Antibacterial Activity of RU44790, a New N-Tetrazolyl Monocyclic 1-Lactam

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1992, p /92/ $02.00/0 Copyright 1992, American Society for Microbiology Vol. 36, No. 8 Antibacterial Activity of, a New N-Tetrazolyl Monocyclic 1-Lactam J. F. CHANTOT,l* M. KLICH,1 G. TEUTSCH,1 A. BRYSKIER,l P. COLLETT'E,' A. MARKUS,2 AND G. SEIBERT2 Centre de Recherches, Roussel-Uclaf, Romainville, France,1 and Hoechst Aktiengesellschaft, D-6230 Frankfurt am Main 80, Gennany2 Received 11 June 1991/Accepted 6 April 1992 belongs to a new class of synthetic monocyclic 1-lactam antibiotics which feature a bioisosteric tetrazole moiety instead of the more classical acidic functions at the N-1 position of the 1-lactam ring. Its antibacterial activity was evaluated against some 900 strains and was compared with those of other recent 13-lactam derivatives, especially aztreonam. is endowed with potent activity against gram-negative bacteria. At.0.6,ug/ml, inhibited 90% of all strains of the family Enterobacteriaceae with the exception of Citrobacter spp. (MIC for 90% of strains tested, 1.2,ug/ml). The activity was similar to that of aztreonam against strains that are normally susceptible to expanded-spectrum cephalosporins. On the other hand, the new compound was 10 to 100 times more potent than aztreonam and most of the other antibiotics tested against enterobacteria that produce chromosome-encoded or plasmid-mediated extended-spectrum,-lactamases. Pseudomonas aeruginosa isolates were equally susceptible to both monobactams. was inactive against staphylococci and had only marginal activity against streptococci (MIC for 50% of strains tested, 2.5,ug/ml). was highly resistant to hydrolysis by various 13-lactamases, particularly cephalosporinases such as P99. The latter enzyme was also inhibited by the compound. showed a high affinity for penicillin-binding protein 3 ofescherichia coli. The results suggest that has good potential in the treatment of infections caused by gram-negative microorganisms. Monocyclic,B-lactam antibiotics were originally described in 1981 by two independent teams (6, 19). They were found to be the products of saprophytic bacteria, and since their discovery, thousands of the so-called monobactams have been synthesized. A characteristic structural feature of these compounds is the presence of a sulfonic function on the,-lactam ring nitrogen which is essential for antibacterial activity. Well-known representatives of this family are aztreonam (21), carumonam (8, 15), and compound BO-1165 (14). Various electron-withdrawing acidic substituents other than the sulfonic moiety have also been introduced onto the P-lactam ring, resulting in the synthesis of different types of biologically active monobactams such as monophosphams (4), oxamazins (23), monosulfactams (5), or monocarbams (4). Tigemonam is a highly active monosulfactam (12, 22) that is administered orally, while pirazmonam (20, 25) and compound U (25) are among the most active parenterally administered monocarbams. (Fig. 1) has been shown to be the most active compound in a novel series of (3S)-N-(tetrazol-5-yl)-azetidinones synthesized at Roussel-Uclaf (9, 10). In, the tetrazole nucleus is successfully used as a 13-lactam ringactivating group and confers potent antibacterial activity to many 4-substituted analogs. The molecule contains a fluoromethyl group in a cis configuration at the 4 position and a carboxycyclobutoxyimino moiety in the lateral chain. These two substituents are responsible for increased antibacterial activity. The synthesis of the racemic form of has been reported by Yoshida et al. (24). In this report, we describe the in vitro antibacterial properties of in comparison with those of aztreonam and some expanded-spectrum cephalosporins. * Corresponding author (This work was presented in