In Vitro Activity of CP-65,207, a New Penem Antimicrobial Agent, in ...

Vol. 33, No. 8

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1989, p. 1160-1166

0066-4804/89/081160-07$02.00/0 Copyright X 1989, American Society for Microbiology

In Vitro Activity of CP-65,207, a New Penem Antimicrobial Agent, in Comparison with Those of Other Agents THOMAS GOOTZ,* JAMES RETSEMA, ARTHUR GIRARD, ERNEST HAMANAKA, MARGE ANDERSON, AND SHARON SOKOLOWSKI Pfizer Central Research, Groton, Connecticut 06340 Received 28 December 1988/Accepted 28 April 1989

CP-65,207 is a new parenteral penem antibiotic with a broad spectrum that includes gram-positive, gram-negative, and anaerobic microorganisms, with MICs for 90% (MIC90s) of the majority of 1,101 clinical pathogens tested being '1 ,ig/ml. The compound was from 10- to 100-fold more active than cefoxitin and broad-spectrum cephalosporins against gram-positive bacteria and anaerobes. CP-65,207 was less active than imipenem for staphylococci, group A streptococci, and Enterococcus faecalis. Against members of the family Enterobacteriaceae, CP-65,207 was in general 100-fold more active than cefoxitin, 5- to 10-fold more active than broad-spectrum cephalosporins, and 2-fold more active than imipenem. Fresh clinical isolates that were resistant to broad-spectrum cephalosporins were highly susceptible to CP-65,207 and imipenem (MIC90, 1 ,ug/ml). Isolates of Enterococcusfaecalis, Serratia marcescens, and anaerobic Peptococcus spp. had MIC9,s of 8, 2, and 3.12 ,ug/mI, respectively. CP-65,207 was not very active against methicillin-resistant staphylococci or Pseudomonas aeruginosa. Killing kinetics showed that against some strains CP-65,207 is rapidly bactericidal at concentrations well below those required to achieve a similar degree of killing with cefotaxime, ceftazidime, and ceftriaxone. CP-65,207 was only slightly susceptible to hydrolysis by type I cephalosporinases and TEM-1, SHV-1, and PSE-2 plasmid-encoded enzymes. It had the highest affinity for penicillin-binding proteins 2, 1A, 1B, and 3 in cell-free preparations of Escherichia coli W-7. Penems are synthetic beta-lactams that, as a class, possess a number of interesting properties which include a broad antibacterial spectrum and low susceptibility to hydrolysis by ,B-lactamases but, at the same time, are inactivated by renal dipeptidases found in the brush border of the kidney (20). A number of novel penems have been evaluated in the past decade; SCH-29482 and SCH-34343 (2, 10, 11) have rivaled carbapenems like imipenem in antibacterial activity, yet in clinical trials they were found to have undesirable side effects. The broad antibacterial spectrum of penems, however, has encouraged continued research in order to find clinically useful compounds. Two of these penems, FCE22101 and SUN-5555, are in clinical trials (20). CP-65,207 (Fig. 1) is a new parenteral penem that demonstrates high activity against gram-positive, gram-negative, and anaerobic bacteria that in some itistances is significantly imtproved over cefoxitin and the broad-spectrum cephalosporins. The current study characterizes the in vitro activity of CP-65,207 compared with those of both cephalosporins and imipenem. (This work was presented in part at the 28th Interscience Conference on Antimicrobial Agents and Chemotherapy [J. Retsema, A. Girard, M. Anderson, L. Brennan, C. Cimochowski, J. Faiella, E. Hamanaka, and T. Gootz, Program Abstr. 28th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 221, 1988].)

