). Strains of H. influenzae were given to us by P. Cavanagh of the Public Health Laboratory, Stokeon-Trent. Disk susceptibility testing. Strains of bacteria were tested for susceptibility to the antibiotics by a standardized disk technique described by Ericsson
and Sherris (2), using Oxoid agar no. 1 (Oxoid Ltd., London). MIC determinations. Minimum inhibitory concentrations (MICs) were determined by an agar dilution technique, in parallel tests with cephalothin. Serial twofold dilutions of freshly prepared standard antibiotic solutions were made into Oxoid no. 1 nutrient agar with or without added enrichment and poured into petri dishes. Plates were inoculated with a replicate inoculating device (Denley Instruments Ltd., Bolney, Sussex) with inocula containing approximately 105 or 107 colonyforming units. The MIC, in micrograms per milliliter, was read after 18 h of incubation at 37 C as the lowest concentration that inhibited growth. Correlation of MIC values with disk test results. The MIC values obtained by the agar plate technique were plotted against zone diameters obtained with disks containing 30 ,ug of antibiotic. From these experimental points a regression curve, calculated by the method of least squares, was drawn for each compound. Growth studies. Various concentrations of antibiotics were added to actively growing cultures in a Jouan biophotometer, and growth was followed by optical density measurements and viable counts. Samples removed at various times were examined under the microscope for morphological variants. The medium used was mainly Oxoid nutrient broth, but casein-yeast extract (CY) medium was used for strains of Proteus. Detection of f8-lactamase activity. All bacterial cultures were examined for 1-lactamase production using the chromogenic cephalosporin described by O'Callaghan et al. (9). Stability studies with f-lactamases. 13-Lactamases were derived from several bacterial species, 511
512
O'CALLAGHAN ET AL. S
rL
C_CONH
;/
l
\CH3
COONa+
Cefuroxime
FIG. 1. Structure of cefuroxime, sodium salt.
including Bacillus cereus, Staphylococcus aureus, Escherichia coli, Enterobacter spp., Proteus spp., Pseudomonas aeruginosa, and Klebsiella aerogenes. All enzymes were used as partially purified preparations, and activity against each of the cephalosporins was measured by a previously described method (9). Stability to mammalian liver homogenates. Fresh liver from mice, rats, and rabbits was homogenized with 25 times its weight of 0.1 M phosphate buffer (pH 7.0) in a Colworth stomacher (A. J. Seward, London). Human liver was homogenized with five times its weight of the same buffer. The homogenates were mixed with equal volumes of antibiotic solutions, and the mixtures were incubated at 37 C for 4 h. After incubation, samples were assayed for antibacterial activity by the largeplate assay against B. subtilis NCIB 8993. Samples were also chromatographed on Whatman no. 1 sheets buffered to pH 7.0 with 0.1 M phosphate buffer. Zones of antibacterial activity were revealed by bioautography on B. subtilis plates. Effect of human serum. The amount of antibiotic bound to human serum protein was determined by an ultrafiltration technique using an Amicon 8MC ultrafiltration unit. MICs were measured in nutrient broth and in nutrient broth containing 50% human serum. RESULTS
Antibacterial activity. The in vitro antibacterial activities of cefuroxime and cephalothin against representative gram-positive bacteria are shown in Table 1. Cefuroxime was highly active against S. aureus, regardless of whether the strains produced a penicillinase. It was, however, much less active against strains resistant to 8 ,ug or more of methicillin per ml. All strains of Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus viridans tested were very susceptible to cefuroxime, but strains of Streptococcus faecalis were resistant. The strains of Clostridia tested were mainly susceptible to cefuroxime. In some instances, cefuroxime was slightly less active than cephalothin. Against gram-negative bacteria cefuroxime was the more active compound (Table 2). More than 98% of the strains of E. coli tested were susceptible to 8 ,ug or less of cefuroxime per ml, compared with 76% for cephalothin. Strains
ANTIMICROB. AGENTS CHEMOTHER.