part at the 26th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, La., 28 September to 1 October 1986 [3a].) MATERIALS AND METHODS Antibiotics., carumonam, and cefotaxime were synthesized by Roussel-Uclaf (Romainville, France). Moxalactam was obtained from Eli Lilly & Co., Indianapolis, Ind.). Ceftriaxone (Hoffmann-La Roche), aztreonam (Azactam, E. R. Squibb & Sons, New Brunswick, N.J.), ceftazidime (Glaxo, Greenford, United Kingdom), and cefmenoxime (Takeda) are commercially available standard powders. Antibiotic solutions were prepared in Mueller-Hinton broth (susceptibility testing) or in 0.05 M phosphate buffer (biochemical studies). Bacterial strains. About 900 strains of gram-negative and gram-positive bacteria were used in this study. Most of them were aerobic or anaerobic clinical isolates collected from various European hospitals, and some of them were laboratory strains used in our department. Special attention was paid to strains which showed decreased susceptibility (MICs, 21.2,ug/ml) or resistance (MICs, >32,ug/ml) to recent cephalosporins. To this end, 110 strains not inhibited by < 1.2,ug of cefotaxime per ml were selected. This panel of strains included microorganisms that produce chromosomally encoded 1-lactamases (cephalosporinases) or plasmidencoded enzymes (extended-spectrum 13-lactamases). Both clinical and standard strains were maintained in deep agar at room temperature or as frozen stocks (-80 C) in glycerolsupplemented broth. Susceptibility testing. In vitro susceptibility tests were

2 VOL. 36, 1992 H2N</ I34 X1jr.OH N 0 NN FIG. 1. Chemical structure of, (35,4S)-1-[[[1-(2-amino- 4 - thiazolyl) [[(4 - fluoromethyl - 2- oxo- 1 - tetrazolyl azetidinyl)] amino]-2-oxo-ethylidene]amino]oxy]cyclobutane carboxylic acid. performed by a twofold agar dilution method. Mueller- Hinton agar medium (ph 7.4; Institut Pasteur Production [IPP]) was used throughout the study. The medium was appropriately supplemented to support the growth of some fastidious microorganisms (5% globular extract for Haemophilus influenzae; 5% horse blood for anaerobes and non-group D streptococci). Inocula were prepared by incubating bacteria in Mueller- Hinton broth (IPP), but non-group D streptococci and anaerobes were grown in Schaedler broth (IPP). H. influenzae was grown in brain heart infusion (IPP) supplemented with 5% globular extract. Test tubes were incubated at 36 C for 20 h. GasPak jars (Biomerieux, Marcy l'etoile, France) with 10% CO2 were used to support the growth of H. influenzae and non-group D streptococci. The overnight broth cultures usually yielded viable counts at about 109 CFU/ml. A standard inoculum of 104 CFU that was prepared from a 1:200 dilution of the bacterial broth suspension was spotted onto agar plates containing the test compound by using a Denley multipoint inoculator (Denley-Tech Ltd.). All plates were incubated at 36 C for 20 h under the conditions described above. The MIC was defined as the lowest concentration of antibiotic at which no visible growth could be detected on agar plates. Effects of medium and inoculum size on MICs of monobactams. Broth macrodilution tests were used to investigate the influence of several parameters on the inhibition of bacterial growth by antibiotics. They were all carried out in tubes containing twofold dilutions of a drug in 5 ml of medium. The effects of 40% serum (in Mueller-Hinton broth) and urine (used as the growth medium) on the MICs of monobactams were studied on a panel of 10 strains of Pseudomonas aeruginosa and enterobacteria, some of which produced a constitutive cephalosporinase. Various other media were also used to study the effect of the composition of the medium on the antibacterial activities of monobactams against one strain each of Eschenichia coli, Enterobacter cloacae, and Klebsiella pneumoniae (,B-lactamase positive), as