High-Level Expression of Chromosomally Encoded SHV-1 β-Lactamase and an Outer Membrane Protein Change Confer Resistance to Ceftazidime and Piperacillin- Tazobactam in a Clinical Isolate of
            <i>Klebsiella pneumoniae</i>

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In an effort to devise strategies for overcoming bacterial ␤-lactamases, we studied LN-1-255, a 6-alkylidene-2-substituted penicillin sulfone inhibitor. By possessing a catecholic functionality that resembles a natural bacterial siderophore, LN-1-255 is unique among ␤-lactamase inhibitors. LN-1-255 combined with piperacillin was more potent against Escherichia coli DH10B strains bearing bla SHV extended-spectrum and inhibitor-resistant ␤-lactamases than an equivalent amount of tazobactam and piperacillin. In addition, LN-1-255 significantly enhanced the activity of ceftazidime and cefpirome against extended-spectrum cephalosporin and Sme-1 containing carbapenem-resistant clinical strains. LN-1-255 inhibited SHV-1 and SHV-2 ␤-lactamases with nM affinity (K I ‫؍‬ 110 ؎ 10 and 100 ؎ 10 nM, respectively). The rapidly increasing number of antibiotic-resistant Gramnegative microorganisms, including the Enterobacteriaceae family and the Pseudomonas, Acinetobacter, and Klebsiella genera, represents a grave threat to human health. The first-line treatments for such infections are ␤-lactam antibiotics, and the most common mechanism of resistance to such agents is bacterial production of ␤-lactamases. Regrettably, Enterobacteriaceae resistant to penicillins and extended-spectrum cephalosporins (e.g. ceftazidime, ceftriaxone and cefepime) are continuing to threaten the efficacy of our available ␤-lactam antibiotics (1-4). Currently, up to 30% of the Enterobacter spp., Escherichia coli, and Klebsiella spp. recovered from patients with infections in hospitals, long-term care facilities, and intensive care units in the United States are resistant to ceftazidime (1, 2, 5-9). Of greatest concern is the emerging number of community-acquired E. coli and Klebsiella spp. that are resistant to these cephalosporins (3, 4, 8 -11). This latter group presents a very significant future danger to the current use of ␤-lactam antibiotics to treat common infections in the ambulatory setting (11).Resistance to extended-spectrum cephalosporins in E. coli and Klebsiella pneumoniae is typically caused by a plasmid or chromosomal class A bla gene that encodes for extended-spectrum ␤-lactamases (ESBLs) 4 (1, 5, 12, 13). Among the ␤-lactam class of antibiotics, only carbapenems are recommended for the therapy of infections caused by ESBL-producing bacteria (1, 2, 7). Many ESBL-producing bacteria possess ␤-lactamases that are susceptible to ␤-lactamase inhibitors, but the clinical efficacy of ␤-lactam/␤-lactamase inhibitor combinations still remains to be established conclusively (5, 12). ␤-Lactam/␤-lactamase inhibitor combinations offer an extremely attractive approach to combating infections caused by ESBL-producing bacteria. Unfortunately, current commercial ␤-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) narrowly target class A enzymes. Thus, there is an urgent need for the * This work was supported, in whole or in part, by National Institutes of Health Grants R01 AI062968 (to F. V. D. A.), RO1 AI063517-01 (to R. A. B.), and the R...

Section: Methodsmentioning

confidence: 99%

Strategic Design of an Effective β-Lactamase Inhibitor

Pattanaik

Bethel

Hujer

et al. 2009

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“…Plasmids and Mutagenesis-bla SHV-1 , subcloned into phagemid vector pBC SK(Ϫ) (Stratagene, La Jolla, CA) and maintained in ElectroMAX TM DH10B TM T1 R cells (Invitrogen) as previously described (17) served as the template for all mutagenesis described herein. Site-saturation mutagenesis was performed on the bla SHV-1 construct using the QuikChange II site-directed mutagenesis kit (Stratagene) and primers degenerate at Ambler position 244.…”