that produced a (3-lactamase had much the same susceptibility to cefuroxime as those that did not, whereas the 18-lactamase-producing strains were much more resistant to cephalothin. Klebsiella spp. were also susceptible to cefuroxime, with more than 80% of strains tested susceptible to 16 ,ug/ml or less. Its activity against various Enterobacter spp. was particularly good; most of these organisms were f8-lactamase producers, rendering them unsusceptible to cephalothin. However, members of the Serratia group were resistant to both antibiotics. Strains of Proteus mirabilis were very susceptible to both compounds, 75% of the strains being inhibited by 2 ,ug/ml or less. Cefuroxime had a geometric mean MIC of 27 ,ug/ml for indole-positive strains of Proteus; these organisms produced an inducible 83-lactamase and were completely unsusceptible to cephalothin. The activity of cefuroxime and cephalothin was essentially the same against Salmonella spp., but cefuroxime was the more active compound against the strains of Shigella tested, even when the organisms were not (3-lactamase producers. Both compounds had relatively poor activity against Bacteroides fragilis and none against P. aeruginosa. Unlike most cephalosporins, cefuroxime had good activity against H. influenzae, including the strains resistant to ampicillin because they produce a class III (3-lactamase (11), which is very similar to that mediated by the transmissible R -factor RrEM (11). A further interesting feature of cefuroxime is its high activity against Neisseria gonorrhoeae and N. meningitidis strains. Effect of inoculum size. Increasing the size of the inoculum from 105 to 107 colony-forming units had little effect on the activity of cefuroxime against the majority of strains tested (Table 3), although against the two proteus strains the inoculum effect was quite large. The activity of the other cephalosporins was considerably diminished when high inoculum levels were used. Regression line analysis. Regression lines and equations correlating MIC values with zone diameters by the agar dilution method are shown in Fig. 2. The values for cefuroxime against most of the organisms tested fall close to the line, so that in general there is good correlation between zone size and MICs. Growth studies. Cefuroxime was bactericidal to most organisms at a concentration identical with, or close to, the MIC. This was confirmed by viable counts, which were done with a range of organisms, the counts being made at 0, 2, 4, and 6 h after inoculation. Table 4 shows that
CEFUROXIME, A NEW CEPHALOSPORIN ANTIBIOTIC
VOL. 9, 1976
513
TABLz 1. Comparative in vitro activities ofcefuroxime (CXM) and cephalothin (CET) against gram-positive organisms No. of strains with MIC (,Ag/mlI' No. of Organism O mstrains
Staphylococcus aureus, methicillin susceptible S. aureus, methicillin resistant Streptococcus pyogenes S. pneumoniae
S. viridans S. faecalis
Clostridium spp.
pound
0.125
0.25-
1-4
56 56 25 25 7 7 6 6 7
CXM CET CXM CET CXM CET CXM CET CXM
8 45 3 1 7 7 6 6 7
45 7 2 4
3 4 2 16
7
CET CXM CET CXM CET
4
3
6 6 7 7
a
Com-
8-16
>16
12 4
6
2
4
6
1
1
2
5
4
1
Geometric MIC
mean
0.25 0.125 5.9 2.0 0.125 z0.125 :0.125 c O. 125 20.125 20.125 >125.0 25.0 1.2 0.23
Inoculum, 105 colony-forming units.
cefuroxime was rapidly bactericidal. Like most cephalosporins, its action was comparatively slow against the strains of S. aureus but, even so, over 99% of the initial inoculum was killed by 6 h. The gram-negative organisms were killed rapidly, and in most cases over 99% of the very large inocula were killed within 2 h; the 3-lactamase-producing strains were killed as quickly as non-enzyme-producing strains. Turbidimetric measurements on growing cultures showed that there was always a period of increase in opacity after the compound had been added, which varied from species to species. Lysis then occurred rapidly, with the opacity of the culture falling below the sensitivity of the instrument. The rate of lysis was slower for staphylococci than for gram-negative organisms, which is consistent with the effect on the viable counts. Some examples are shown in Fig. 3 and 4. All susceptible organisms tested were lysed by cefuroxime, including N. gonorrhoeae and H. influenzae. The rate of lysis of gram-negative organisms by 13-lactam antibiotics has been shown to depend to varying degrees upon the osmolality of the medium used (4), and strains of Proteus are particularly susceptible to this effect. The example shown in Fig. 4 shows that cefuroxime lysed the organisms much more effectively in CY medium than in nutrient broth, which has a higher osmolality. Microscopic examination of various gramnegative organisms growing in low concentrations of cefuroxime showed that many of them produced filamentous forms over a concentration range between one-fourth and four times the MIC.
Stability to f3-lactamases. Eleven partially purified (8-lactamase preparations were tested for their ability to hydrolyze cefuroxime and three other cephalosporin antibiotics. The results in Table 5 show that cefuroxime was more stable to all the enzymes than the other compounds tested. Only three of the enzymes tested were able to hydrolyze cefuroxime relatively rapidly; these were the class IV enzyme produced by certain strains of K. aerogenes, a class I type of enzyme produced by some strains of B. fragilis (1), and the enzyme complex produced by B. cereus 659/H9 (5). Cefuroxime was unaffected by the ,8-lactamase from S. aureus, whereas enzyme hydrolysis of the other compounds was detected. Effect of liver esterases on cefuroxime. Cefuroxime has an ester group at position 3 (Fig. 1) but, unlike cephalothin and the 3acetoxymethyl analogues of cefuroxime, it does not lose any antibacterial activity when incubated for 4 h with homogenates of mouse, rat, rabbit, or human liver (Table 6). Chromatography of the reaction mixtures, followed by bioautography, confirmed that cefuroxime remained unchanged, whereas both cephalothin and the 3-acetoxymethyl analogue cefuroxime were converted into different compounds with reduced antibacterial activity. This stability is similar to that shown by cefoxitin, which also has a carbamate group at position 3 (C. S. Goodwin, E. B. Raftery, H. Skeggs, A. E. Hill, and C. M. Martin, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 13th, Washington, D.C., Abstr. 51, 1973). Effect of human serum. Thirty-three per-
514
ANTIMICROB. AGENTS CHZMOTHZR.