follows: nutrient broth, nutrient broth no. 2, antibiotic medium no. 3, brain heart infusion, Trypto casein soy broth, and cooked meat medium, all from IPP. The influence of ph on the activity of monobactams against these strains was studied at a ph range of 6.5 to 8.5 in Mueller-Hinton broth. The ph was adjusted by adding 1 M HCl or 1 M NaOH to the growth medium. In all tests, tubes were seeded at a final standard inoculum size of 105 CFU/ml. ANTIBACTERIAL ACTIVITY OF 1757 The effect of inoculum size was determined in Mueller- Hinton broth by using inocula that varied from 102 to 106 CFU/ml. Half of the 14 strains that were used constitutively produced a 1-lactamase. All tubes were incubated at 36 C for 20 h. The MIC was defined as the lowest concentration of antibiotic at which no visible growth could be detected in tubes. Killing curves. The time course of bactericidal activity was determined by enumerating viable cells in 20 ml of a bacterial suspension exposed to the drug. The initial inoculum was adjusted to about 105 CFU/ml, and the culture was incubated for an additional 2 h on a rotatory shaker at 36 C. The test compound was then added to the culture at a final concentration corresponding to a multiple of the MIC. Samples of 0.1 ml were removed at fixed times and were plated onto drug-free Mueller-Hinton agar after they were suitably diluted. Colonies were counted after incubation at 36 C for 24 h. At the highest concentration of antibiotic (2x the MIC) and for an inoculum of 500 CFU/ml, the drug carryover effects accounted for less than 33% of the cell counts. Stability to and inhibition of 13-lactamases. The enzymes were released from the bacteria by ultrasonication and were partially purified by chromatography on Sephacryl S200 superfine (Pharmacia). For the spectrophotometric UV test (16), 2 ml of a 0.1 mm solution of antibiotic and 50,ul of enzyme solution were mixed. The A317 (aztreonam) and A290 () were monitored for 10 min at room temperature. The relative rates of hydrolysis were compared with that of cephaloridine, the hydrolysis of which was measured at 255 nm and was taken as 100%. The inhibitory effects of the compounds on,b-lactamases were tested by the same spectrophotometric assay by using 1 x 10-' to 3 x 10' M Padac (Calbiochem-Behring, La Jolla, Calif.) as the substrate and various amounts of the inhibitor. The rate of hydrolysis of Padac was measured at 550 nm (18). Under our conditions, monobactams were not preincubated with the test enzymes. Affinity for PBPs. The binding of antibiotics to penicillinbinding proteins (PBPs) of E. coli K-12 was determined as described previously (17). After the bacterial membrane preparations were incubated with the test monobactam for 10 min and then with 1251I-labeled ampicillin, PBPs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were detected by subsequent autoradiography of the gels. The pattern of PBPs obtained with highly I251-labeled ampicillin differed from that known for '4C-labeled penicillin G, in that some additional bands were visible. However, the main PBPs (PBP 1, PBP 2, and PBP 3) detected by this method were identical to the PBPs detected with '4C-labeled penicillin G (17). RESULTS In vitro antibacterial activity. Table 1 shows the in vitro antibacterial activity of against various clinical isolates in comparison with those of aztreonam and cefotaxime. As is usually observed with most monobactams, and aztreonam displayed poor activity against gram-positive bacteria, particularly staphylococci. In contrast to aztreonam, however, exhibited marginal activity against Streptococcus pyogenes; the MIC of for 50% of S. pyogenes isolates tested (MIC50) was 2.5,ug/ml. was slightly more active than aztreonam against H. influenzae, with all strains being inhibited by 0.02,ug/ml., however, remained the most active compound.