mixture of glycerol and brain heart infusion broth until needed. Anaerobic organisms were obtained from R. C. Tilton, University of Connecticut at Farmington. Antibiotics. CP-65,207 was synthesized at Pfizer Central Research as a sodium salt. The compound is a diastereomeric mixture composed of approximately equal ratios of isomers. Standard laboratory antibiotic reference powders were provided by the following: cefoxitin and imipenem, Merck Sharp & Dohme, Rahway, N.J.; clindamycin, The Upjohn Co., Kalamazoo, Mich.; ceftriaxone, Hoffmann-La Roche Inc., Nutley, N.J.; cephaloridine, cefamandole, and moxalactam, Eli Lilly & Co., Indianapolis, Ind.; cefotaxime, Hoechst-Roussel Pharmaceuticals Inc., Somerville, N.J.; and methicillin, Sigma Chemical Co., St. Louis, Mo. All compounds were weighed and dissolved in buffer or growth medium on the day of use. Susceptibility studies. Antibiotic susceptibility studies were performed in microdilution trays containing 0.2 ml of unsupplemented Mueller-Hinton broth (MHB) per well with twofold dilutions of antibiotic (3). Inocula were grown overnight in MHB and diluted in broth to give a final inoculum of approximately 7.5 x 105 CFU/ml. Inoculated plates were incubated at 37°C in air for 18 h. The MIC was defined as the lowest concentration of drug inhibiting visible growth by 18 h. The MBC was determined for 193 isolates by subculturing 0.01 ml from all clear MIC wells onto Mueller-Hinton agar.

MATERIALS AND METHODS

Organisms. A total of 1,101 clinical isolates collected since 1983 were used for susceptibility testing. These included methicillin-resistant staphylococci, carbenicillin-resistant members of the family Enterobacteriaceae, and organisms resistant to broad-spectrum cephalosporins. All strains were clinical isolates obtained from microbiology laboratories across the United States and stored at -70'C in a 50:50 *

Corresponding author.

FlG. 1. Chemical structure of CP-65,207. 1160

VOL. 33, 1989

IN VITRO ACTIVITY OF CP-65,207

TABLE 1. Comparative in vitro activity of CP-65,207

Organism (no. of isolates tested)

Staphylococcus autreus, ampicillin and methicillin susceptible (20)

Staphylococcus auireits, ampicillin resistant, methicillin susceptible (33)

Staphylococcus alr-euts, methicillin resistant (21)

MIC (pg/ml)

CP-65,207 Imipenem Cefoxitin Cefotaxime Ceftazidime Methicillin CP-65,207 Imipenem Cefoxitin Cefotaxime Ceftazidime Methicillin

Staphlylococcus epiderunidis, ampicillin and methicillin susceptible (22)

CP-65,207 Imipenem

Staphylococcus epidermnidis, ampicillin resistant, methicillin susceptible (20)

CP-65,207 Imipenem Cefoxitin Cefotaxime Ceftazidime Methicillin

Staphylococcuis epidermidis, methicillin resistant (15)

TABLE 1-Continuted

Agent0 CP-65,207 Imipenem Cefoxitin Cefotaxime Ceftazidime Methicillin

Cefoxitin Cefotaxime Ceftazidime Methicillin

.0.03 .0.03 1


0.06 2 4 8 4

0.125

0.25 0.06 4 4 8 4

.0.03 4 4 8 2 >64 16 >64 >64 >64 >64

.0.03 .0.03

0.5 0.125 4 4 8 4

1 1 4 2

2 CP-65,207 Imipenem 0.5 Cefoxitin >64 Cefotaxime >64 Ceftazidime >64

Streptococclus pneuimoniae (32)

Streptococcus agalactiae (35)

CP-65,207 Imipenem Cefoxitin Cefotaxime Ceftazidime Penicillin G CP-65,207 Imipenem Cefoxitin Cefotaxime Ceftazidime Penicillin G

CP-65,207 Imipenem Cefoxitin Cefotaxime Ceftazidime Penicillin G

Enterococcuts ficecalis (34)

0.016 .0.004

0.5 0.016 0.125 0.008

0.5 0.016 0.25 0.008

0.008 0.008 2 0.016 0.25 0.0156

0.125 0.063

Escherichia coi, ampicillin susceptible (29)