O'CALLAGHAN ET AL.
TABLE 2. Comparative in vitro activities of cefuroxime (CXM) and cephalothin (CET) against gram-negative
organisms Organism
E. coli
Klebsiella spp. Acinetobacter spp. Enterobacter spp. (E. aerogenes, E. cloacae, E. Iiquefaciens) Enterobacteriaceae, others (Hafnia, Citrobacter, Providencia) Serratia spp.
No. of strains with MIC (Ag/ml) 8-16 32-62 0.25- 1-4 0.5
No. of strains
Compound
129 129 73 73 6 6 138 138
CXM CET CXM CET CXM CET CXM CET
7 4
13 13
CXM CET
1
:0.125
1
CXM CET Proteus mirabilis CXM CET P. morganii CXM CET P. vulgaris CXM CET P. rettgeri 1 CXM CET Salmonella spp. CXM CET Shigella spp. CXM CET B. fragilis CXM CET H. influenzae, ampicillin CXM AMPc susceptible H. influenzae, ampicillin CXM resistant AMP N. gonorrhoeae 22 CXM CET N. meningitidis CXM CET Pseudomonas aeruginosa CXM CET a Inoculum, 105 colony-forming units. b MICs spread too widely; average not meaningful. r AMP, Ampicillin. 8 8 27 27 9 9 21 21 4 4 40 40 10 10 16 16 16 16 15 15 22 22 2 2 12 12
2 1 23
1 6 8 19
8 106
2.9 7.0 8.2 24.3 3.6 58 10.2 >125
1
2 12
11.5 >125
1
1
6 8
2 3 3
6
1 4 3 1 12
116 >125 2.2 6.3 16 >125 39 >125
1
1
12 63 22 22 1
31 6
91 5
4
5 1
1
38 34 9 4 1
Geometric mean MIC
2 12 13 10
108 48 37 18 3
24 20
>125
1 6
2 8 3 21 1
-b
4
>125 3.6 3.1 4.6 17 53 60 0.5 0.25 0. 5 250 0.005 0.4 0.125 0.125 >125 >125
1 1
3
3
11 14
4 2
16
16 15 15
22 2 2 12 12
TABLE 3. Effect of inoculum size on the activity of cefuroxime and other cephalosporins MIC (,ug/ml) of: Organism
Strain
Escherichia coli E. coli Klebsiella sp. K. aerogenes Enterobacter cloacae E. cloacae
SM209 1301 48283 47423 PH660 PH781 248 423
Proteus mirabilis P. morganii a
Colony-forming units.
Cefuroxime
Cephalothin
Cephaloridine
Cefazolin
0___071______
no.
2 4 8 8 2 8 1 8
4 8 16 8 2 8 32 125
32 16 62 16 4 8 4 >250
>250 250 >250 62 125 125 >250 >250
62 62 4 32 4 250 125 >250
125 250 >250 >250 16 >250 250 >250
8 8 16 8 2 62 8 16
62 250 250 62 62 >250 16 16
Log2N
CEFUROXIME 30pg DISCS
MIC
(M/ml)
sr"
>256
o Staphylococcus aureus * Streptococcus faecalis o Streptococcus species * Haemophilus influenzae \ Bacteroides tragilis
00
8
-,Zr
7
128-
w&
6
64-
7t
*
.
0
\ Enterobacter sp. \ * Klebsiella sp. * Escherichia coli v Proteus mirabilis , Indole positive proteus , * * * Serratia sp. * Salmonella sp. * Shigella sp. .---^ -.* *-
I
*-&A 't
321 4
16
3
8
2
4
1
2
0
1
-1
0-5
Least squares line for y * k+a kz--55=skope a*15sintercept
.
£;
£
* @0
* °Uv
v °
8
8
°
\
o
\V
-2
0-25 -
-3
0-125[ 6
8
10
12
16
14
18
20
I \ ~~~~~~ nnI ~~~~~~~~I
n
n
I
" V s
-4
24
22
28
26
30
34
32
38
36
>38
Zone diameter (mm)
CEPHALOTHIN DISCS 30
MIC
(mg/ml)
Least squares line for y- kx+a k- -.40-skbpe a-tl 4-inrpt
>256r 8
2561-
7
128-
areus *StaphylococCUs Streptococus faecalis Steptococcus species
6
64 -
5
32
4
16
3
8
2
4
1
2
0
1
-1
05
-2
0-25
-3
0-125
-4
a
A
1#
oKJebsielasp. o Eschericha
*
\
|I
A
Proteus mrirabilis ~~~~~~~~~~~~v kdoie positeb poeus a Serrata sp. * Salmonela sp.
