ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, MaY 1979, p. 646-650 0066-4804/79/05-0646/05$02.00/0
Vol. 15, No. 5
In Vitro Antibacterial Activity and fB-Lactamase Stability of SCE-129, a New Cephalosporin HAROLD C. NEU* AND KWUNG P. FU
Division of Infectious Diseases, Departments of Medicine and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032
Received for publication 8 February 1979
SCE-129 [3-4-carbamoyl- 1-pyridiniomethyl-7fi-(D-a-sulfophenylacetamido)ceph-3-em-4-carboxylate] is a cephalosporin that inhibits Pseudomonas aeruginosa and Staphylococcus aureus. SCE-129 is tenfold more active than carbenicllin in inhibiting P. aeruginosa. SCE-129 has poor activity against Enterobacteriaceae compared with other cephalosporins and is 16-fold less active than the cephalosporins against streptococci. The activity of SCE-129 does not correlate with ,8-lactamase stability, although SCE-129 is resistant to hydrolysis by grampositive and gram-negative bacteria. The compound does not inhibit the hydrolysis of other cephalosporins. Although SCE-129 acts synergistically with gentamicin to inhibit some Pseudomonas, this cannot be predicted based on knowledge of resistance to one or more compounds.
Although cephalosporin antibiotics have a wide range of antibacterial activity, including activity against aerobic and anaerobic gram-positive and gram-negative bacteria, except for HR 756 (2, 3), there has been none that has inhibited Pseudomonas aeruginosa. The lack of susceptibility of Pseudomonas to cephalosporins has been attributed to the type of /B-lactamases present in these bacteria as well as to permeability factors which prevent entry of these fi-lactams to their receptor site (5). Evidence for both resistance factors in Pseudomonas has been demonstrated by the synergistic inhibition of Pseudomonas by semisynthetic antistaphylococcal penicillins combined with f,-lactamasesusceptible penicillins and by the enhanced activity of cephaloridine in the presence of ethylenediaminetetraacetic acid. SCE-129 [3-4-car-
collection. The fi-lactamase clification is based on substrate profile, inhibition of isoxazoyl penicillins, clavulanic acid and p-chloromercuribenzoate (5). Minimal inhibitory concentrations (MICs) were determined by using serial twofold dilutions of antibiotic in Mueller-Hinton agar and an inoculum of 105 colonyforming units (CFU). Incubation was at 35°C for 18 h. Broth dilutions were performed in Mueller-Hinton broth with 105 CFU and incubated for 18 h at 35°C. Bactericidal levels were determined by plating 0.01 ml from clear tubes to agar. Synergy studies were performed by using twofold dilutions of the antibiotics in agar combined in a ratio of four parts cephalosporin to one part aminoglycoside. The inoculum was 105 CFU. Synergy was defined as a fourfold reduction in inhibitory concentration of both agents. ,B-Lactamase assays of cephalosporins were performed by using a spectrophotometric assay. Potassium phosphate buffer (0.05 M; pH 7.0), containing substrate at a final concentration of 0.1 mM was used. bamoyl - 1- pyridinio - methyl - 7,8 - (D-a-sulfo- The reaction was run at 300C in temperature-conphenyl, acetamido) ceph- 3 - em- 4 - carboxylate ] trolled cuvettes. Assays were performed at the extinchas been reported to inhibit P. aeruginosa (6, tion maximum of the particular agent. Inhibition of 7). We have investigated its antibacterial activity cephalosporin hydrolysis was determined with cephaas substrate and addition of compounds to the as well as its resistance to ,B-lactamase hydroly- loridine reaction at varying concentrations. Change in absorbsis. ance at 255 nm was followed. /i-Lactamases were purified to homogeneity by pubMATERIALS AND METHODS lished techniques or extracts subjected to centrifugaSCE-129 was a generous gift of Takeda Chemical tion and Sephadex G50 chromatography to yield parIndustries, Japan. Carbenicillin was a gift from Bee- tially purified preparations (4). cham Laboratories. Piperacillin was a gift of Toyama. RESULTS Aminoglycosides were obtained from their respective manufacturers, and cefoxitin was a gift from Merck The activity of SCE-129 compared to that of Sharp & Dohme. Bacteria were clinical isolates from blood, urine, carbenicillin, piperacillin, gentamicin, and amiand sputum of patients hospitalized at the Columbia- kacin against 123 recent clinical isolates of P. Presbyterian Medical Center, New York, N.Y. The aeruginosa is shown in Fig. 1. SCE-129 was as Bl-lactamase-containing isolates were those from our active as the aminoglycosides. SCE-129 in46
SCE-129: ANTI-PSEUDOMONAS CEPHALOSPORIN
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hibited 50% of isolates at 3.1 jig/ml and 90% at 50 jig/ml. Carbenicillin at 50 ,g/ml inhibited only 62% of the isolates. These isolates included a number of ,B-lactamase-producing strains as
well as strains that contained aminoglycosideinactivating enzymes. SCE-129 was six times as active as carbenicillin at a concentration of 12.5
C(bw3iNNn P.Faerug-io 0.4
FIG. 1. Comparative activity of SCE-129 against P. aeruginosa (123 isolates).