CP-65,207 Imipenem Cefoxitin Cefotaxime Ceftazidime Ceftriaxone

.0.031 0.125 4

0.063 0.25 8 0.063 0.125 0.063

64 Cefotaxime >64

.0.031 0.25 .0.031

Enterobacter aerogenes (35)


0.25 1 >64 0.125 0.25 0.125

0.5 1

32 64 64

Serratia inarcescens (32)"

CP-65,207 Imipenem Cefoxitin Cefotaxime Ceftazidime

Klebsiella pneinmoniiae (31)


.0.31

Klebsiella oxytoca (32)


0.063 0.25 4 .0.31 0.125 0.125

Citr-obacter fteundii (18)

CP-65,207 Imipenem

0.125 0.063 0.25 0.5 64 >64 0.25 1 0.5 1 0.25 1

Cefoxitin Cefotaxime Ceftazidime Ceftriaxone

Citrobacter diversus (9)

0.25 2 0.5 1 16 >64 0.25 1 0.125 1

0.125 0.25 4 0.5 0.063

CP-65,207 Imipenem

0.063 0.25 4

Cefoxitin Continuied

0.063 0.25 8 0.25 0.5 0.063 1 1

8 1

C0.031 0.125 2

0.25 0.5 >64 >64 >64 >64

2 0.125

.0.031

0.063

.0.031


0.063

64 >64 0.25 1 16 '0.031 0.125 '0.031

>64 >64

0.016 .0.004

90%

0.125 1 4 .0.031 0.063 '0.031

Methiciliin >64 Streptococcus pyogenes (34)

(pg/ml)


Escherichia (oli, ampicillin resistant (31) 0.25 0.063 4 2 16 4

50%

Haetnophiluts inflienzae, ampicillin resistant (28)

32

0.125 .0.03 2 1 4 2

MIC Agent

Ceftazidime Ceftriaxone

.0.03

2 8 2

1161

Continuted

on

0.25 0.5 16 0.125 1 0.125

0.25 0.5 8 1 0.5 8

0.063 0.25 8

following page

1162

GOOTZ ET AL.

ANTIMICROB. AGENTS CHEMOTHER.

TABLE 1-Continued Organism (no. of isolates tested)

Salmonella enteritidis (32)

Shigella sonnei (36)

Yersinia enterocolitica (32)

Morganella morganii (34)

Proteus vulgaris (38)

Proteus mirabilis (35)

Providencia stuartii (32)

Agent

0.063 0.125


0.062 3 0.25 4 0.12! 5 0.25 0.12! 5

0.125


'0.03]1 0.12!5 2 .0.0311 0.0633 .0.03]1

0.063 0.25 4


0.0633 0.25 4 .0.03]1 0.12! 5 0.12! 5

0.125 0.25


0.5 2 8

CP-65,207 Imipenem Cefoxitin Cefotaxime Ceftazidime Ceftriaxone CP-65,207 Imipenem Cefoxitin Cefotaxime Ceftazidime Ceftriaxone

CP-65,207 Imipenem Cefoxitin Cefotaxime


Branhamella catarrhalis (14)

MIC (jig/ml) 50% 90%

Cefotaxime Ceftazidime Ceftriaxone

Ceftazidime Ceftriaxone Providencia alcalifaciens (20)

TABLE 1-Continued


0.063

0.5 0.25 0.25 0.5 8 0.25 0.5

0.25 2 2

'0.0311

.0.03]1 .0.03]1

0.063

8 0.125 0.25 0.25 4 16 4 1 0.5

0.5 4 0.25

0.063

0.25

.-0.031 0.063 .0.031

0.25 1 2 0.25 0.25

0.25 2 16 2 2 0.5

0.12! 5 0.5 2

'0.03]1I 0.063 '0.03]1

0.25 2 0.25

0.5 0.063

.0.032 1

0.125

'0.03]1 0.12! 5 .50.032 1 '0.03] 0.063

0.063

0.125 0.063

31 0.03 1.0 Continued

Agent


MIC (jg/ml) 50% 90%

0.06 0.06 1 '0.03 '0.03 0.03

0.125 0.06 >16

.0.03 2 0.06

CP-65,207 32 Imipenem 1 Cefoxitin >64 Cefotaxime >64 Ceftazidime 2 Ceftriaxone >64