\ A
o-o A&\ GA
00\
oL. OA
oS
o
*
A
vv
80 * o *
v
v
n
oIV
Li
8
, , , , , 10
12
14
16
,
18
,
20
,
22
,
24
,
26
Shkola sp.
a
0o
6
Haemophius irfluenzae Bactroedes fragilis
Enterobacter sp.
, o a
28
vv
8
8
I
30
32
, o8 . 34 36
Zone diameter (mm)
FIG. 2. Regression lines for cefuroxime and cephalothin using 30-pug disks. 515
i
38
>38
516
ANTiMICROB. AGENTS CHEMOTHER.
O'CALLAGHAN ET AL.
TABLE 4. Bactericidal effect of cefuroxime Initial count (0 Original inoculum killed (%) concn (pug/ h) Antibiotic
Test organism
ml)
Staphylococcus aureus 663 S. aureus 11127a Escherichia coli TEME. coli TEM+a Klebsiella aerogenes 46189a Enterobacter hafnia 1325Ea Proteus vulgaris 52740a P. morganii 1375a a
b
10 10 10 50 100 50 50 25
1.4 2.2 3.3 5.2 5.8 4.8 5.2 2.2
x 108 x 107 x 107 x 107 x 107 x 108 x 107 x 107
2h
4h
6h
98.7 88.8 99.94 99.9 99.83 x 1.3b 99.62 98.14
99.90 95.19 >99.99 >99.99 99.91 37.0 99.85 98.91
99.29 99.41 >99.99 >99.99 99.91 99.01 99.91 99.50
8-Lactamase-producing strain. +, Indicates some multiplication. (a) Staphylococcus aureus 11127
m
cefuroxime against the three organisms tested; in contrast, the very high binding of cefazolin was associated with much reduced antibacterial activity in the presence of serum. DISCUSSION Cefuroxime is a new cephalosporin antibiotic which offers a number of advantages over other (a) in nutrient broth 100 _
Control
2
0
Hours after antibiotic addition
o
60 X
/
SOpQrnl~~~~~~~~~~~~~1
(b) Eacherichia coli (R TEM)
(b) in CY medium
Hours after antibiotic addition
2h ma cl
FIG. 3. (a) Effect of different levels of cefuroxime on the growth of S. aureus. (b) Effect of different levels of cefuroxime on the growth of /3-lactamase-
0
producing E. coli RTEM.
cent of cefuroxime was bound to human serum proteins, which is a little higher than the 0 2 4 6 serum binding of cephaloridine but much lower Hours after antibiotic addition than that of cephalothin and cefazolin, at 70 and 80%, respectively (Table 7). The presence FIG. 4. Effect of different levels of cefuroxime on of human serum in the growth medium had P. mirabilis grown in: (a) nutrient broth; (b) CY little effect on the antibacterial activity of medium.
VOL. 9, 1976
CEFUROXIME, A NEW CEPHALOSPORIN ANTIBIOTIC
517
TABLE 5. Hydrolysis of cefuroxime and other cephalosporins by a range of ,&lactamases Ezm Enzyme enzyme Source of of Source enzyme Cefuroxime classa
Escherichia coli (R+TEM) E. coli (R GN238) E. coli D31 Proteus mirabilis Klebsiella aerogenes Kl Enterobacter cloacae P99 Proteus vulgaris Bacteroides fragilis 1600 Pseudomonas aeruginosa 1822 Bacillus cereus 659/H9 Staphylococcus aureus PClb
III V I III IV I I I I
ytg of antibiotic Cephaloridine 100 50 265 210 315 315 143 663 315 168 2.9
Copyright C 1976 American Society for Microbiology
Cefuroxime,
a
Vol. 9, No. 3 Printed in U.SA.