3 1758 CHANTOT ET AL. TABLE 1. Comparative in vitro antibacterial activities of, aztreonam, and cefotaxime Organism group, organism MIC (Ig/ml) (no. of isolates), and drug Range 50% 90% Gram-positive microorganisms Staphylococcus spp. (23) Streptococcus spp., betahemolytic (38) Gram-negative microorganisms Escherichia coli (106) Klebsiella pneumoniae (81) Klebsiella oxytoca (11) Citrobacter spp. (40) Enterobacter spp. (73) Morganella morganii (22) Serratia spp. (69) Providencia rettgeri (22) Providencia stuartii (28) Proteus mirabilis (62) Proteus vulgaris (16) < s s s s s s s Continued ANTIMICROB. AGENTS CHEMOTHER. TABLE 1-Continued Organism group, organism MIC (,Lg/ml) (no. of isolates), and drug Range 50% 90% Salmonella spp. (27) Shigella spp. (11) Enterobactenaceae,,B-lactamase positivea (85) Enterobacteriaceae, TEM-3 P-lactamase positiveb (9) Cefmenoxime Moxalactam Enterobactenaceae, SHV-2,-lactamase positiveb (12) Cefmenoxime Moxalactam Enterobacteriaceae, SHV-4 P-lactamase positiveb (3) Cefmenoxime Moxalactam Haemophilus influenzae (25) Acinetobacter calcoaceticus (40) Pseudomonas aeruginosa (46) Pseudomonas cepacia (7) Pseudomonas diminuta (5) s s S S S Continued on following page

4 VOL. 36, 1992 TABLE 1-Continued Organism group, organism MIC (pg/ml) (no. of isolates), and drug Range 50% 90% Pseudomonas fluorescens (8) Pseudomonas mendocina (4) Pseudomonas paucimobilis (5) Pseudomonas picketli (5) Pseudomonas putida (10) Pseudomonas stutzeri (10) Xanthomonas maltophilia (10) a Chromosomally encoded constitutive j3-lactamases. Producing strains included Enterobacter spp. (n = 52), Citrobacter spp. (n = 13), Eschenichia coli (n = 13), Morganella morganii (n = 3), Proteus vulgaris (n = 3), and Klebsiella oxytoca (n = 1), all of which showed resistance or decreased susceptibility to cefotaxime (MICs,.1.2,ug/ml). b Plasmid-encoded constitutive extended-spectrum P-lactamases. Producing strains included Kiebsiella pneumoniae (n = 13), Escherichia coli (n = 7), and Salmonella spp. (n = 4), all of which showed resistance or decreased susceptibility to cefotaxime (MICs.1.2 xg/ml). With MIC50s of 5,ug/ml, and aztreonam displayed very similar activities against P. aeruginosa, which were significantly higher than that of cefotaxime. Both monobactams were generally less active against other species of the genus Pseudomonas. They were inactive against Xanthomonas maltophilia. None of the monobactams possessed any activity against Acinetobacter calcoaceticus or Bacteroides fragilis at concentrations below 20,ug/ml. Members of the family Enterobacteriaceae were highly susceptible to, because the MIC90s for those organisms never exceeded 1.2 p,g/ml. Microorganisms which were highly susceptible to cefotaxime were equally susceptible to the monobactams. Conversely, was significantly more active than aztreonam against bacterial isolates within the genera Escherichia, Kiebsiella, Citrobacter, Enterobacter, Serratia, Proteus, and Morganella which showed decreased susceptibility (MICs, >1.2,ug/ml) or resistance (MICs, 232,ug/ml) to cefotaxime. The new mono- ANTIBACTERIAL ACTIVITY OF 1759 TABLE 2. Comparative in vitro antibacterial activities of aztreonam and cefotaxime against strains with decreased susceptibilities to z Strain MIC (,ug/ml) Kebsiella oxytoca GR4 Citrobacter spp. 261GR GR GR HT DU Enterobacter cloacae 293GR GR C C CO Enterobacter agglomerans HT1 Enterobacter sp. strain DU19 Morganella morganii GR16 Serratia sp. strain GR16 a The MIC of was.1.2,ug/ml. bactam was active against enterobacteria which produced chromosomally encoded cephalosporinases. Ninety percent of strains were inhibited by 2.5,ug of per ml, a value 10 to 100 times lower than that obtained for aztreonam, carumonam, and all expanded-spectrum cephalosporins which were tested (data not shown). Similar differences were observed with microorganisms that produce plasmid-encoded extended-spectrum,b-lactamases (TEM-3, SHV-2, and SHV-4). at 1.2 plg/ml inhibited all of the strains tested. Most of these bacterial isolates were resistant to aztreonam, ceftazidime, ceftriaxone, and, to a lesser extent, cefmenoxime, but they were susceptible to moxalactam. Table 2 shows the comparative antibacterial activities of aztreonam and cefotaxime against strains with decreased susceptibilities to (MICs,.1.2,ug/ml). was systematically more active against these strains, all of which were P-lactamase producers, than were the other compounds tested. The only exception was a strain of Klebsiella oxytoca. The effects of the composition of the medium on antibacterial activity were investigated with seven commercial culture media. There were no significant changes (not more than 1 dilution) in the MICs of and aztreonam for the three strains tested (data not shown). The activities of both monobactams were not significantly affected by the ph of the growth medium. For the three strains tested, the MICs did not vary by more than 1 or 2 dilution steps within a ph range of 6.5 to 8.5 (data not shown). The addition of 40% normal human serum to the medium or the use of urine as the sole growth medium-for bacterial cultures had negligible effects on the antibacterial activities of the compounds (data not shown).

