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Antimicrobial Agents and Chemotherapy, September 2007, p. 3089-3095, Vol. 51, No. 9
0066-4804/07/$08.00+0     doi:10.1128/AAC.00218-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Interactions of Ceftobiprole with ß-Lactamases from Molecular Classes A to D{triangledown}

Anne Marie Queenan,1* Wenchi Shang,1 Malgosia Kania,2 Malcolm G. P. Page,2 and Karen Bush1

Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 1000 Route 202 South, Raritan, New Jersey 08869,1 Basilea Pharmaceutica Ltd., Grenzacherstrasse 487, P.O. Box, CH-4005 Basel, Switzerland2

Received 13 February 2007/ Returned for modification 5 April 2007/ Accepted 14 June 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The interactions of ceftobiprole with purified ß-lactamases from molecular classes A, B, C, and D were determined and compared with those of benzylpenicillin, cephaloridine, cefepime, and ceftazidime. Enzymes were selected from functional groups 1, 2a, 2b, 2be, 2d, 2e, and 3 to represent ß-lactamases from organisms within the antibacterial spectrum of ceftobiprole. Ceftobiprole was refractory to hydrolysis by the common staphylococcal PC1 ß-lactamase, the class A TEM-1 ß-lactamase, and the class C AmpC ß-lactamase but was labile to hydrolysis by class B, class D, and class A extended-spectrum ß-lactamases. Cefepime and ceftazidime followed similar patterns. In most cases, the hydrolytic stability of a substrate correlated with the MIC for the producing organism. Ceftobiprole and cefepime generally had lower MICs than ceftazidime for AmpC-producing organisms, particularly AmpC-overexpressing Enterobacter cloacae organisms. However, all three cephalosporins were hydrolyzed very slowly by AmpC cephalosporinases, suggesting that factors other than ß-lactamase stability contribute to lower ceftobiprole and cefepime MICs against many members of the family Enterobacteriaceae.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cephalosporins are effective first-line therapies for many bacterial infections. Ceftobiprole, a parenteral investigational cephalosporin in phase III clinical trials, has notable activity against gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). In several studies, the MICs for MRSA ranged from 0.12 µg/ml to 4 µg/ml, with MIC90 values of 2 µg/ml generally observed (3, 15, 19). In addition, ceftobiprole has activity against gram-negative bacteria, including Pseudomonas aeruginosa, similar to those of the extended-spectrum cephalosporins (17, 18, 40).

The mechanism of action for ceftobiprole is common to that of the ß-lactam class in general, where binding of drug to the penicillin-binding proteins (PBPs) results in the inhibition of cell wall synthesis, followed by cell death. A distinguishing feature of ceftobiprole is that, in addition to inhibiting the normal complement of PBPs in most species, it also binds with a high affinity to the acquired PBP 2a (PBP 2') of MRSA strains, PBP 2a of Staphylococcus epidermidis, and PBP 2x of penicillin-resistant Streptococcus pneumoniae (14, 17, 22, 26). Ceftobiprole does not bind to PBP 5 of Enterococcus faecium and therefore has high MICs when it is tested with this organism (17).

Approximately 90% of the clinical isolates of S. aureus are penicillin resistant, with the majority of these strains carrying the blaZ gene, which codes for staphylococcal ß-lactamases (23, 24). Four major gram-positive organism ß-lactamases, primarily penicillinases, are found in Staphylococcus spp. and in some rare Enterococcus faecalis isolates (32, 41). Previous studies have shown that ceftobiprole is very poorly hydrolyzed by the PC1 ß-lactamase of S. aureus (17), resulting in a ceftobiprole MIC90 value of 0.5 µg/ml for a collection of methicillin-susceptible S. aureus strains with 90% penicillin resistance (19) and indicating that the presence of ß-lactamases in S. aureus does not affect the in vitro activity of ceftobiprole.