The activity of SCE-129 compared to that of carbenicillin, cephalothin, and cefoxitin against gram-positive cocci is given in Table 1. SCE-129 was less active than cephalothin or cefoxitin against S. pneumoniae and S. pyogenes, and it did not inhibit S. faecalis or S. faecium (Table 1). SCE-129 was 8- to 16-fold less active than cephalothin against staphylococci, but similar to cefoxitin in activity against staphylococci. It had poor activity against all members of the Enterobacteriaceae. There was no correlation between the MICs of SCE-129 and those of cephalothin, carbenicillin, or cefoxitin; the MICs of SCE-129 were in the range of 50 to 100 ,ug/ml for isolates of Escherichia coli, Salmonella, Shigella, and Proteus mirabilis in contrast to MICs of the other three agents in the range of 0.6 to 6.3 ,ug/ml. SCE-129 also failed to
TABLE 1. Activity of SCE-129 compared to the activity of cephalothin, carbenicillin, and cefoxitin MIC and range (pAg/ml) flv-fror%;cvQtem urgan (no. of isolates) Cefoxitin SCE-129 Cephalothin Carbenicillin 3.1-25 0.1 0.05-0.4 0.4-0.8 Streptococcus pneumoniae
(21) S. pyogenes (4) S. agalactiae (4) S. faecalis (4) S. faecium (2) Staphylococcus aureus (7) S. epidermidis (5) Listeria monocytogenes (4) Escherichia coli
6.3 3.1-12.5 >400 >400 1.6-12.5
0.1-0.2 0.1-0.8 25-100 >400 0.1-0.4
0.05-0.8 0.2-1.6 25 50 6.3-25
0.4-0.8 0.8-3.2 >400 >400 0.8-3.1
Salmonella species (4)
100-200 50->400 3.1-200 50-100
>400 >400 >400 >400
0.4-25 1.6->400 12->400 >400
Proteus mirabilis (32)
Citrobacter freundii (2) Enterobacter cloacae (4) E. aerogenes (2) Klebsiella pneumoniae (3) Serratia marcescens (6) P. morganii (5) P. rettgeri (4) P. vulgaris (2) Providencia stuartii (5) a -, Not detemiined.
25->400 3.1-12.5 3.1-100 3.1-12.5 .6.3-200
ANTIMICROB. AGENTS CHEMOTHER.
NEU AND FU
inhibit Serratia and Enterobacter resistant to both penicillins and cephalosporins by virtue of both /3-lactamase and permeability mechanisms. Figure 2 demonstrates that the inhibitory levels of SCE-129 did not correlate with the presence or absence of,-lactamase activity for any of the organisms tested. For example, the MICs of both the fl-lactamase-positive S. aureus OD OD OD g31 and the ft-lactamase-negative strain were 6.3 ,tg/ ml. Similar results were obtained with P. aerugic O / 1.6-_ oB nosa and E. coli. We did not find a correlation between the MICs of carbenicillin and SCE-129 against our MIC (Ag/nr) CAENKLL isolates of Pseudomonas; that is, isolates inActivity of SCE-129 against carbenicillinFIG. 3. were ml -100 of hibited by ,ig carbenicillin per not always inhibited by low MICs of SCE-129. susceptible and -resistant P. aeruginosa (56 isolates). The MIC of carbenicillin was below 100 ug/ml with 63% of the isolates (Fig. 3) which required 10 for inhibition MICs of SCE-129 equivalent to 25 DNM A~INA ,tg or less per ml. Among 21 of the isolates tested and found resistant to carbenicillin (MICs '100 // ~~~~~0 inhibited by MICs of *0eo0- ~ smi ,tg/ml), 14 isolates were SCE-129 equivalent to 800 ,ug/ trations of SCE-129 against P. aeruginosa (10 isoml. The MIC required to inhibit one isolate was lates). 