5 1760 CHANTOT ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 3. Effect of inoculum size on the MICs of and aztreonam MIC (1Lg/ml) of the following drugs with the indicated inoculum size (CFU/ml) Organism group and strain Cephalosporinase negative Escherichia coli 250HT Escherichia coli 250HTS Escherichia coli 250HT Eschenichia coli 250HT Klebsiella pneumoniae 281HT Enterobacter cloacae 293HT Pseudomonas aeruginosa 391HT Cephalosporinase positivea Escherichia coli 250GR Escherichia coli 250GRS Kiebsiella oxytoca 283HT >80 >80 Citrobacterfreundii 261GR Citrobacterffreundii 261GR Citrobacterffreundii 261HT Enterobacter cloacae 293GR >80 >80 a The strains constitutively produce chromosomal,-lactamases. Table 3 shows the inoculum effect on the MICs of the two monobactams. For most strains, the MICs remained unaffected when the bacterial inoculum was raised from 102 to 106 CFU/ml. However, MICs increased fourfold or greater with P. aeruginosa, two 3-lactamase-producing strains of E. cloacae and Citrobacterfreundii, and two cephalosporinasenegative strains of enterobacteria. The bactericidal activity of against E. coli, K pneumoniae, Providencia stuartii, and P. aeruginosa is shown in Fig. 2. A rapid decrease in the number of viable cells was observed at a concentration of 1 x or 2 x the MIC, and the bacterial population was reduced to less than 1% within 2 to 4 h. Regrowth was observed with K pneumoniae at lx the MIC but not at 2x the MIC after 24 h of incubation. Similar results were obtained with aztreonam against these strains. The susceptibilities of and aztreonam to,b-lactamase hydrolysis were tested by using various chromosomally or plasmid-encoded enzymes (Table 4). Both monobactams appeared to be resistant to enzymatic hydrolysis by the common plasmid-mediated 1-lactamases. appeared to be more susceptible to hydrolysis by TEM-3, SHV-2, and SHV-4 extended-spectrum enzymes. was also markedly less susceptible to the action of constitutive chromosomally mediated enzymes belonging to groups 1, 2b', and 2e of the classification of Bush (1-3). inhibited the hydrolysis of Padac by chromosomal cephalosporinases to a lesser extent than did aztreonam, contrary to the trend observed with the plasmid-mediated TEM-1 enzyme (Table 5). When it was measurable, inhibition was of the competitive type. and aztreonam displayed similar patterns of affinity for the PBPs of E. coli K-12 (Fig. 3). The highest affinity of was for PBP-3. DISCUSSION is a novel monobactam with an original structure featuring a tetrazole heterocycle on the nitrogen of the azetidinone ring. In addition, a fluoromethyl group replaces the methyl function at position 4, and a carboxycyclobutyl substituent is present on the oxime moiety of the well-known aminothiazolylmethoxyimino side chain at position 3 of the 1-lactam ring. The result of these structural changes is that the drug is different from aztreonam, leading to significant variations in its spectrum of activity and potency. Thus, differentiates itself from aztreonam in that it displays noticeable activity against 13-hemolytic streptococci, an unusual feature that it shares with tigemonam (22). Although this result is remarkable for a monobactam, the antibacterial activity achieved might not be sufficient for therapeutic efficacy against these pathogens. As has already been observed with all monobactams which bear an aminothiazolylalkoxyimino moiety in the 3-acylamido side chain (13), the compound has no detectable activity against other gram-positive species of bacteria, e.g., staphylococci. inhibited most members of the family Enterobactenaceae at low concentrations, and the MIC90s were usually well below 1,ug/ml. The spectrum of activity of encompasses cefotaxime-resistant gram-negative rods that produce chromosomally or plasmid-encoded,b-lactamases. Indeed, the drug displayed good activity against bacteria which produce derepressed chromosomal P-lactamases, unlike aztreonam, ceftriaxone, ceftazidime, and, to a lesser extent, moxalactam, which