New Cephalosporin Antibiotic: Activity In Vitro
CYNTHIA H. O'CALLAGHAN,* R. B. SYKES, A. GRIFFITHS, AND J. E. THORNTON Glaxo Research Ltd., Greenford, Middlesex, England
Received for publication 25 September 1975
Cefuroxime is a new broad-spectrum cephalosporin antibiotic with increased stability to f8-lactamases. This stability, although no absolute in all cases, has the effect of widening the antibacterial spectrum of the compound so that many organisms resistant to the established cephalosporins are susceptible to cefuroxime. It is active against gram-positive organisms, including penicillinaseproducing staphylococci, but it is less active against methicillin-resistant strains. In addition to its high activity against non-,8-lactamase-producing gram-negative bacteria, cefuroxime effectively inhibits the growth of many 1Blactamase-producing strains, including Enterobacter, Klebsiella, and indole-positive Proteus spp. It is highly active against Neisseria gonorrhoeae, Neisseria meningitidis, and also Haemophilus influenzae, including ampicillin-resistant strains. Cefuroxime is rapidly bactericidal and induces the formation and subsequent lysis of filamentous forms over a small concentration range. All the cephalosporins in current clinical use have some limitations in their spectrum of antibacterial activity. None is very active against Neisseria or Haemophilus infZuenzae, whereas poor activity against many gramnegative bacilli can often be attributed to destruction by 4-lactamases. Other deficiencies encountered among this group of antibiotics include metabolic instability and high binding to serum proteins. Cefuroxime, 6R,7R-3-carbamoyloxymethyl-7-(2Z)-2-methoxyimino (fur2-yl) acetamido-ceph-3-em-4-carboxylate, is a new semisynthetic cephalosporin analogue that overcomes some of these disadvantages. Its structure is given in Fig. 1. MATERIALS AND METHODS Antibiotics. Cefuroxime sodium was prepared in our own laboratories as a creamy white crystalline solid with a molecular weight of 446.4; it is approximately 20% soluble in water. Cephaloridine, cephalothin, and cephalexin were commercially available materials made by Glaxo Laboratories Ltd.; cefazolin (Totacef, Bristol Laboratories) and ampicillin were purchased. Organisms. Most of the organisms tested were recent clinical isolates from Westminster Hospital (London), London Hospital, Papworth Hospital (Cambridge), and Southmead Hospital (Bristol). Strains of H. influenzae were given to us by P. Cavanagh of the Public Health Laboratory, Stokeon-Trent. Disk susceptibility testing. Strains of bacteria were tested for susceptibility to the antibiotics by a standardized disk technique described by Ericsson
and Sherris (2), using Oxoid agar no. 1 (Oxoid Ltd., London). MIC determinations. Minimum inhibitory concentrations (MICs) were determined by an agar dilution technique, in parallel tests with cephalothin. Serial twofold dilutions of freshly prepared standard antibiotic solutions were made into Oxoid no. 1 nutrient agar with or without added enrichment and poured into petri dishes. Plates were inoculated with a replicate inoculating device (Denley Instruments Ltd., Bolney, Sussex) with inocula containing approximately 105 or 107 colonyforming units. The MIC, in micrograms per milliliter, was read after 18 h of incubation at 37 C as the lowest concentration that inhibited growth. Correlation of MIC values with disk test results. The MIC values obtained by the agar plate technique were plotted against zone diameters obtained with disks containing 30 ,ug of antibiotic. From these experimental points a regression curve, calculated by the method of least squares, was drawn for each compound. Growth studies. Various concentrations of antibiotics were added to actively growing cultures in a Jouan biophotometer, and growth was followed by optical density measurements and viable counts. Samples removed at various times were examined under the microscope for morphological variants. The medium used was mainly Oxoid nutrient broth, but casein-yeast extract (CY) medium was used for strains of Proteus. Detection of f8-lactamase activity. All bacterial cultures were examined for 1-lactamase production using the chromogenic cephalosporin described by O'Callaghan et al. (9). Stability studies with f-lactamases. 13-Lactamases were derived from several bacterial species, 511
512
O'CALLAGHAN ET AL. S
rL
C_CONH
;/
l
\CH3
COONa+
Cefuroxime
FIG. 1. Structure of cefuroxime, sodium salt.
including Bacillus cereus, Staphylococcus aureus, Escherichia coli, Enterobacter spp., Proteus spp., Pseudomonas aeruginosa, and Klebsiella aerogenes. All enzymes were used as partially purified preparations, and activity against each of the cephalosporins was measured by a previously described method (9). Stability to mammalian liver homogenates. Fresh liver from mice, rats, and rabbits was homogenized with 25 times its weight of 0.1 M phosphate buffer (pH 7.0) in a Colworth stomacher (A. J. Seward, London). Human liver was homogenized with five times its weight of the same buffer. The homogenates were mixed with equal volumes of antibiotic solutions, and the mixtures were incubated at 37 C for 4 h. After incubation, samples were assayed for antibacterial activity by the largeplate assay against B. subtilis NCIB 8993. Samples were also chromatographed on Whatman no. 1 sheets buffered to pH 7.0 with 0.1 M phosphate buffer. Zones of antibacterial activity were revealed by bioautography on B. subtilis plates. Effect of human serum. The amount of antibiotic bound to human serum protein was determined by an ultrafiltration technique using an Amicon 8MC ultrafiltration unit. MICs were measured in nutrient broth and in nutrient broth containing 50% human serum. RESULTS
Antibacterial activity. The in vitro antibacterial activities of cefuroxime and cephalothin against representative gram-positive bacteria are shown in Table 1. Cefuroxime was highly active against S. aureus, regardless of whether the strains produced a penicillinase. It was, however, much less active against strains resistant to 8 ,ug or more of methicillin per ml. All strains of Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus viridans tested were very susceptible to cefuroxime, but strains of Streptococcus faecalis were resistant. The strains of Clostridia tested were mainly susceptible to cefuroxime. In some instances, cefuroxime was slightly less active than cephalothin. Against gram-negative bacteria cefuroxime was the more active compound (Table 2). More than 98% of the strains of E. coli tested were susceptible to 8 ,ug or less of cefuroxime per ml, compared with 76% for cephalothin. Strains
ANTIMICROB. AGENTS CHEMOTHER.
