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Antimicrobial Agents and Chemotherapy, December 1999, p. 2904-2909, Vol. 43, No. 12
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Biochemical Characterization of Novel Tetrahydrofuranyl 1beta -Methylcarbapenems: Stability to Hydrolysis by Renal Dehydropeptidases and Bacterial beta -Lactamases, Binding to Penicillin Binding Proteins, and Permeability Properties

Youjun Yang,* Raymond T. Testa, Niraja Bhachech, Beth A. Rasmussen, and Karen Bushdagger

Infectious Disease Research Section, Wyeth-Ayerst Research, Pearl River, New York 10965

Received 1 June 1999/Returned for modification 12 August 1999/Accepted 21 September 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The biochemical properties of tetrahydrofuranyl (THF) carbapenems, carbapenems with THF substituents, were evaluated with respect to enzyme stability, binding to penicillin-binding proteins (PBPs), and penetration into gram-negative organisms. THF carbapenems showed increased stability to hog renal dehydropeptidases (DHPs) compared to that of imipenem or meropenem and were more stable to human DHP than imipenem (<10% hydrolysis compared to that for imipenem). THF carbapenems were stable to hydrolysis by all serine beta -lactamases tested. CL 191,121, a prototype THF carbapenem, was more stable to hydrolysis by carbapenem-hydrolyzing serine beta -lactamases such as IMI-1 and Sme-1 than imipenem, with a relative kcat value of <20% for imipenem. Similar to imipenem and meropenem, THF carbapenems were not stable to the metallo beta -lactamases CcrA and L1. However, CL 191,121 bound to all Staphylococcus aureus PBPs at concentrations that were less than or equal to the MICs. The THF carbapenems bound to PBPs from Escherichia coli and Pseudomonas aeruginosa, with the highest affinities being for PBPs 2 and 4, as noted with imipenem. The affinities for PBPs 1a and 1b in E. coli were reduced for the THF carbapenems compared to that for imipenem, even though the MICs of the THF carbapenems for E. coli strains were lower than those of imipenem. The penetrability of the THF carbapenems into Serratia marcescens S6, which produces the Sme-1 carbapenem-hydrolyzing beta -lactamase, was 2.4 to 7.8 times less than that of imipenem. Compounds CL 190,294 and CL 188,624 showed good penetrability, with permeability coefficient values comparable to those of the rapidly penetrating agents cephaloridine, imipenem, meropenem, and biapenem. Decreased penetration into wild-type P. aeruginosa was suggested by the high MICs of the THF carbapenems (MICs, 16 to 32 µg/ml), despite equivalent or better binding to P. aeruginosa PBPs than that of imipenem. However, the MICs of the THF carbapenems for wild-type P. aeruginosa compared to that for an OprD2 mutant generally varied no more than 2-fold, but those of imipenem and other carbapenems differed 16-fold. These data indicated that THF carbapenems do not appear to enter through protein OprD2. In conclusion, the THF carbapenems exhibited stability to hydrolysis by renal DHPs and serine beta -lactamases, exhibited strong binding to essential PBPs from E. coli and S. aureus, and penetrated gram-negative enteric bacteria at rates comparable to those for meropenem and biapenem.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Carbapenems, such as imipenem, meropenem, and biapenem, have the broadest spectrum of antimicrobial activity among the beta -lactams and exhibit fewer common resistance problems (2, 20, 23). This activity is due to the combined effects of good stability to hydrolysis by most beta -lactamases, strong binding to essential penicillin-binding proteins (PBPs), and good penetrability into gram-negative organisms (8, 29, 30). The excellent activity of imipenem against Pseudomonas aeruginosa has also been attributed to entry into the organisms via a specific carbapenem uptake pathway involving the OprD protein channel (30). However, imipenem can easily be destroyed by mammalian dehydropeptidases (DHPs) (13, 19) and must be administered with the DHP inhibitor cilastatin (11). In addition, imipenem has a relatively short half-life in vivo and is not orally active. With a 1,beta -methyl group attached to the beta -lactam nucleus, meropenem and biapenem showed significantly improved stability to DHP hydrolysis (9, 35). However, none of these carbapenems are orally active.