are at least 30 times less active. In agreement with the results of others (15), carumonam did not possess particular advantages over aztreonam against these strains. Likewise, BO-1165, the carboxycyclopropyl analog of in the N-sulfonated series of monocyclic 3-lactams, has been shown to exhibit decreased activity against cephalosporinase-producing enterobacteria (14). The tetrazole moiety seems to play an essential role in enhancing the activity of against these bacteria. also displays antibacterial activity against plasmid-encoded extended-spectrum,b-lactamase-producing (TEM-3, SHV-2, or SHV-4) strains. With many strains of E. coli, K pneumoniae, and Salmonella spp., the ratio of activity of the new monobactam can be as high as 100 with regard to ceftriaxone and ceftazidime. Unexpectedly, cefmenoxime, with an S-methyltetrazole moiety at position 3 of the cephem nucleus, is significantly more active than the

6 VOL. 36, Time (hours) AZTREONAM FIG. 2. Bactericidal activities of and aztreonam against E. coli 250HT2 (A and B), K pneumoniae 283UC2 (C and D), P. stuartii 320UC1 (E and F) and P. aeruginosa 391HT2 (G and H). 0, control; A, 1/2x the MIC; +, lx the MIC; 0, 2x the MIC. The MICs of monobactams are given in the upper left of each panel. The minimum accurately countable bacterial population was 300 CFU/ml (---). congeneric cephalosporins against these microorganisms. In agreement with the results of others (7), moxalactam is even more potent against these strains, probably because of its cephamycin-type structure. Those investigators have also demonstrated significant improvement in the activity of carumonam over that of aztreonam against E. coli strains that produce extended-spectrum 3-lactamases (7). It is worth noting that both carumonam (with a 4-carbamoylmethyl substituent) and (with a 4-fluoromethyl substituent) have their substituents at positions 3 and 4, in a cis configuration, as opposed to the trans configuration in aztreonam. This structural difference might explain in part the increased antibacterial activity of, as has ANTIBACTERIAL ACTIVITY OF 1761 TABLE 4. Hydrolysis of and aztreonam by several 1-lactamases Rate (%) of Enzyme source Typea enzymatic hydrolysis' Plasmid Escherichia coli 2136E 2b (TEM-1) <1 <1 Escherichia coli 2137E 2b (TEM-2) <1 <1 Escherichia coli 250BE7 2b' (TEM-3) <1 4.6 Klebsiella pneumoniae 2b (SHV-1) <1 <1 1976E Klebsiella pneumoniae 2b' (SHV-2) < BE2 Klebsiella pneumoniae 2b' (SHV-4) <1 <1 283BE1 Escherichia coli 2138E 2d (OXA-1) <1 2.5 Escherichia coli 2139E 2d (OXA-2) <1 <1 Escherichia coli 2140E 2d (OXA-3) <1 <1 Pseudomonas aeruginosa 2c (PSE-1) <1 <1 1937E Pseudomonas aeruginosa 2d (PSE-2) <1 <1 1973E Pseudomonas aeruginosa 2c (PSE-3) <1 <1 1920E Pseudomonas aeruginosa 2c (PSE-4) <1 <1 1559E Chromosome Escherichia coli 250GR2 1 <1 <1 Citrobacterfreundii GN Enterobacter cloacae P99 1 <1 <1 Morganella morganii 1 <1 <1 313GR16 Pseudomonas aeruginosa 1 <1 8.9 GN918 Klebsiella oxytoca 1082E Kl 2b' <1 21 Citrobacterfreundii J20 2e <1 2.5 Proteus vulgaris GN76 2e <1 <1 a Bush classification (1-3). b Rate of enzymatic hydrolysis relative to that of cephaloridine, which was arbitrarily taken at 100. already been shown among N-tetrazole-substituted monocyclic 1-lactams (9). displays resistance to hydrolysis by chromosomally encoded 1-lactamases, a property which contributes to the overall activity of the new monobactam against microorganisms that constitutively produce these enzymes. On the other hand, is less effective than aztreonam in inhibiting chromosomal,b-lactamases. shows a high level of stability to plasmid-encoded enzymes, particularly to extended-spectrum,b-lactamases. With the exception of SHV-4, aztreonam appears to be slightly more labile TABLE 5. Enzyme source 3-Lactamase inhibition by and aztreonam Typea Ki (M) Escherichia coli 2136E 2b (TEM-1) >1.0 x 10-3 >1.0 X 10-3 Enterobacter cloacae P x x 10-6 Citrobacterfreundii J20 2e >1.0 x X 10-5 Kiebsiella oxytoca 1082E 2b' >1.0 x X 10-4 Kl a Bush classification (1-3).