that produced a (3-lactamase had much the same susceptibility to cefuroxime as those that did not, whereas the 18-lactamase-producing strains were much more resistant to cephalothin. Klebsiella spp. were also susceptible to cefuroxime, with more than 80% of strains tested susceptible to 16 ,ug/ml or less. Its activity against various Enterobacter spp. was particularly good; most of these organisms were f8-lactamase producers, rendering them unsusceptible to cephalothin. However, members of the Serratia group were resistant to both antibiotics. Strains of Proteus mirabilis were very susceptible to both compounds, 75% of the strains being inhibited by 2 ,ug/ml or less. Cefuroxime had a geometric mean MIC of 27 ,ug/ml for indole-positive strains of Proteus; these organisms produced an inducible 83-lactamase and were completely unsusceptible to cephalothin. The activity of cefuroxime and cephalothin was essentially the same against Salmonella spp., but cefuroxime was the more active compound against the strains of Shigella tested, even when the organisms were not (3-lactamase producers. Both compounds had relatively poor activity against Bacteroides fragilis and none against P. aeruginosa. Unlike most cephalosporins, cefuroxime had good activity against H. influenzae, including the strains resistant to ampicillin because they produce a class III (3-lactamase (11), which is very similar to that mediated by the transmissible R -factor RrEM (11). A further interesting feature of cefuroxime is its high activity against Neisseria gonorrhoeae and N. meningitidis strains. Effect of inoculum size. Increasing the size of the inoculum from 105 to 107 colony-forming units had little effect on the activity of cefuroxime against the majority of strains tested (Table 3), although against the two proteus strains the inoculum effect was quite large. The activity of the other cephalosporins was considerably diminished when high inoculum levels were used. Regression line analysis. Regression lines and equations correlating MIC values with zone diameters by the agar dilution method are shown in Fig. 2. The values for cefuroxime against most of the organisms tested fall close to the line, so that in general there is good correlation between zone size and MICs. Growth studies. Cefuroxime was bactericidal to most organisms at a concentration identical with, or close to, the MIC. This was confirmed by viable counts, which were done with a range of organisms, the counts being made at 0, 2, 4, and 6 h after inoculation. Table 4 shows that
CEFUROXIME, A NEW CEPHALOSPORIN ANTIBIOTIC
VOL. 9, 1976
513
TABLz 1. Comparative in vitro activities ofcefuroxime (CXM) and cephalothin (CET) against gram-positive organisms No. of strains with MIC (,Ag/mlI' No. of Organism O mstrains
Staphylococcus aureus, methicillin susceptible S. aureus, methicillin resistant Streptococcus pyogenes S. pneumoniae
S. viridans S. faecalis
Clostridium spp.
pound
0.125
0.25-
1-4
56 56 25 25 7 7 6 6 7
CXM CET CXM CET CXM CET CXM CET CXM
8 45 3 1 7 7 6 6 7
45 7 2 4
3 4 2 16
7
CET CXM CET CXM CET
4
3
6 6 7 7
a
Com-
8-16
>16
12 4
6
2
4
6
1
1
2
5
4
1
Geometric MIC
mean
0.25 0.125 5.9 2.0 0.125 z0.125 :0.125 c O. 125 20.125 20.125 >125.0 25.0 1.2 0.23
Inoculum, 105 colony-forming units.
cefuroxime was rapidly bactericidal. Like most cephalosporins, its action was comparatively slow against the strains of S. aureus but, even so, over 99% of the initial inoculum was killed by 6 h. The gram-negative organisms were killed rapidly, and in most cases over 99% of the very large inocula were killed within 2 h; the 3-lactamase-producing strains were killed as quickly as non-enzyme-producing strains. Turbidimetric measurements on growing cultures showed that there was always a period of increase in opacity after the compound had been added, which varied from species to species. Lysis then occurred rapidly, with the opacity of the culture falling below the sensitivity of the instrument. The rate of lysis was slower for staphylococci than for gram-negative organisms, which is consistent with the effect on the viable counts. Some examples are shown in Fig. 3 and 4. All susceptible organisms tested were lysed by cefuroxime, including N. gonorrhoeae and H. influenzae. The rate of lysis of gram-negative organisms by 13-lactam antibiotics has been shown to depend to varying degrees upon the osmolality of the medium used (4), and strains of Proteus are particularly susceptible to this effect. The example shown in Fig. 4 shows that cefuroxime lysed the organisms much more effectively in CY medium than in nutrient broth, which has a higher osmolality. Microscopic examination of various gramnegative organisms growing in low concentrations of cefuroxime showed that many of them produced filamentous forms over a concentration range between one-fourth and four times the MIC.