Tetrahydrofuranyl (THF) carbapenems, such as CL 191,121, CL 188,624, and CL 190,294, are a new class of carbapenems with THF substituents (15-17). These carbapenems exhibit broad-spectrum antibacterial profiles that combines the good activities of meropenem and biapenem against gram-negative bacteria with the excellent activity of imipenem against gram-positive bacteria (15, 31). A series of peptidic THF carbapenem prodrugs such as CL 191,638 and CL 191,983 are based on the parent compound CL 191,121 and exhibit activity when they are administered orally (16, 32). They are especially attractive because both imipenem and meropenem are parenterally administered drugs. The present study outlines the biochemical properties of THF carbapenems for their stabilities to the hydrolytic activities of DHPs from the hog, mouse, rat, and human and to bacterial serine and metallo beta -lactamases. The PBP affinities for THF carbapenems were compared with those for other carbapenems. The penetration properties of THF carbapenems into gram-negative bacteria have also been evaluated.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Antibiotics. Biapenem, piperacillin, THF carbapenems, and their derivatives were synthesized by the Chemistry Group of Wyeth-Ayerst Research. The structural characteristics of each THF carbapenem are indicated in Fig. 1 and Table 1 (also, see references 15 to 17). Imipenem was obtained from Merck (Rahway, N.J.), meropenem was obtained from Zeneca (Maccelesfield, England), cephaloridine was obtained from Sigma (St. Louis, Mo.), and cefotaxime was obtained from Hoechst-Roussel Pharmaceuticals Inc. (Frankfurt, Germany). Working solutions of antimicrobial agents were freshly prepared on each day of assay.


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  		FIG. 1.   Structures of the aminomethyl THF 1-beta -methyl carbapenems. Stereochemistries were as follows (R and S define the configuration of the chiral carbon atom): CL 191,121, 3R, 2R; CL 188,624, 3S, 5R and 3R, 5S; CL 190,294, 3R, 5R and 3S, 5S.

                              
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  		TABLE 1.   Stabilities of THF carbapenems to hydrolysis by renal DHPs and metallo beta -lactamasesa   

Microorganisms. Serratia marcescens S6, a carbapenem-hydrolyzing serine beta -lactamase producer (33), was used as the test organism for the penetration assays. P. aeruginosa 27853 was used to derive the OprD2-deficient mutants. A standard P. aeruginosa OprD2-deficient isolate, isolate GC 1543, originated by Quinn et al. (24), was also used for comparison. Staphylococcus aureus 29213, Escherichia coli MC4100, and P. aeruginosa 27853 were used as test organisms for the PBP assays.

Enzymes. DHPs from fresh hog and mouse kidney and from rat intestine were extracted with butanol and were precipitated with ammonium sulfate in 10 mM HEPES (N-[2-hydroxyethyl]piperazine-N'-2 ethanesulfonic acid) buffer (pH 7.2) (7). The enzymes were solubilized from frozen precipitates on each day of assay. A human DHP gene was cloned from a human kidney cDNA gene bank and was expressed in human kidney 293 cells (25). DHP was partially purified as described previously (1).

Bacterial metallo beta -lactamase CcrA from Bacterioids fragilis was purified to homogeneity from the CcrA cloned in E. coli DH5alpha (34). Metallo beta -lactamase L1 from Stenotrophomonas maltophilia was purified by CM-C50 cation-exchange chromatography (4). The serine beta -lactamases, P99 from Enterobacter cloacae, PC1 from S. aureus, TEM-2 from E. coli, TEM-26 from Klebsiella pneumonia, and Sme-1 from S. marcescens, were purified to homogeneity by previously described methods (35). The IMI-1 beta -lactamase from E. cloacae was purified to homogeneity by column chromatography (26).