The ceftobiprole MICs for gram-negative clinical isolates are frequently ≤4 µg/ml, with the exception of strains producing derepressed AmpC or rare class A cephalosporinases or strains producing extended-spectrum ß-lactamases (ESBLs), suggesting that ceftobiprole is hydrolyzed by these enzymes (17-19). ß-Lactamase-mediated resistance is a growing threat among the gram-negative bacteria, with expansion of ESBLs of the TEM, SHV, and CTX-M types throughout the world (4, 27), in addition to the production of AmpC ß-lactamases from plasmids, serine carbapenemases, metallo-ß-lactamases, and OXA ß-lactamases (1, 5, 6, 29, 36, 37). The purpose of these studies was to evaluate the hydrolysis of ceftobiprole by a spectrum of ß-lactamases from almost every functional group (8) and to compare its hydrolysis parameters to those obtained for ceftazidime and cefepime.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antimicrobial agents. Ceftobiprole was obtained from Johnson & Johnson Pharmaceutical Research and Development L.L.C. (Raritan, NJ). Benzylpenicillin, cephaloridine, and cefotaxime were purchased from Sigma (St. Louis, MO); and ceftazidime was obtained from the U.S. Pharmacopeia (Rockville, MD). Cefepime was a gift from Bristol-Meyers Squibb (Princeton, NJ).

MIC determinations. The MICs were determined by the broth microdilution methodology, as described by the Clinical and Laboratory Standards Institute (CLSI) (13). The interpretive criteria were defined according to the CLSI (12).

ß-Lactamase purification. The strains used are listed in Table 1. The ß-lactamases were purified to >90% homogeneity by fast-performance liquid chromatography. Cultures of tryptic soy broth (1 to 2 liters) were inoculated and grown overnight at 37°C. Strains that encoded the ß-lactamase on a plasmid were grown under selective conditions of 100 µg ampicillin per ml. Some clinical isolates expressed low levels of the ß-lactamase; in these cases the complete ß-lactamase-coding regions were cloned into a pET expression vector (pET24a or pET29a, with kanamycin selection) and expression was induced in Escherichia coli BL21(DE3) cells with 400 µM isopropyl-ß-D-thiogalactopyranoside, according to the manufacturer's instructions (Novagen, EMD Biosciences, San Diego, CA). The cells were harvested by centrifugation and washed with 50 mM phosphate buffer (pH 7.0). The cells were resuspended in 5 ml of the same buffer and subjected to five freeze-thaw cycles on dry ice-ethanol. Following centrifugation, the supernatants were filtered through 0.45-µm-pore-size filters and passed through a Superdex 100 gel filtration column (GE Healthcare, Piscataway, NJ). Active fractions eluted in 50 mM phosphate (pH 7.0) were pooled and further purified by using HiTrap SP cation and Q anion-exchange columns (GE Healthcare). The choices of the column and the buffer used depended on the isoelectric point of the ß-lactamase. The proteins were checked for purity on NuPAGE 10% bis-tris gels stained with colloidal blue (Invitrogen, Carlsbad, CA) and were quantitated by the Micro BCA assay (Pierce, Rockford, IL). Some AmpC enzymes were purified according to the method described by Page (25).


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  		TABLE 1. Description of strains and ß-lactamases