>64 4

Pseudomonas spp. (41)b


>64 16 >64 >64 16 >64

Acinetobacter calcoaceticus subsp. anitratus (22)

0.5 CP-65,207 Imipenem 0.125 Cefoxitin >64 8 Cefotaxime Ceftazidime 4 Ceftriaxone 16

0.063

.0.03]1 .50.03] 1

'0.03]1

Pseudomonas aeruginosa (39)

0.125

0.5 4 4

0.5 2 4 '0.03]

Neisseria gonorrhoeae (15)

0.25

1

0.0633 0.25 .0.03]1


8 0.25 64 8 1 8

16

1 0.125 16 4 32

65% of strains were carbenicillin resistant.

bSpecies (number of isolates tested): P. putida (12); P. fluorescens (11); P. stutzeri (10); P. cepaciai (4); P. acidovorans (2); P. putrefaciens (2).

The MBC was defined as the lowest concentration of drug inhibiting 99.9% of growth. All Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae were tested in MHB plus 5% bovine serum. Tests with Haemophilus influenzae were performed with MHB plus 5% Fildes and 2% Cofactor enrichment (Quiger Labs, Davis, Calif.). Methicillin-resistant staphylococci were incubated with test drugs at 30°C to allow full expression of methicillin resistance. Anaerobes were tested by using a 1:100 dilution of densely turbid overnight cultures grown in prereduced chopped meat medium (Scott Laboratories, Inc., Fiskeville, R.I.). MICs were determined by using the agar dilution technique (3) with Wilkins-Chalgren agar (Difco Laboratories, Detroit, Mich.). Inoculation and incubation with drug were performed in an anaerobic chamber. Killing kinetics. The rate of bacterial killing for CP-65,207 and comparative beta-lactams was determined at the respective MIC for a number of strains in 50 ml of MHB (5). Broth bottles with drug were inoculated from overnight cultures of strains after dilution in MHB to give a final inoculum of between 5 x 105 and 1 x 106 CFU/ml. Bottles were incubated without shaking, and samples were removed at timed intervals, diluted in saline, and plated in duplicate on Mueller-Hinton agar. To prevent drug carryover, 1 ml each of the undiluted samples was centrifuged for 5 min at 15,000 rpm in an Eppendorf centrifuge (model 5414; Brinkmann Instruments, Inc., Westbury, N.Y.). The resulting cell pellet was suspended in drug-free medium and centrifuged again. The washed pellet was suspended in the original volume of drug-free medium prior to plating. Colonies were counted after overnight incubation to determine the number of viable CFU in each culture. The minimal accurately detectable number of CFUs per milliliter was 30 (3 colonies in 0.1 ml).


VOL. 33, 1989

1163

TABLE 2. Comparative in vitro activity of CP-65,207 against anaerobic organisms MIC9(, (,ug/ml)


CP-65,207

Imipenem

Cefoxitin

Clindamycin

Metronidazole

Bacteroides fragilis (26) Clostridium perfringens (12) Clostridiurn difficile (17) Peptococcuts spp. (19) Peptostreptococcus spp. (10)