Section: Methodsmentioning

confidence: 99%

Probing Active Site Chemistry in SHV β-Lactamase Variants at Ambler Position 244

Thomson

Distler

Prati

et al. 2006

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109

Inhibitor-resistant class A ␤-lactamases are an emerging threat to the use of ␤-lactam/␤-lactamase inhibitor combinations (e.g. amoxicillin/clavulanate) in the treatment of serious bacterial infections. In the TEM family of Class A ␤-lactamases, single amino acid substitutions at Arg-244 confer resistance to clavulanate inactivation. To understand the amino acid sequence requirements in class A ␤-lactamases that confer resistance to clavulanate, we performed site-saturation mutagenesis of Arg-244 in SHV-1, a related class A ␤-lactamase found in Klebsiella pneumoniae. Twelve SHV enzymes with amino acid substitutions at Arg-244 resulted in significant increases in minimal inhibitory concentrations to ampicillin/ clavulanate when expressed in Escherichia coli. Kinetic analyses of SHV-1, R244S, R244Q, R244L, and R244E ␤-lactamases revealed that the main determinant of clavulanate resistance was reduced inhibitor affinity. In contrast to studies in the highly similar TEM enzyme, we observed increases in clavulanate k inact for all mutants. Electrospray ionization mass spectrometry of clavulanate inhibited SHV-1 and R244S showed nearly identical mass adducts, arguing against a difference in the inactivation mechanism. Testing a wide range of substrates with C 3-4 carboxylates in different stereochemical orientations, we observed impaired affinity for all substrates among inhibitor resistant variants. Lastly, we synthesized two boronic acid transition state analogs that mimic cephalothin and found substitutions at Arg-244 markedly affect both the affinity and kinetics of binding to the chiral, deacylation transition state inhibitor. These data define a role for Arg-244 in substrate and inhibitor binding in the SHV ␤-lactamase.␤-Lactams have been the cornerstone of antibacterial chemotherapy since the introduction of penicillin. Although this family of antibiotics now contains numerous classes (penicillins, cephalosporins, and carbapenems, see Fig. 1), their use is threatened by the expansion and spread of ␤-lactamase enzymes (1). These bacterial enzymes hydrolyze ␤-lactam antibiotics before reaching their target, the penicillin-binding proteins. In an effort to retain the utility of several generations of life-saving ␤-lactam antibiotics, ␤-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) have been developed. These mechanism-based inhibitors are ␤-lactam compounds that experience multistep reactions within the active site of ␤-lactamase enzymes (2-4). Typically the inhibitors lack antimicrobial activity and are formulated with a ␤-lactam antibiotic to act as shields against ␤-lactamase enzymes. This allows the ␤-lactam to bypass the ␤-lactamase and inactivate the penicillin binding proteins.Unfortunately, inhibitor-resistant class A ␤-lactamases are emerging in the clinic and undermining the use of ␤-lactam/␤-lactamase inhibitor therapy (5-7). Interest has been renewed in the discovery of novel ␤-lactamase inactivators to circumvent these inhibitor-resistant enzymes (8 -11). In this context, detailed analy...

“…Bacterial Strains and Plasmids-The chromosomal SHV-1 ␤-lactamase with its native promoter and ribosomal binding site was cloned into the recombinant phagemid vector, pBC SK(Ϫ) (Stratagene, La Jolla, CA) as described previously (14). Plasmid DNA was obtained from Epicurean coli™ XL1 Blue (Stratagene, La Jolla, CA) and Escherichia coli DH10B (Invitrogen) cells using Wizard™ Purification kits (Promega, Madison, WI).…”

Section: Methodsmentioning

confidence: 99%

Understanding Resistance to β-Lactams and β-Lactamase Inhibitors in the SHV β-Lactamase

Helfand

Bethel

Hujer

et al. 2003

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Bacterial resistance to ␤-lactam/␤-lactamase inhibitor combinations by single amino acid mutations in class A ␤-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A ␤-lactamases, alterations at Ser-130 confer resistance to inactivation by the ␤-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 ␤-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G ␤-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G ␤-lactamase demonstrated a significant elevation in apparent K m and a reduction in k cat /K m for ampicillin. Marked increases in the dissociation constant for the preacylation complex, K I , of clavulanic acid (SHV-1, 0.14 M; S130G, 46.5 M) and tazobactam (SHV-1, 0.07 M; S130G, 4.2 M) were observed. In contrast, the k inact s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for ␤-lactams and ␤-lactamase inhibitors. These results underscore the mechanistic versatility of class A ␤-lactamases and have implications for the design of novel ␤-lactamase inhibitors.