100 ,tg/ml with inocula of 103, 105, and 107 CFU. Figure 4 shows that there were differences between MICs and miniimal bactericidal concen- difference in the MICs and minimal bactericidal trations of SCE-129 against P. aeruginosa at an concentrations. fi-Lactamase stability. The resistance of inoculum of 105 CFU. These differences ranged SCE-129 to hydrolysis by gram-positive and from 2-fold to 64-fold. At 103 CFU there was no gram-negative /3-lactamases is shown in Table 2. None of the enzymes hydrolyzed SCE-129, in contrast to the hydrolysis of cephaloridine, cefazolin, and cephalothin. There was no correla, 400 vv tion between the SCE-129 MIC and the /3-lac200tno * tamase activity of the isolate. Furthermore, the lOO1MICs of SCE-129 did not correlate with the 00 activity of a particular enzyme against the other 50 cephalosporins hydrolyzed. SCE-129 was as stable to hydrolysis as was cefoxitin. However, 12.5 t25 SCE-129 was an ineffective inhibitor of the hy121 Staph sumu a E. coli drolysis by any of the f,-lactamases as compared Provkec to dicloxacillin or even cefoxitin (Table 3). SCEKhblob lbf 9 3.1 A Pseudmk,wsw 129 partially inhibited the hydrolysis of cephaProdaw loridine by Richmond type I /3-lactamses which *v ShigWla t. are chromosomally mediated enzymes. However, it had no effect on the plasmid-mediated 0Q . type III and type V enzymes. Figure 5 shows that 2 mM cefoxitin produced a 60% inhibition A P bPooftj of both the P. morganii and C. freundii enFIG. 2. MICsofSCE-129against,/-lactamase-pos- zymes, whereas it took 100 yM SCE-129 to proitive and -negative organisms. duce a 60% inhibition of the activity of the 90-
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SCE-129: ANTI-PSEUDOMONAS CEPHALOSPORIN
TABLE 2. Comparative /3-lactamase hydrolysis of SCE-129 with other known cophalosporins' Rate of hydrolysis (pmol/min)b Organism Type of ,-lactamase
Cephaloridine Cephalothin Cefazolin Cefoxitin SCE-129 4.9 (>400) I; Constitutive 11.1 (>400) 2.58 (>400) 0 (>400) 0 (100) I; Induced 4.76 (100) 4.70 (50) 2.98 (50) 0 (100) 0 (25) 5.1 (>400) 0 (>400) 0 (>400) I; Induced 8.3 (>400) 6.5 (>400) 12.06 (200) 3.03 (100) 0.21 (200) 0 (>400) II; Induced 0 (25) III; Plasnid consti- 3.56 (>400) 0.60 (>400) 0.39 (>400) 0 (>400) 0 (6.3) tutive 13.7 (200) 0 (25) 0 (>400) Klebsiella IV; Induced 8.6 (100) 3.8 (50) 3.9 (50) 0.91 (200) 0.4 (3.1) 0 (6.3) 0 (100) Shigella V; Plasmid aB-Lactamase activity was determined by a spectrophotometric method measuring optical density decreases at 255 nm at 300C. Reaction mixture contained 0.5 ml of 0.4 mM cephalosporin substrate in 0.5 ml of phosphate buffer (0.05 M; pH 7). MIC, in parentheses (ug/ml), were detennined by agar dilution method in Mueller-Hinton agar (Baltimore Biological Laboratories). Values do not indicate the relative activity of different organisms. P. morganii Citrobacter Pseudomonas P. mirabilis Pseudomonas
TABLE 3. Inhibition of 13-lactamase hydrolysis of cephaloridine by SCE-129 compared with cefoxitina Relative hydrolysis rate (%)' Organism
Type of fl-lactamase
Cehaoidn Cephaloridine/ Cephaloridine/ Dicoxcili DicloxacWin Cephalondine SCE-129 cefoxitin Pseudomonas I; Induced 100 46 74 0 Citrobacter I; Induced 100 0 78 0 P. morganii I; Constitutive 100 0 72 0 P. mirabilis II; Induced 100 100 100 _ Pseudomonas III; Constitutive 100 106 100 0 Klebsiella IV; Induced 100 100 78 Shigella V; Constitutive 100 85 100 100 a Reaction mixture contained 0.5 ml of 0.2 mM cephaloridine plus 0.5 ml of 0.05 M phosphate buffer (pH 7) as control, or plus 0.5 ml of 0.2 mM cefoxitin, SCE-129, and dicloxacillin, respectively. b The 18-lactamase hydrolysis rate of cephaloridine was given a value of 100. c-, Not determined.