0.10 0.025 1.56 3.12 0.10

0.20 0.10 3.12 0.78 0.05

12.5 0.78 >100 6.25 6.25

0.20 0.05 6.25 0.10 0.20

0.78 0.78 0.78 0.78 >25

,I-Lactamase activity in sonic extracts. The relative susceptibility to hydrolysis of CP-65,207 and several other betalactams by 1-lactamase prepared from sonic extracts was tested by standard methods (5). Briefly, sonic extracts were prepared from log-phase cultures of isolates producing Plactamase. The protein content of each sonic extract was determined by the method of Lowry et al. (7). The specific 3-lactamase activity of cleared sonic extracts was measured against beta-lactam substrates by the spectrophotometric assay of O'Callaghan et al. (12). Hydrolysis of substrates at a final concentration of 100 pM was assayed at 37°C in phosphate buffer (pH 7.2) by measuring the decrease in absorbance of the beta-lactam ring of each compound. The hydrolysis of CP-65,207 was monitored at 322 nm, and the extinction coefficient was 7,600. Affinity for PBPs. The affinity of CP-65,207 for the penicillin-binding proteins (PBPs) of Escherichia coli W-7 was determined by using the cell-free membrane-labeling technique of Spratt (16). Membranes were prepared by sonication and centrifugation (16). The relative affinity of CP65,207 for the PBPs of E. coli at 30°C was determined by a competitive binding assay measuring the displacement of 3H-penicillin G (Amersham Corp., Arlington Heights, Ill.). RESULTS Susceptibility studies. The in vitro activity of CP-65,207 compared with those of other beta-lactams against aerobic and facultative organisms is shown in Table 1. CP-65,207 was less active than imipenem against methicillin-susceptible isolates of Staphylococcuts auireuis and Staphylococcuts epidermidis, with MICs for 90% of strains tested (MICQ0s) in the range of 0.25 p.g/ml. This level of activity is improved, however, over those of cefoxitin and the broad-spectrum cephalosporins tested, where the MIC90s ranged up to 4 and 8 pg/ml, respectively (Table 1). None of the beta-lactams tested were active against methicillin-resistant staphylococci tested at 30°C. CP-65,207 was less active than imipenem against strains of streptococci but was 100-fold more active than cefoxitin against isolates of Streptococcus agalactiae. CP-65,207 was eightfold less active than imipenem against isolates of Enterococcus faecalis but was highly active compared with broad-spectrum cephalosporins (Table 1). CP-65,207 was slightly less active than broad-spectrum cephalosporins against ampicillin-resistant H. intfluenzae, with an MIC90 of 0.25 ,ug/ml. It was similar to broadspectrum cephalosporins in activity against E. coli and was the most active compound tested against Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freuindii, and Citrobacter diversius, which possess inducible type I cephalosporinases (4). The MIC90 for all of these isolates was 's1 p.g/ml. CP-65,207 was nearly equivalent in activity to imipenem and broad-spectrum cephalosporins against Serratia marc escens (65% carbenicillin resistant). CP-65,207 was

the most active agents against Klebsiella spp., Salmonella enteritidis, Shigella sonnei, and Yersinia enterocolitica (MIC90 range, 0.063 to 0.25 p.g/ml). CP-65,207 was four- to eightfold more active than imipenem against both Proteuts mirabilis and indole-positive Proteus spp. It was significantly more active than cefoxitin and equivalent in action to or slightly less active than cefotaxime, ceftazidime, and ceftriaxone against these organisms (Table 1). CP-65,207 was also highly active against isolates of Providencia spp., Branhamella catarrhalis, and Neisseria gonorrhoeae, with MIC9ts of s0.25 p.g/ml. Imipenem and ceftazidime were the most active agents against strains of Pseuldomonas spp. The CP-65,207 MICs for 50% (MIC50s) of Pseuidomonas aeruiginosa and Pseuidotnonas spp. were 32 and 8 p.g/ml, respectively. CP-65,207 and imipenem were the two most active compounds tested against Acinetobacter calcoaceticus, being significantly more active than cefoxitin and the broadspectrum cephalosporins. MBCs of CP-65,207 were determined with 193 clinical isolates. These included 15 ampicillin-resistant and methicillin-susceptible Staphylococcus alureus isolates, 10 ampicillin-resistant and methicillin-susceptible Staphylococci.s epider-midis isolates, 17 Enterococcus faecalis isolates, 21 ampicillin-resistant H. inflienzae isolates, 31 ampicillinamong