Enzyme stability and kinetic studies. Carbapenems were prepared at a concentration of 1.0 mg/ml in water and were assayed at a final concentration of 50 µg/ml. The relative hydrolysis rates for carbapenems were determined spectrophotometrically at UV wavelengths of 290 to 300 nm on the basis of the maximum change in absorbance in the difference spectrum following enzymatic hydrolysis by the CcrA enzyme. The hydrolysis of carbapenems by the DHPs and metallo beta -lactamases was measured in 10 mM HEPES buffer (pH 7.2). Hydrolysis of carbapenems by serine beta -lactamases was determined in 50 mM phosphate buffer (pH 7.0). Two different volumes of enzyme (10 to 50 µl) were used in a total volume of 1,000 µl, and rates were determined as nanomoles of substrate hydrolyzed per microliter of enzyme solution added. Imipenem and meropenem were included as reference compounds for each set of assays. Relative hydrolysis rates were calculated by normalizing the specific molar hydrolysis rates to those observed with imipenem on the same day. Kinetic parameters (Km and Kcat) of the carbapenem-hydrolyzing enzymes were derived with the ENZPACK (Biosoft) program on the basis of two independent experiments with at least six concentrations of the substrate at a single enzyme concentration. The maximum hydrolytic activity was reported as a kcat value (second-1) for each substrate. The total protein concentration in each enzyme preparation was determined by a bicinchoninic acid assay (Pierce Chemical Co., Rockford, Ill.).

Assay for PBP binding. PBP binding was assessed by competition assays based on the method of Spratt (28). In standard assays, carbapenems were incubated with solubilized membranes for 10 min at 30°C. [14C]benzylpenicillin was added to give a final concentration of 10 µg/ml. The membranes were incubated for another 10 min at 30°C, and the reaction was terminated with cold acetone. For mechanistic studies the timing and temperatures of the incubations were varied (3). PBPs were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. X-ray films were analyzed on a Shimadzu CS9000U densitometer, with the 50% inhibitory concentrations (IC50s) determined graphically.

Penetration studies. The penetrabilities of the beta -lactams into gram-negative bacteria were determined by the method introduced by Zimmermann and Rosselet (37) and modified by Nikaido et al. (22). S. marcescens S6, the Sme-1 carbapenem-hydrolyzing serine beta -lactamase producer, was used as the test organism. Details of the permeability assay were described previously (35). The antibiotic diffusion rate (VE) was obtained with whole-cell suspensions. The hydrolysis rates were determined with sonicated cells and were used to derive Km and Vmax values. Permeability coefficients were calculated by the formula originated by Nikaido et al. (22).

Derivation and identification of P. aeruginosa OprD2 mutants. P. aeruginosa 27853 was cultured (108 CFU) on a tryptic soy agar plate containing 4, 8, and 16 µg of imipenem per ml. Plates containing 4 and 8 µg of imipenem per ml were incubated at 37°C for 24 h, and plates containing 16 µg of imipenem per ml were incubated for 48 h. Resistant selection frequency was calculated on the basis of the growth of colonies on the imipenem-containing plates. The imipenem resistance of the isolated colonies was confirmed by disc diffusion testing and MIC assays. Outer membrane proteins of the parent and selected resistant isolates, as well as those of the control OprD2 deficient strain, were extracted with 1% laurylsarcosine and identified by SDS-PAGE with 12% polyacrylamide gels (27).

Microbiological assays. MICs were determined by the broth microdilution method in Mueller-Hinton II broth as recommended by the National Committee for Clinical Laboratory Standards (21).


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Stabilities of THF carbapenems to DHPs. The relative hydrolysis rates of DHP and metallo beta -lactamases for the THF carbapenems, together with their structural characteristics, are summarized in Table 1. For hog DHPs, all the THF carbapenems were hydrolyzed at rates slower than those observed for imipenem and meropenem. The stabilities of carbapenems to hydrolysis by mouse and human DHPs indicated that all compounds tested were less stable to mouse DHPs than to hog DHPs, especially meropenem and CL 191,638. However, for human DHP, all THF carbapenems showed excellent stability compared to that of imipenem, with a rate <20% that for imipenem. The hydrolysis of the THF carbapenems CL 191,121, CL 191,638, and CL 191,983 by rat intestine enzymes proceeded at a rate comparable to or faster than that for hydrolysis of imipenem (Table 1).

Stabilities to carbapenem-hydrolyzing beta -lactamases. Many metallo beta -lactamases can hydrolyze all classes of beta -lactams including carbapenems. The THF carbapenems, including the hydrophilic compounds with a hydrogen or amino group at the terminus of one or both of the side chains, were also not stable to the hydrolysis by the two metallo beta -lactamases CcrA and L1 (Table 1).