7 1762 CHANTOT ET AL. RV Ktr 00 0,1 I 0,001 0,01 0,1 1I FIG. 3. Affinity of and aztreonam for PBPs of E. coli K-12. Ktr, control. to these enzymes. This differential behavior of the two monobactams toward enzymatic degradation is reflected and even amplified at the level of the MICs for the 13-lactamaseproducing strains. The antibacterial activity of or aztreonam was generally unaffected by varilations of the ph within physiological values or--by the addition of human serum at ph 7.4. With most strains, the activity of and aztreonam did not appear to be very dependent on the inoculum size up to a value of 106 CFU/ml. Nevertheless, it would be desirable to examine the activities of the drugs when using a higher inoculum, particularly in the case of P-lactamaseproducing isolates. The new antibiotic has a bactericidal type of action, as deduced from examination of the killing curves determined against several pathogens. Time-kill curves are almost superimposed on those obtained with aztreonam for concentrations corresponding to the same multiple of the MIC. Indeed, the binding patterns of both monobactams to bacterial targets seem to be very similar, at least in E. coli K-12, with a very high affinity for PBP 3. Unusual additional bands were revealed upon autoradiography of the gels when we used 1'I5-labeled ampicillin to label the PBPs. Such a high-energy radioactive marker might label some PBP-related fragments which can be formed during membrane purification or storage (11). In conclusion, is an original and promising new monocyclic 13-lactam antibiotic that has potent in vitro antibacterial activity against gram-negative microorganisms. Further pharmacological and clinical evaluations seem to be warranted. ACKNOWLEDGMENT We thank Nicole Claussner for excellent technical assistance. REFERENCES 1. Bush, K Characterization of P-lactamases. Antimicrob. Agents Chemother. 33: Bush, K Classification of P-lactamases: groups 1, 2a, 2b, and 2b'. Antimicrob. Agents Chemother. 33: ANTIMICROB. AGENTS CHEMOTHER. 3. Bush, K Classification of,-lactamases: groups 2c, 2d, 2e, 3, and 4. Antimicrob. Agents Chemother. 33: a.Chantot, J. F., G. Seibert, M. Klich, and G. Teutsch Program Abstr. 26th Intersci. Conf. Antimicrob. Agents Chemother., abstr Cimarusti, C. M., D. P. Bonner, H. Breuer, H. W. Chang, A. W. Fritz, D. M. Floyd, T. P. Kissick, W. H. Koster, D. Kronenthal, F. Massa, R. H. Mueller, J. Pluscec, W. A. Lusarchyk, R. B. Sykes, M. Taylor, and E. R. Weaver Alkylated monobactams. Chiral synthesis and antibacterial activity. Tetrahedron 39: Gordon, E. M., M. A. Ondetti, J. Pluscec, C. M. Cimarusti, D. P. Bonner, and R. B. Sykes sulfated P-lactam hydroxamic acids monosulfactams, novel monocyclic,-lactam antibiotics of synthetic origin. J. Am. Chem. Soc. 104: Imada, A., K. Kitano, K. Kintaka, M. Muroi, and M. 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