TABLE 3. In vitro activity of CP-65,207 against gram-negative isolates containing well-characterized 3-lactamases or showing resistance to cephalosporins 13-Lactamase class and agent MIC90 (,ug/ml) Type I derepressed cephalosporinases" CP-65,207 .0.39 >12.5 Moxalactam. >12.5 Cefotaxime. Imipenem .1.56 Plasmid-encoded penicillinaseh CP-65,207 .1.56 Moxalactam .1.56 Cefotaxime .0.20 Ampicillin .>12.5 Cephalosporin-resistant clinical isolates (n = 59)' 1.0 CP-65,207.. Imipenem .1.0 Cefoxitin .>64 Cefotaxime .32 Ceftazidime .32 Ceftriaxone .64 "

Eight mutant strains composed of Enterobacter cloacae, Citrobacter

and C. diversus producing type I cephalosporinases constitutively. freiindii, " Seventeen isolates composed of E. c(oli, K. pneioinoniioe. P. miirabilis. and Enterohacter OggloeOrnOIs containing TEM-1, TEM-2, OXA-1, OXA-2,

OXA-3, or SHV-1 penicillinases. Composed of 14 E,iterEohb(cter cloacae isolates, 16 C. fireundlii isolates, 6 teraeiogenies isolates. 2 C. dfiversius Serra(litia n1a(rcescens isolates, 7 Enterobacr isolates, 1 M. itnorganiii isolate, 2 E. coli isolates, 6 K. paealnalonliae isolates, 1 K. oxvtoca isolate, 2 Provideleniaw stiaiautii isolates, and 2 A. cal(oaceticis isolates. "

1164

GOOTZ ET AL.


E I

U. C.

8

TIME (hrs.) TIME

(hrs.)

8

IC)

I U. C)

8 0 TIME (hr..)

2

4

8 6 TIME (h..)

24

FIG. 2. Killing kinetics curves of CP-65,207, ceftazidime, ceftriaxone, cefoxitin, and cefotaxime in 50 ml of MHB against Staphylococcus aureus 01A708 (A), E. coli 51A503 (B), Enterobacter cloaccae 67B130 (C), and Providencia stuartii 77A020 (D). Numbers in parentheses refer to the concentration of drug tested and indicate the MIC of each drug determined in MHB by a microdilution technique.

resistant E. coli isolates, 16 K. pneumoniae isolates, 16 Serratia marcescens isolates, 16 Enterobacter cloacae isolates, 18 C. freundii isolates, 17 Morganella morganii isolates, and 16 Providencia stuartii isolates. In all cases, the MBCs of CP-65,207 agreed with the MICs or were no more than twofold higher. The activity of CP-65,207 against anaerobes is compared with those of imipenem, cefoxitin, clindamycin, and metronidazole in Table 2. CP-65,207 was more active than imi-

penem and clindamycin against isolates of Bacteroides fragilis and Clostridium perfringens. It was essentially equivalent in activity to metronidazole and imipenem against isolates of Clostridium difficile. CP-65,207 was also highly

active against Peptostreptococcus spp. but was less active than imipenem for Peptococcus spp. CP-65,207 was active against organisms with derepressed, type I cephalosporinases or plasmid-encoded penicillinases (Table 3). The MIC90 for 17 strains containing TEM, OXA,

VOL. 33, 1989


IA B

PENICILLIN BINDING PROTEINS

3

5/6 Lane#

1

0

2 0.004

3 0.02

4 0.04

5 0.2

6 0.4

7

1.1

8 2.1

9 3.2

10 4.2

11

CONCENTRATION OF UNLABELED CP-65,207 (ug/mI)

FIG. 3. Autoradiograph showing the competitive binding of unlabeled CP-65,207 versus 3H-penicillin G for the PBPs of E. coli W-7. Lanes 1 through 10, Test system with concentrations of CP-65,207 between 0 and 4.2 pLg/ml; lane 11, radiolabeled molecular weight standards. or SHV penicillinases was 1.56 p.g/ml. Bacterial strains that are resistant to the broad-spectrum cephalosporins because of decreased permeability or various 1-lactamase-mediated mechanisms are being isolated clinically (1, 13, 14, 17, 19). Fifty-nine isolates moderately or highly resistant to cepha-

losporins, consisting predominantly of Enterobacter, Citrobacter, Serratia, and Klebsiella spp., were collected from four hospitals in the United States and tested for their susceptibility to CP-65,207 and other beta-lactams. As shown in Table 3, CP-65,207 and imipenem were the most active agents against this resistant collection, with an MIC90 of 1 ,ug/ml.