 
Determination of kinetic parameters. Hydrolysis was measured with a Shimadzu UV-1601 spectrophotometer at 25°C in 50 mM phosphate buffer (pH 7.0) (35). Extinction coefficients ({Delta}{varepsilon} values) were determined for all substrates by averaging the values obtained from the change in the absorbance divided by the molarity for complete hydrolysis of three concentrations of compound. The wavelengths and {Delta}{varepsilon} values were as follows for the indicated substrates: for benzylpenicillin, {Delta}{varepsilon}240 = –575 M–1 cm–1; for cephaloridine {Delta}{varepsilon}295 = –1,217 M–1 cm–1; for cefepime, {Delta}{varepsilon}265 = –11,670 M–1 cm–1; and for ceftazidime {Delta}{varepsilon}260 = –11,220 M–1 cm–1. All substrates except ceftobiprole were prepared as 1-mg/ml stocks in 50 mM phosphate buffer (pH 7.0); ceftobiprole ({Delta}{varepsilon}290 = –6,970 M–1 cm–1) was prepared at a concentration of 0.32 mg/ml. The kinetic parameters Km and Vmax were determined from a Hanes plot of the hydrolysis data or by directly weighted fits to the Michaelis-Menten equation (20). When the hydrolysis of a substrate was too slow to determine Km, a Ki value was obtained by preincubating the enzyme and the inhibitor for 5 min at 25°C and starting the reaction by using 25 µM nitrocefin as a substrate. The 50% inhibitory concentrations and Kis were determined graphically (9, 11). In general, the substrates were tested on two separate days, with variations of less than or equal to 20% of the average value reported in Table 2. Standard deviations of more than 20% are provided in the footnotes to Table 2.


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  		TABLE 2. ß-Lactamase hydrolysis parameters for ceftobiprole and comparator antibiotics

 

   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ceftobiprole, cefepime, and ceftazidime MICs are shown in Table 1 for the strains used as sources of ß-lactamases. Susceptibility to comparator cephalosporins was observed for the S. aureus strain expressing the PC1 penicillinase, the Escherichia coli and Klebsiella pneumoniae strains expressing the TEM-1 and SHV-1 ß-lactamases, and the Serratia marcescens strain expressing the SME-3 class A carbapenemase. The ceftobiprole MICs for these organisms were ≤0.5 µg/ml. Nonsusceptibility to cefepime and ceftazidime was observed for strains expressing the CTX-M-15 ESBL and the VIM-2 and KPC-2 carbapenemases, as well as some P. aeruginosa strains with high levels of expression of class C ß-lactamases. Ceftobiprole MICs ≥16 µg/ml were reported for many of the same strains and also for the P. aeruginosa strains producing high levels of class C ß-lactamases and the OXA-10 ß-lactamase.

Cephalosporinase-expressing strains of P. aeruginosa, S. marcescens, Morganella morganii, Enterobacter cloacae, and Providencia stuartii were generally susceptible to ceftobiprole, cefepime, and ceftazidime, with ceftobiprole MICs often being ≤4 µg/ml. Notably, MICs were ≤4 µg/ml for both ceftobiprole and cefepime against the AmpC-hyperproducing Enterobacter strains, such as E. cloacae strains 908R, 4092, and 4081, which had ceftazidime MICs of ≥64 µg/ml.

The rates of hydrolysis of ceftobiprole, ceftazidime, and cefepime were compared to those of the reference standards cephaloridine and benzylpenicillin (Table 2) by using the enzymes purified from the strains listed in Table 1. The hydrolysis rate of ceftobiprole by the penicillinase from gram-positive strain S. aureus PC1 was almost 3,000-fold lower than the rate obtained for benzylpenicillin and 5-fold lower than the rate obtained for cephaloridine. Both cefepime and ceftazidime had lower kcat values compared to those of ceftobiprole by approximately 10-fold and 100-fold, respectively. All of the cephalosporins were hydrolyzed poorly (<0.2%) compared to the hydrolysis of benzylpenicillin.

The class A ß-lactamases from gram-negative bacteria were represented by TEM-1 and SHV-1 (broad-spectrum ß-lactamases); CTX-M-15, K1 and TEM-26 (ESBLs); and KPC-2 and SME-3 (serine carbapenemases). Ceftobiprole and cefepime had similarly low rates of hydrolysis by the TEM-1 enzyme, with kcat values of 8.8 s–1 and 4.4 s–1, respectively. The relative kcat values for these two substrates were less than 1% of that for cephaloridine. Ceftazidime was a poor substrate for the TEM-1 enzyme, with a kcat value of 0.0023 s–1.