The kinetic parameters for hydrolysis of selected THF carbapenems with carbapenem-hydrolyzing beta -lactamases are summarized in Table 2. As initially indicated by the relative hydrolysis rate (Table 1) for the metallo beta -lactamase CcrA, all THF carbapenems except CL 191,121 were less stable than imipenem on the basis of the kcat value; CL 191,121 was almost twofold more stable than imipenem. For the metallo beta -lactamase L1, all THF carbapenems were as stable as or slightly more stable than imipenem on the basis of the kcat value. Km values with the CcrA enzyme were lower for THF carbapenems than for imipenem, resulting in higher kcat/Km values. However, on the basis of kcat values, THF carbapenems were much more stable than imipenem to hydrolysis by the carbapenem-hydrolyzing serine beta -lactamases Sme-1 and IMI-1. The kcat values of Sme-1 and IMI-1 for the THF carbapenems were 5 to 16 times slower than those for imipenem. Although lower Km values were observed for THF carbapenems than for imipenem with IMI-1 and Sme-1 (Table 2), the catalytic efficiencies (kcat/Km values) were still lower for the THF carbapenems than for imipenem with the Sme-1 enzyme.

                              
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  		TABLE 2.   Kinetic parameters of carbapenem-hydrolyzing beta -lactamases for THF carbapenemsa

Stabilities to other serine beta -lactamases. All THF carbapenems showed excellent stability to hydrolysis by serine beta -lactamases in functional groups 1, 2a, 2b, and 2e (molecular classes C and A) (Table 3), with the relative hydrolysis rate for the THF carbapenems never exceeding 1% of that for the reference compound. The extended-spectrum beta -lactamase TEM-26 hydrolyzed all the carbapenems slowly.

                              
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  		TABLE 3.   Stabilities of THF carbapenems to hydrolysis by serine beta -lactamases

Affinities of THF carbapenems for PBPs. THF carbapenems bound to PBPs from both gram-positive and gram-negative organisms, including P. aeruginosa, with affinities comparable to those of other carbapenems (Table 4). The good antimicrobial activities of the THF carbapenems against S. aureus (31) were consistent with tight binding to PBP 1 (Table 4). Moreover, CL 191,121 bound well to all PBPs from S. aureus. None of the THF carbapenems bound well to PBP 2a from methicillin-resistant S. aureus BB270 (MICs, >16 µg/ml), with the IC50s of all compounds being >100 µg/ml (data not shown). With E. coli all three THF carbapenems exhibited similar PBP profiles, with excellent binding to PBPs 2 and 4 (IC50s, <0.1 µg/ml). CL 191,121 had better affinity for PBPs 1a and 1b than CL 188,624 and CL 190,294, but it still bound to these two PBPs less effectively than imipenem or meropenem did. The last two THF carbapenems bound to PBP 3 better than any of the carbapenems tested other than meropenem. Despite the reduced antimicrobial activity against P. aeruginosa, the THF carbapenems exhibited strong binding to the PBPs in P. aeruginosa, having comparable or better IC50s than those of imipenem and biapenem for PBPs 1b, 1c, 2, and 3 (Table 4).

                              
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  		TABLE 4.   Binding of carbapenems to PBPs of S. aureus, E. coli, and P. aeruginosa

When assayed at 30°C, poor binding to PBP in S. aureus was seen for CL 188,624 and CL 190,294 (IC50s were estimated to be 5 and 20 µg/ml, respectively), whereas IC50s were 0.5 and 0.15 µg/ml, respectively, at 0°C. However, the IC50 of CL 191,121 (0.06 µg/ml) was much lower at 30°C and was comparable to the IC50 determined at 0°C (0.12 µg/ml). Apparent affinities were improved 10- to 100-fold for CL 188,624 and CL 190,294 when assay temperatures were decreased to 0°C. As seen previously with selected monobactams (3), this differential in IC50s as a function of temperature suggests that rapid deacylation of the first two isomers can occur at 30°C. Better binding to PBP 1 of S. aureus was observed at the higher assay temperature. This is consistent with direct kinetic measurements indicating a slow acylation rate for PBP 1 (half-life of 2.5 to 3.2 min at 30°C).