Kinetics of bacterial killing. To more fully characterize the antibacterial activity of CP-65,207, killing kinetics studies were performed in 50 ml of broth with a small number of representative clinical isolates of gram-positive and gramnegative organisms. The activity of CP-65,207 was compared with those of cephalosporins at the MIC of each drug. Figure 2A shows the killing kinetics obtained with a penicillinresistant Staphylococcus aureus strain in the presence of CP-65,207, cefoxitin, cefotaxime, or ceftazidime. While all drugs produced a bactericidal response up to 8 h, CP-65,207 was effective at concentrations 8- to 16-fold lower than those of the other beta-lactams tested. Against ampicillin-resistant strains of E. coli, Enterobacter cloacae, and Providencia

1165

stuartii, CP-65,207 produced rapid and complete killing at concentrations 8- to 100-fold lower than those observed with cefoxitin, cefotaxime, ceftazidime, and ceftriaxone (Fig. 2B through D). With Enterobacter cloacae 67B130, significant growth was observed by 24 h with ceftazidime, ceftriaxone, and cefotaxime at the MICs determined from the broth microdilution technique (Fig. 2C). This result was not observed with CP-65,207. Similar studies indicated that the bactericidal effect of CP-65,207 was unaffected in the presence of 90% human serum (data not shown). Binding of CP-65,207 to PBPs of E. coli W-7. The affinity of CP-65,207 for the PBPs of E. coli was determined by using cell-free membrane preparations of E. coli W-7 in a competitive binding assay using 3H-penicillin G. Unlabeled CP65,207 was preincubated with E. coli membranes in separate tubes containing increasing concentrations of penem. Figure 3 (lane 1) shows an exposed film of the PBP bands after addition of 'H-penicillin G alone. Competitive studies with cold CP-65,207 indicate that this compound has the highest affinity for PBP 2 since as little as 0.004 ,Lg/ml prevents binding of label to this protein in the cell-free membrane preparation. PBP 1A and 1B were saturated at 0.4 ,ug/ml, 100-fold higher levels than that of PBP 2 (Fig. 3, lane 6). The relative order of affinity of CP-65,207 for PBPs in E. coli W-7 is PBP 2> PBP lA > PBP lB > PBP 4> PBP 3 > PBP 5 or 6. The concentrations of CP-65,207 required to saturate the lethal PBPs (1A, 1B, 2, and 3) in broken-cell preparations of E. coli W-7 agree well with the concentrations required to inhibit the growth of wild-type strains of E. coli (Table 1). This suggests that CP-65,207 penetrates the gram-negative outer membrane efficiently and achieves necessary bactericidal concentrations at the lethal PBP targets in the cell. Relative susceptibility to hydrolysis by I8-lactamases. The relative susceptibility of CP-65,207 and other beta-lactams to hydrolysis by crude P-lactamases prepared from sonic extracts is shown in Table 4. CP-65,207 was highly stable to hydrolysis by representative type I cephalosporinases, along with TEM-1, SHV-1, and PSE-2 plasmid-encoded enzymes. CP-65,207 was not susceptible to hydrolysis by derepressed type I enzymes and was the most stable compound tested against the PSE-2 1-lactamase that showed significant hydrolysis of several cephalosporins (Table 4). DISCUSSION This study shows that penem CP-65,207 was active against the majority of aerobic, facultative, and anaerobic clinical pathogens, with the exception of methicillin-resistant staph-

TABLE 4. Relative hydrolysis by P-lactamases Strain

Relative hydrolysis"