The SHV-1 ß-lactamase displayed a kcat value of 30 s–1 for ceftobiprole; this rate of hydrolysis was 15-fold faster than the rate for cefepime and over 2,000-fold faster than the rate for ceftazidime. Ceftobiprole had a kcat/Km value of 0.23 s–1 µM–1, whereas the values were 0.025 s–1 µM–1 and 0.000026 s–1 µM–1 for cefepime and ceftazidime, respectively. These hydrolytic differences did not correspond to the differences in the MICs observed for the SHV-1-expressing strain, for which the ceftazidime MIC was 2 µg/ml and the ceftobiprole and cefepime MICs were 0.5 µg/ml and 0.25 µg/ml, respectively. This may be due in part to the ceftobiprole Km value of 130 µM (70 µg/ml), indicating that the enzyme would not be saturated under physiological conditions to achieve its kcat value.

Because the CTX-M-15, K1, and TEM-26 enzymes are characterized by their ability to hydrolyze cefotaxime, this substrate was added to their hydrolytic profiles. A high kcat value of 270 s–1 and a low Km value of 19 µM for ceftobiprole hydrolysis gave ceftobiprole the highest kcat/Km value of all of the substrates tested with CTX-M-15. Cefotaxime was hydrolyzed at a rate of 180 s–1, with a Km value of 26 µM, resulting in the second highest value of kcat/Km. Cefepime was hydrolyzed sixfold more slowly than ceftobiprole, and ceftazidime had the lowest hydrolysis rate measured for this enzyme. The K1 enzyme possessed a hydrolytic profile similar to that of CTX-M-15, with the hydrolysis rates and the kcat/Km values higher for ceftobiprole than for cefepime and cefotaxime, and ceftazidime was a poor substrate for this enzyme. In contrast, TEM-26 preferentially hydrolyzed ceftazidime and cefepime; the hydrolytic efficiency of ceftobiprole was the lowest among the extended-spectrum cephalosporins. These trends were reflected in the MICs for the TEM-26 producer, where the ceftazidime MIC was >256 µg/ml, the cefepime MIC was 4 µg/ml, and the ceftobiprole MIC was 0.25 µg/ml.

Serine carbapenemases were represented by the potent KPC-2 enzyme and the less active SME-3 ß-lactamase (29, 39). The KPC-2 carbapenemase hydrolyzed ceftobiprole, cefepime, and ceftazidime more rapidly than the SME-3 enzyme did. The KPC-2 enzyme demonstrated a ceftobiprole kcat value of 110 s–1, which was 28% of the rate for cephaloridine. Cefepime was hydrolyzed at a rate of 12 s–1, and ceftazidime was hydrolyzed at a rate of 0.38 s–1, making it the only cephalosporin with a relative hydrolysis rate of <1% compared to the rate for cephaloridine. In contrast, for the SME-3 enzyme, the relative kcat and kcat/Km values for all of the substrates were <1% of the value for cephaloridine, and of these, ceftazidime was the substrate with the slowest hydrolysis. The different hydrolytic profiles between the two enzymes matched the trend in the MIC profiles, with the KPC-2-expressing strain having higher cephalosporin MICs than the SME-3-expressing strain.

Most of the AmpC cephalosporinases demonstrated kcat and kcat/Km values for ceftobiprole, cefepime, and ceftazidime that were <1% of the rate for cephaloridine. In many cases, the rates observed were too slow to determine accurate kcat values. Subtle differences in the activities of enzymes from different species were observed. For example, cefepime was hydrolyzed at a rate of 3 s–1 by the P. stuartii AmpC cephalosporinase, which is 15-fold higher than the rates of hydrolysis for ceftobiprole and ceftazidime; and ceftobiprole was hydrolyzed 5-fold faster than cefepime by the S. marcescens AmpC enzyme. Cefepime usually had the highest Km values with the AmpC enzymes, followed by ceftobiprole, frequently resulting in lower kcat/Km values for these substrates compared to those for ceftazidime. As a general rule, ceftobiprole, cefepime, and ceftazidime were hydrolyzed very slowly by the AmpC ß-lactamases, with variability in both kcat and Km values observed among the ß-lactamases from different bacterial species.