Penetration of THF carbapenems into S. marcescens S6. The permeability coefficients of representative beta -lactams and THF carbapenems CL 188,624, CL 190,294, and CL 191,121 are summarized in Table 5. Cephaloridine and imipenem are compounds that penetrate gram-negative organisms fast, as demonstrated here and by other researchers (22, 36). Piperacillin and cefotaxime penetrated the organisms more slowly. The THF carbapenems, especially CL 190,294 and CL 188,624, showed good penetrability through the porin channels of S. marcescens S6, with permeability coefficients comparable to that of the rapidly penetrating compound cephaloridine. CL 191,121 penetrated more slowly than the other two THF carbapenems tested but still exhibited a rate approximately 30% of that for cephaloridine. The THF carbapenems CL 190,294 and CL 188,624 were transported into S. marcescens at rates similar to those for meropenem and biapenem.

                              
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  		TABLE 5.   Penetration of novel THF carbapenems into S. marcescens S6a

Selection and identification of OprD2-deficient mutants. Imipenem-resistant mutants of P. aeruginosa 27853 were selected at a frequency of 10-7. Resistance to imipenem with the selected mutants was confirmed by disc diffusion tests. Outer membrane extraction with laurylsarcosine and by protein identification by SDS-gel electrophoresis indicated that these imipenem-selected mutants shared the same outer membrane profile as the control OprD2-deficient strain: a loss of the OprD2 protein compared to the outer membrane protein profile of the parent strain (Fig. 2). The MICs of imipenem, meropenem, biapenem, and THF carbapenems for the OprD-deficient mutants in comparison with those for the wild-type parent are summarized in Table 4. For imipenem, meropenem, and biapenem, the MICs for the resistant mutants were elevated 8- to 32-fold compared to that for the parent strain, P. aeruginosa 27853. The MICs of the THF carbapenems were never more than twofold higher for the OprD2 mutants compared to those for the wild-type strain.


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  		FIG. 2.   Outer membrane protein profiles of P. aeruginosa strains. Lanes A and E, molecular mass markers (Bio-Rad, Richmond, Calif.); lane B, isolate B of an OprD2-deficient strain (24); lane C, P. aeruginosa ATCC 27853; lane D, OprD2-deficient mutant from P. aeruginosa 27853 selected with imipenem. Numbers on the left are in kilodaltons.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Early carbapenems such as imipenem were not stable to hydrolysis by the zinc-containing enzymes such as mammalian renal DHPs and metallo-beta -lactamases. One of the most important features that a novel carbapenem needs to compete in the commercial marketplace is improved stability to DHPs. As with meropenem, the addition of a 1-beta -methyl group to the beta -lactam nucleus of THF carbapenems such as CL 191,121 and CL 191,983 increased the stability to mammalian DHPs.

beta -Lactamase production is one of the major mechanisms of bacterial resistance to beta -lactams. Although carbapenems are generally quite stable to hydrolysis by most common beta -lactamases, they are generally not stable to the class B metallo beta -lactamases. Therefore, it is still essential that the stabilities of novel carbapenems to a battery of beta -lactamases from various sources be evaluated. The evaluation of THF carbapenems for hydrolysis by the metallo-beta -lactamases CcrA and L1 indicated that hydrophilic compounds containing a free carboxylic group were less stable to hydrolysis by the metallo beta -lactamases. Carbapenem-hydrolyzing serine beta -lactamases such as IMI-1 and Sme-1 were capable of hydrolyzing imipenem and meropenem. However, THF carbapenems such as CL 191,121 were more stable than imipenem to hydrolysis by IMI-1 and Sme-1. In addition, THF carbapenems such as CL 191,121 were stable to hydrolysis by the other serine beta -lactamases tested.

The three compounds selected for more extensive evaluation demonstrated that the good penetrability of the THF carbapenems, especially CL 190,294 and CL 188,624, through the porin channels of S. marcescens S6, combined with their high degrees of affinity to PBPs 2 and 4 of gram-negative organisms (5), contributed to their good activities against gram-negative bacteria.