3-Lactamase

CEP

CET

CTZ

MOX

CP-65,207

0.2

1.2

0.3

0.5

0.1 0.5

0.6 4.4

0.3 6.8

0.5 1.2

0.1 0.1

0.05 0.1

0.3

0.08

0.1 89 1.6

66 110 54.4

0.9 136 0.7

2.8 327 1.5

CEF

CTX

CFX

0.8 0.3 78

0.2 0.1 16

30 110 51.1

1.5 111 2.0

Enterobacter cloacae 55W

Type

lb

P99 (+) Citrobacter diversus 15M Escherichia coli J153(RSF 1010)

Type I' Type I'

J153(PUZ8-R151A1) HB101(pBR322)

PSE-2"

SHV-1" TEM-1d

0.5 33 0.6

0.2 25 0.2

" Relative hydrolysis compared with that of cephaloridine, which is assigned a value of 100. Abbreviations: CEF, cefamandole; CTX, cefotaxime; CFX, cefoxitin; CEP, cefoperazone; CET, ceftriaxone; CTZ, ceftazidime; MOX, moxalactam. All compounds were tested in a spectrophotometric assay at 100 ,uM. bRichmond and Sykes type I cephalosporinase, inducible (5). ' Richmond and Sykes type I cephalosporinase. constitutively produced. " Plasmid-mediated enzymes.

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ylococci and Pseudomonas spp. CP-65,207 was 16- to 21-fold more active than cefoxitin and broad-spectrum cephalosporins against methicillin-susceptible strains of staphylococci. Like the cephalosporins, CP-65,207 was not highly active against methicillin-resistant staphylococci, with MIC90s of >64 pg/ml. Imipenem and certain penems like FCE-22101, with a carbon-linked side chain at position C-2 of the nucleus, have some activity against methicillin-resistant strains, and this is likely due to an improved affinity of these agents for PBP 2' found in these isolates (6). CP-65,207 demonstrated minimal activity against such staphylococci, and this is consistent with that observed with other sulfurlinked penems at C-2 such as SCH-34343 (11). CP-65,207 had activity comparable to that of imipenem against streptococci, with an MIC90 for all strains of sO.125 >xg/ml. The penem was significantly more active than cephalosporins against Enterococcus faecalis (MICg, 8 ,ug/ml) but was less active than imipenem against this organism. CP-65,207 was more active than imipenem and clindamycin against isolates of Bacteroides fragilis and Clostridium perfringens. Against the members of the family Enterobacteriaceae tested, CP-65,207 was generally more active than imipenem, with an MIC%0 of ' 1 g±g/ml for isolates of E. coli, Enterobacter spp., Citrobacter spp., Klebsiella spp., Proteus spp., Providencia spp., Salmonella spp., and Shigella spp. It was also more active than broad-spectrum cephalosporins, except for isolates of Shigella sonnei, Proteus mirabilis, and Providencia alcalifaciens. This degree of activity is also improved over those of penems CGP-31608, FCE-22101, and HRE-664 reported in the literature (8, 9, 15, 18). Like other penems (15), CP-65,207 shows high affinity for PBP 2 in cell-free studies with E. coli. This high affinity for PBPs, combined with its stability to hydrolysis by a wide variety of P-lactamases, may explain the high antibacterial activity of CP-65,207 against members of the Enterobacteriaceae. In killing kinetics studies, CP-65,207 was rapidly bactericidal against selected isolates that were not highly susceptible to cephalosporins. High activity, equivalent to that of imipenem, was also observed against a collection of 59 clinical isolates that demonstrated moderate or high-level resistance to cephalosporins. The results of this study suggest that further pharmacological and clinical studies should be conducted with CP-65,207 to determine the clinical utility of this penem antimicrobial agent. LITERATURE CITED 1. Beckwith, D. G., and J. A. Jahre. 1980. Role of cefoxitininducible P-lactamase in a case of breakthrough bacteremia. J. Clin. Microbiol. 12:517-520. 2. Brown, R. M., R. Wise, and J. M. Andrews. 1982. SCH-29,482a novel penem antibiotic: an in vitro comparison of its activity with other beta-lactams. J. Antimicrob. Chemother. 9(Suppl. C):17-23. 3. Ericcson, H. M., and J. C. Sherris. 1971. Antibiotic sensitivity

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