The hydrolysis of ceftobiprole was clearly differentiated from the hydrolysis of cefepime and ceftazidime for the IMP-1 and VIM-2 metallo-ß-lactamases and the OXA-10 group 2d ß-lactamase. The IMP-1 kcat/Km value was fivefold higher for ceftobiprole than for cefepime and eightfold higher than the value obtained for ceftazidime. The VIM-2 enzyme demonstrated kcat/Km values 100- to 200-fold higher for ceftobiprole than those obtained for cefepime and ceftazidime. For the OXA-10 ß-lactamase, the kcat value for ceftobiprole was the highest of those for all the substrates tested, and the kcat/Km value for ceftobiprole was 6,000-fold higher than that for cefepime. Ceftazidime hydrolysis was below the detectable limits for this enzyme.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Ceftobiprole is an investigational, parenteral cephalosporin with a wide spectrum of activity against gram-negative and gram-positive bacteria, including MRSA. While ceftobiprole MICs were ≤4 µg/ml for many clinically important pathogens such as S. aureus, S. pneumoniae, E. coli, and K. pneumoniae, a decrease in antibacterial activity has been observed for ceftazidime-resistant P. aeruginosa and ESBL-producing members of the family Enterobacteriaceae (17, 19). Because ß-lactamase-mediated hydrolysis is a major cause of resistance, especially in gram-negative pathogens, it was important to characterize the interaction of ceftobiprole with ß-lactamases from a spectrum of functional and molecular classes. Enzymes were purified from representative clinical isolates so that their hydrolytic contributions to the activity of ceftobiprole against organisms known to cause clinical disease could be evaluated.

The signature activity of ceftobiprole is its activity against MRSA, due to a combination of potent binding to PBP 2a and stability to the staphylococcal penicillinases (17). The hydrolysis parameters obtained with the gram-positive organism PC1 ß-lactamase in this study confirmed the previous results of Hebeisen et al., where this staphylococcal penicillinase was ineffective in hydrolyzing ceftobiprole (17). The published kcat value of 0.016 s–1 (recorded as 0.93 min–1 in that study) was similar to the value of 0.024 s–1 obtained in our experiments. Our data also included Km values, which were similar for all of the cephalosporins tested, and showed that the PC1 penicillinase had at least a 10-fold lower affinity for cephalosporins than for benzylpenicillin. Although the rates of hydrolysis for ceftazidime and cefepime were lower than those obtained for ceftobiprole, the MICs for ceftobiprole were four- to eightfold lower than those for these comparators, due to the more potent activity of ceftobiprole in staphylococci.

Although only a few ESBLs have been studied directly for their interaction with ceftobiprole, it appears that these enzymes generally hydrolyze ceftobiprole, similar to their action on cefepime and ceftazidime. While ceftobiprole MICs were generally ≤4 µg/ml for wild-type members of the family Enterobacteriaceae, previous publications have reported an increase in ceftobiprole MICs when an organism expressed an ESBL (17, 19); ceftobiprole was also shown by Hebeisen et al. (17) to be measurably hydrolyzed by the TEM-3 and TEM-4 ESBLs. In addition, a CTX-M-expressing strain of K. pneumoniae was not eliminated by ceftobiprole treatment in an in vivo murine pneumonia model (30).