Stability to f3-lactamases. Eleven partially purified (8-lactamase preparations were tested for their ability to hydrolyze cefuroxime and three other cephalosporin antibiotics. The results in Table 5 show that cefuroxime was more stable to all the enzymes than the other compounds tested. Only three of the enzymes tested were able to hydrolyze cefuroxime relatively rapidly; these were the class IV enzyme produced by certain strains of K. aerogenes, a class I type of enzyme produced by some strains of B. fragilis (1), and the enzyme complex produced by B. cereus 659/H9 (5). Cefuroxime was unaffected by the ,8-lactamase from S. aureus, whereas enzyme hydrolysis of the other compounds was detected. Effect of liver esterases on cefuroxime. Cefuroxime has an ester group at position 3 (Fig. 1) but, unlike cephalothin and the 3acetoxymethyl analogues of cefuroxime, it does not lose any antibacterial activity when incubated for 4 h with homogenates of mouse, rat, rabbit, or human liver (Table 6). Chromatography of the reaction mixtures, followed by bioautography, confirmed that cefuroxime remained unchanged, whereas both cephalothin and the 3-acetoxymethyl analogue cefuroxime were converted into different compounds with reduced antibacterial activity. This stability is similar to that shown by cefoxitin, which also has a carbamate group at position 3 (C. S. Goodwin, E. B. Raftery, H. Skeggs, A. E. Hill, and C. M. Martin, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 13th, Washington, D.C., Abstr. 51, 1973). Effect of human serum. Thirty-three per-
514
ANTIMICROB. AGENTS CHZMOTHZR.
O'CALLAGHAN ET AL.
TABLE 2. Comparative in vitro activities of cefuroxime (CXM) and cephalothin (CET) against gram-negative
organisms Organism
E. coli
Klebsiella spp. Acinetobacter spp. Enterobacter spp. (E. aerogenes, E. cloacae, E. Iiquefaciens) Enterobacteriaceae, others (Hafnia, Citrobacter, Providencia) Serratia spp.
No. of strains with MIC (Ag/ml) 8-16 32-62 0.25- 1-4 0.5
No. of strains
Compound
129 129 73 73 6 6 138 138
CXM CET CXM CET CXM CET CXM CET
7 4
13 13
CXM CET
1
:0.125
1
CXM CET Proteus mirabilis CXM CET P. morganii CXM CET P. vulgaris CXM CET P. rettgeri 1 CXM CET Salmonella spp. CXM CET Shigella spp. CXM CET B. fragilis CXM CET H. influenzae, ampicillin CXM AMPc susceptible H. influenzae, ampicillin CXM resistant AMP N. gonorrhoeae 22 CXM CET N. meningitidis CXM CET Pseudomonas aeruginosa CXM CET a Inoculum, 105 colony-forming units. b MICs spread too widely; average not meaningful. r AMP, Ampicillin. 8 8 27 27 9 9 21 21 4 4 40 40 10 10 16 16 16 16 15 15 22 22 2 2 12 12
2 1 23
1 6 8 19
8 106
2.9 7.0 8.2 24.3 3.6 58 10.2 >125
1
2 12
11.5 >125
1
1
6 8
2 3 3
6
1 4 3 1 12
116 >125 2.2 6.3 16 >125 39 >125
1
1
12 63 22 22 1
31 6
91 5
4
5 1
1
38 34 9 4 1
Geometric mean MIC
2 12 13 10
108 48 37 18 3
24 20
>125
1 6
2 8 3 21 1
-b
4
>125 3.6 3.1 4.6 17 53 60 0.5 0.25 0. 5 250 0.005 0.4 0.125 0.125 >125 >125
1 1
3
3
11 14
4 2
16
16 15 15
22 2 2 12 12
TABLE 3. Effect of inoculum size on the activity of cefuroxime and other cephalosporins MIC (,ug/ml) of: Organism
Strain
Escherichia coli E. coli Klebsiella sp. K. aerogenes Enterobacter cloacae E. cloacae
SM209 1301 48283 47423 PH660 PH781 248 423
Proteus mirabilis P. morganii a
Colony-forming units.
Cefuroxime
Cephalothin
Cephaloridine
Cefazolin
0___071______
no.
2 4 8 8 2 8 1 8
4 8 16 8 2 8 32 125
32 16 62 16 4 8 4 >250
>250 250 >250 62 125 125 >250 >250
62 62 4 32 4 250 125 >250
125 250 >250 >250 16 >250 250 >250
8 8 16 8 2 62 8 16
62 250 250 62 62 >250 16 16
Log2N
CEFUROXIME 30pg DISCS
MIC
(M/ml)
sr"
>256
o Staphylococcus aureus * Streptococcus faecalis o Streptococcus species * Haemophilus influenzae \ Bacteroides tragilis
00
8
-,Zr
7
128-
w&
6
64-
7t
*
.