Citrobacter enzyme and, even at a concentration of 100 tM, there was only a 30% inhibition of the P. morganii enzyme. Synergy studies. SCE-129 was combined with gentamicin and tested against P. aeruginosa isolates. Of 32 isolates tested, synergy was shown for 22% of isolates. Examples of the effect are given in Table 4. Synergy could be demonstrated with a SCE-129-gentamicin combination against susceptible and resistant isolates, regardless of their susceptibility to either compound, suggesting the unpredictability of synergistic activity of the combination. Synergy was shown against isolates that contained f,-lactamases as well as against isolates which contained aminoglycoside inactivating enzymes and isolates which contained plasmids mediating both types of resistance.
DISCUSSION This study has shown that SCE-129 is a unique semisynthetic cephalosporin. It inhibits only P. aeruginosa and some gram-positive microorganisms such as S. aureus at concentrations of 12.5 ,ug or less per ml. SCE-129 is tenfold more active than carbenicillin against P. aeruginosa. This activity does not seem to correlate with resistance to /8-lactamase, although SCE-
40 20 I
I I SCE-129
FIG. 5. Inhibition of ,B-lactamase hydrolysis of cephaloridine by cefoxitin and SCE-129. Final concentration of cephaloridine was 0.1 mM.
ANTIMICROB. AGENTS CHEMOTHER.
NEU AND FU
TABLE 4. Synergistic activity of SCE-129 combined with gentamicin MIC (pg/ml) P. aeruginosa isolate 1 2 3 4 5 6 7
12.5 12.5 6.3 100 25 3.1 200
>50 3.1 >50 >50 >50 0.8 >50
SCE-129 + gentamicin
12.5-3.1 1.6-0.4 6.3-1.6 3.1-0.8 12.5-3.1 0.8-0.2 100-25
129 is resistant to hydrolysis by the ,B-lactamases of all of the gram-positive and gram-negative bacteria tested. The overall activity of SCE-129 against Pseudomonas approaches that of some of the aminoglycosides such as amikacin. These results are consistent with those of Tsuchiya et al. (7). However, we did find that there was a difference between the MICs and the minimal bactericidal concentrations of SCE-129 for some Pseudomonas isolates and that with a high inoculum (107 CFU), the drug was 64-fold less active than with an inoculum of 105 CFU. The fact that resistance to 8-lactamase hydrolysis does not correlate with the inhibitory activity suggests that entry into the cell and binding to /3-lactam receptor sites may be the factors which are unique to this agent. Indeed,
the failure of SCE-129 to inhibit the hydrolysis of other cephalosporins suggests that SCE-129 does not bind to the ,B-lactamase hydrolytic site. SCE-129 acts synergistically with aminoglycosides against Pseudomonas in a manner similar to that of the penicillins, carbenicillin, or ticarcillin. Whether synergy could or could not be demonstrated was not related to the type of resistance found in a particular strain. LITERATURE CIT 1. Fu, K. P., and H. C. Neu. Piperacillin, a new penicillin active against many bacteria resistant to other penicillins. Antimicrob. Agents Chemother. 13:358-367. 2. Heymes, R., A. Lutz, and E. Schrinner. 1977. Experimental evaluation of HR 756, a new cephalosporin derivative: pre-clinical study. Infection 5:259-260. 3. Neu, H. C., N. Aewapokee, P. Aswapokee, and K. P. Fu. 1979. HR 756, a new cephalosporin active against gram-positive and gram-negative aerobic and anaerobic bacteria. Antimicrob. Agents Chemother. 15:273-281. 4. Neu, H. C., and E. B. Winshell. 1970. Purification and characterization of penicillinase from Salmonella typhimurium and E. coli. Arch. Biochem. Biophys. 139: 278-290. 5. Richmond, M. H., and R. B. Sykes. 1973. The,B-lactamases of gram-negative bacteria and their possible physiological role. Adv. Microbiol. Physiol. 9:314-8. 6. Tsuchiya, K., and M. Kondo. 1978. Comparative in vitro activities of SCE-129, sulbenicilhin, gentamicin, and dibekacin against Pseudomonas. Antimicrob. Agents Chemother. 13:536-539. 7. Tsuchiya, K., M. Kondo, and H. Nagatomo. 1978. SCE129, antipseudomonal cephalosporin: in vitro and in vivo antibacterial activities. Antimicrob. Agents Chemother. 13:137-145.