The CTX-M ß-lactamases have rapidly expanded worldwide and may have become the most frequent ESBL type reported (27). CTX-M-15 hydrolyzed ceftobiprole 6-fold faster than it hydrolyzed cefepime and 60-fold faster than it hydrolyzed ceftazidime. This activity was reflected in the MICs obtained for the CTX-M-15-producing strain, for which the ceftobiprole MIC was >128 µg/ml, and those of cefepime and ceftazidime were 16 µg/ml and 32 µg/ml, respectively. Other researchers have shown that the CTX-M-15 ß-lactamase shows an extreme sensitivity to inhibition by clavulanic acid and tazobactam, with 50% inhibitory concentrations of 9 nM and 2 nM, respectively (28). This characteristic could make the use of ß-lactam-ß-lactamase inhibitor combinations a successful strategy for the restoration of susceptibility in organisms producing a single CTX-M enzyme.

Hydrolytic profile analysis demonstrated that the VIM and KPC carbapenemases, the ESBLs, and the OXA-10 ß-lactamase were capable of ceftobiprole hydrolysis and, to a lesser extent, cefepime and ceftazidime hydrolysis. This activity is a major contributor to the high cephalosporin MICs that have occurred in ESBL-expressing strains of E. coli and K. pneumoniae and in organisms possessing carbapenem-hydrolyzing enzymes.

Ceftobiprole and cefepime are clearly differentiated from ceftazidime with respect to their in vitro activities against AmpC cephalosporinase-producing gram-negative pathogens. Many of the Enterobacteriaceae and P. aeruginosa carry a gene for an inducible AmpC cephalosporinase. This chromosomal cephalosporinase is usually expressed at low levels, but in response to certain types of cell wall damage or ß-lactam antibiotics, it may be induced to high levels (16). Mutants that constitutively express high levels of AmpC may also occur, resulting in resistance to ceftazidime and most other ß-lactams (31).

In a recent SENTRY study, 26% of Enterobacter spp. from North American intensive care units were resistant to ceftazidime, while no cefepime resistance was observed (34). In our experiments, the ceftobiprole and cefepime MICs for seven of nine AmpC strains tested were ≤4 µg/ml, even for E. cloacae strains that expressed high ß-lactamase levels and that had ceftazidime MICs of 64 µg/ml. Two P. aeruginosa strains with high levels of AmpC production had MICs of ≥16 µg/ml for all these cephalosporins. The hydrolysis rates for ceftobiprole and comparators by the AmpC enzymes were all low, at rates usually <1% of those for cephaloridine, indicating that these compounds are poor substrates for AmpC cephalosporinases. For the AmpC-producing organisms, the potent antibacterial activities of ceftobiprole and cefepime were not directly correlated with ß-lactamase stability, suggesting that ceftobiprole may possess other properties, such as rapid penetration into the periplasm of gram-negative organisms, which is also the case for cefepime, that can account for its good activity against these organisms. Studies are in progress to examine this possibility.

We have determined that ceftobiprole is resistant to hydrolysis by the gram-positive organism PC1 penicillinase, a class representative of the enzymes present in approximately 90% of S. aureus strains (24). Although ceftobiprole is hydrolyzed by ESBLs and enzymes with carbapenemase activity, non-ESBL class A enzymes, such as the ubiquitous TEM-1 and SHV-1 enzymes and the AmpC chromosomal ß-lactamases from gram-negative species, hydrolyzed ceftobiprole, cefepime, and ceftazidime at low rates. These hydrolysis characteristics of ceftobiprole, combined with its high degree of PBP inhibition, define its overall potent activity against both staphylococci and many gram-negative pathogens.


   ACKNOWLEDGMENTS
 
We thank J. M. Frere for providing the purified S. aureus PC1 ß-lactamase and S. Crespo-Carbone for the purification of the P99 ß-lactamase.


   FOOTNOTES
 
* Corresponding author. Mailing address: Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 1000 Route 202 South, Raritan, NJ 08869. Phone: (908) 704-5515. Fax: (908) 707-3501. E-mail: aqueenan{at}prdus.jnj.com Back

{triangledown} Published ahead of print on 25 June 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Antimicrobial Agents and Chemotherapy, September 2007, p. 3089-3095, Vol. 51, No. 9
0066-4804/07/$08.00+0     doi:10.1128/AAC.00218-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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