0
\ Enterobacter sp. \ * Klebsiella sp. * Escherichia coli v Proteus mirabilis , Indole positive proteus , * * * Serratia sp. * Salmonella sp. * Shigella sp. .---^ -.* *-
I
*-&A 't
321 4
16
3
8
2
4
1
2
0
1
-1
0-5
Least squares line for y * k+a kz--55=skope a*15sintercept
.
£;
£
* @0
* °Uv
v °
8
8
°
\
o
\V
-2
0-25 -
-3
0-125[ 6
8
10
12
16
14
18
20
I \ ~~~~~~ nnI ~~~~~~~~I
n
n
I
" V s
-4
24
22
28
26
30
34
32
38
36
>38
Zone diameter (mm)
CEPHALOTHIN DISCS 30
MIC
(mg/ml)
Least squares line for y- kx+a k- -.40-skbpe a-tl 4-inrpt
>256r 8
2561-
7
128-
areus *StaphylococCUs Streptococus faecalis Steptococcus species
6
64 -
5
32
4
16
3
8
2
4
1
2
0
1
-1
05
-2
0-25
-3
0-125
-4
a
A
1#
oKJebsielasp. o Eschericha
*
\
|I
A
Proteus mrirabilis ~~~~~~~~~~~~v kdoie positeb poeus a Serrata sp. * Salmonela sp.
\ A
o-o A&\ GA
00\
oL. OA
oS
o
*
A
vv
80 * o *
v
v
n
oIV
Li
8
, , , , , 10
12
14
16
,
18
,
20
,
22
,
24
,
26
Shkola sp.
a
0o
6
Haemophius irfluenzae Bactroedes fragilis
Enterobacter sp.
, o a
28
vv
8
8
I
30
32
, o8 . 34 36
Zone diameter (mm)
FIG. 2. Regression lines for cefuroxime and cephalothin using 30-pug disks. 515
i
38
>38
516
ANTiMICROB. AGENTS CHEMOTHER.
O'CALLAGHAN ET AL.
TABLE 4. Bactericidal effect of cefuroxime Initial count (0 Original inoculum killed (%) concn (pug/ h) Antibiotic
Test organism
ml)
Staphylococcus aureus 663 S. aureus 11127a Escherichia coli TEME. coli TEM+a Klebsiella aerogenes 46189a Enterobacter hafnia 1325Ea Proteus vulgaris 52740a P. morganii 1375a a
b
10 10 10 50 100 50 50 25
1.4 2.2 3.3 5.2 5.8 4.8 5.2 2.2
x 108 x 107 x 107 x 107 x 107 x 108 x 107 x 107
2h
4h
6h
98.7 88.8 99.94 99.9 99.83 x 1.3b 99.62 98.14
99.90 95.19 >99.99 >99.99 99.91 37.0 99.85 98.91
99.29 99.41 >99.99 >99.99 99.91 99.01 99.91 99.50
8-Lactamase-producing strain. +, Indicates some multiplication. (a) Staphylococcus aureus 11127
m
cefuroxime against the three organisms tested; in contrast, the very high binding of cefazolin was associated with much reduced antibacterial activity in the presence of serum. DISCUSSION Cefuroxime is a new cephalosporin antibiotic which offers a number of advantages over other (a) in nutrient broth 100 _
Control
2
0
Hours after antibiotic addition
o
60 X
/
SOpQrnl~~~~~~~~~~~~~1
(b) Eacherichia coli (R TEM)
(b) in CY medium
Hours after antibiotic addition
2h ma cl
FIG. 3. (a) Effect of different levels of cefuroxime on the growth of S. aureus. (b) Effect of different levels of cefuroxime on the growth of /3-lactamase-
0
producing E. coli RTEM.
cent of cefuroxime was bound to human serum proteins, which is a little higher than the 0 2 4 6 serum binding of cephaloridine but much lower Hours after antibiotic addition than that of cephalothin and cefazolin, at 70 and 80%, respectively (Table 7). The presence FIG. 4. Effect of different levels of cefuroxime on of human serum in the growth medium had P. mirabilis grown in: (a) nutrient broth; (b) CY little effect on the antibacterial activity of medium.
VOL. 9, 1976
CEFUROXIME, A NEW CEPHALOSPORIN ANTIBIOTIC
517
TABLE 5. Hydrolysis of cefuroxime and other cephalosporins by a range of ,&lactamases Ezm Enzyme enzyme Source of of Source enzyme Cefuroxime classa
Escherichia coli (R+TEM) E. coli (R GN238) E. coli D31 Proteus mirabilis Klebsiella aerogenes Kl Enterobacter cloacae P99 Proteus vulgaris Bacteroides fragilis 1600 Pseudomonas aeruginosa 1822 Bacillus cereus 659/H9 Staphylococcus aureus PClb
III V I III IV I I I I
ytg of antibiotic Cephaloridine 100 50 265 210 315 315 143 663 315 168 2.9