The modest antipseudomonal activities of the THF compounds were also studied. Although the penetrabilities of the THF carbapenems into members of the family Enterobacteriaceae were comparable to those of other carbapenems, penetration into P. aeruginosa appeared to be diminished. The levels of binding to essential PBPs in P. aeruginosa were comparable for biapenem and the THF carbapenems (5), but the MICs of the THF carbapenems were higher. This study demonstrated that the loss of the OprD2 protein channel had little effect upon the antipseudomonal activities of the THF carbapenems but markedly affected the activities of imipenem and biapenem (eightfold or greater increases in MICs). The differences in the MICs of the THF carbapenems for parent strain P. aeruginosa 27853 were not more than twofold compared to those for its OprD2-deficient mutants. These results indicate that THF carbapenems do not appear to use the specific imipenem-penetrating channel OprD2 as their major route for entry into P. aeruginosa. This behavior is similar to that reported for a series of carbapenems bearing a basic group at either position 1 or position 6 (e.g., BMY 45047); imipenem has a single basic group at position 2 (10). Decreased activity in P. aeruginosa appeared to be due to poor uptake through protein OprD2.

Imipenem resistance in P. aeruginosa reflects a complex interplay between inducibility and stability to group 1 (class C) beta -lactamases, uptake through porins, especially OprD2 (18), and efflux potential (12, 14). The THF carbapenem CL 191,121 behaved as a weaker inducer than imipenem, with at least a fivefold lower induction ratio than that for imipenem at a concentration of one-half the MIC (data not shown). Unfortunately, the stabilities of imipenem and CL 191,121 to hydrolysis by crude enzyme extracts from P. aeruginosa ATCC 27852 and the OprD2-deficient mutants were not measurable due to the relative stabilities of both compounds and the low enzyme concentrations in the extracts. However, with a homogeneously purified AmpC beta -lactamase from E. cloacae, CL 191,121 was almost fourfold more stable than imipenem at 100 µM (data not shown). These data suggest that, in addition to not using the OprD2 channel, the better stability of the THF carbapenems to hydrolysis by an AmpC beta -lactamase may contribute to the minimal effect on the THF carbapenem MICs for imipenem-resistant mutants. These findings correspond to Livermore's (18) comments that "the activity of a carbapenem more beta -lactamase stable than imipenem should be less affected by the porin loss." The efflux potentials for THF carbapenems have not been evaluated, whereas those for the carbapenem ER-35786 and for various beta -lactams have been evaluated by Köhler et al. (12) and Li et al. (14), respectively.

The good stability to hydrolysis by DHP and beta -lactamases, efficient binding to the target proteins, and favorable penetrability coupled with their in vitro and in vivo antimicrobial activities support the further evaluation of THF carbapenems. These THF carbapenems are under investigation as orally active compounds.


    ACKNOWLEDGMENTS

We thank Y.-I. Lin, P. Bitha, S. M. Sakya, T. W. Stronhmeyer, and Z. Li for synthesizing the THF carbapenems and W. Weiss for performing the antimicrobial susceptibility tests.


    FOOTNOTES

* Corresponding author. Mailing address: 205/227, Infectious Disease Research Section, Wyeth-Ayerst Research, 401 North Middletown Rd., Pearl River, NY 10965. Phone: (914) 732-4466. Fax: (914) 732-2480. E-mail: Yangy{at}war.wyeth.com.

dagger Present address: R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ 08869.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Antimicrobial Agents and Chemotherapy, December 1999, p. 2904-2909, Vol. 43, No. 12
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Weiss, W. J., Petersen, P. J., Murphy, T. M., Tardio, L., Yang, Y., Bradford, P. A., Venkatesan, A. M., Abe, T., Isoda, T., Mihira, A., Ushirogochi, H., Takasake, T., Projan, S., O'Connell, J., Mansour, T. S. (2004). In Vitro and In Vivo Activities of Novel 6-Methylidene Penems as {beta}-Lactamase Inhibitors. Antimicrob. Agents Chemother. 48: 4589-4596 [Abstract] [Full Text]  

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