Variants of β-Lactamase KPC-2 That Are Resistant to Inhibition by Avibactam

KPC-2 is the most prevalent class A carbapenemase in the world. Previously, KPC-2 was shown to hydrolyze the β-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. In addition, substitutions at amino acid position R220 in the KPC-2 β-lactamase increased resistance to clavulanic acid. A novel bridged diazabicyclooctane (DBO) non-β-lactam β-lactamase inhibitor, avibactam, was shown to inactivate the KPC-2 β-lactamase. To better understand the mechanistic basis for inhibition of KPC-2 by avibactam, we tested the potency of ampicillin-avibactam and ceftazidime-avibactam against engineered variants of the KPC-2 β-lactamase that possessed single amino acid substitutions at important sites (i.e., Ambler positions 69, 130, 234, 220, and 276) that were previously shown to confer inhibitor resistance in TEM and SHV β-lactamases. To this end, we performed susceptibility testing, biochemical assays, and molecular modeling. Escherichia coli DH10B carrying KPC-2 β-lactamase variants with the substitutions S130G, K234R, and R220M demonstrated elevated MICs for only the ampicillin-avibactam combinations (e.g., 512, 64, and 32 mg/liter, respectively, versus the MICs for wild-type KPC-2 at 2 to 8 mg/liter). Steady-state kinetics revealed that the S130G variant of KPC-2 resisted inactivation by avibactam; the k₂/K ratio was significantly lowered 4 logs for the S130G variant from the ratio for the wild-type enzyme (21,580 M⁻¹ s⁻¹ to 1.2 M⁻¹ s⁻¹). Molecular modeling and molecular dynamics simulations suggested that the mobility of K73 and its ability to activate S70 (i.e., function as a general base) may be impaired in the S130G variant of KPC-2, thereby explaining the slowed acylation. Moreover, we also advance the idea that the protonation of the sulfate nitrogen of avibactam may be slowed in the S130G variant, as S130 is the likely proton donor and another residue, possibly K234, must compensate. Our findings show that residues S130 as well as K234 and R220 contribute significantly to the mechanism of avibactam inactivation of KPC-2. Fortunately, the emergence of S130G, K234R, and R220M variants of KPC in the clinic should not result in failure of ceftazidime-avibactam, as the ceftazidime partner is potent against E. coli DH10B strains possessing all of these variants.     

Crystal structures of KPC-2 β-lactamase in complex with 3-nitrophenyl boronic acid and the penam sulfone PSR-3-226

Class A carbapenemases are a major threat to the potency of carbapenem antibiotics. A widespread carbapenemase, KPC-2, is not easily inhibited by β-lactamase inhibitors (i.e., clavulanic acid, sulbactam, and tazobactam). To explore different mechanisms of inhibition of KPC-2, we determined the crystal structures of KPC-2 with two β-lactamase inhibitors that follow different inactivation pathways and kinetics. The first complex is that of a small boronic acid compound, 3-nitrophenyl boronic acid (3-NPBA), bound to KPC-2 with 1.62-Å resolution. 3-NPBA demonstrated a K_m value of 1.0 ± 0.1 μM (mean ± standard error) for KPC-2 and blocks the active site by making a reversible covalent interaction with the catalytic S70 residue. The two boron hydroxyl atoms of 3-NPBA are positioned in the oxyanion hole and the deacylation water pocket, respectively. In addition, the aromatic ring of 3-NPBA provides an edge-to-face interaction with W105 in the active site. The structure of KPC-2 with the penam sulfone PSR-3-226 was determined at 1.26-Å resolution. PSR-3-226 displayed a K_m value of 3.8 ± 0.4 μM for KPC-2, and the inactivation rate constant (k_inact) was 0.034 ± 0.003 s⁻¹. When covalently bound to S70, PSR-3-226 forms a trans-enamine intermediate in the KPC-2 active site. The predominant active site interactions are generated via the carbonyl oxygen, which resides in the oxyanion hole, and the carboxyl moiety of PSR-3-226, which interacts with N132, N170, and E166. 3-NPBA and PSR-3-226 are the first β-lactamase inhibitors to be trapped as an acyl-enzyme complex with KPC-2. The structural and inhibitory insights gained here could aid in the design of potent KPC-2 inhibitors.     

Phenotypic Study of Resistance of β-Lactamase-Inhibitor-Resistant TEM Enzymes Which Differ by Naturally Occurring Variations and by Site-Directed Substitution at Asp276

At this time an amino acid substitution at position 276 in the TEM-1 enzyme is associated with an additional substitution at position 69 in natural β-lactamase-inhibitor-resistant (IRT) β-lactamases. The effect of the Asn²⁷⁶→Asp substitution on resistance was assessed with the Asn276Asp variant, generated by site-directed mutagenesis. The mutant was resistant to β-lactamase inhibitors, but the MICs of amoxicillin combined with clavulanic acid or tazobactam were strikingly different for E. coli strains producing the Asn276Asp variant and those producing naturally occurring IRTs with single or double substitutions. The inhibitory effects of clavulanic acid and tazobactam were the same in IRTs with substitutions at position 69 (IRT-5 and IRT-6). The effect of clavulanic acid on the MICs of amoxicillin for the Asn276Asp variant was greater than that of tazobactam. In IRTs with double substitutions, at positions 69 plus 276 (IRT-4, IRT-7, and IRT-8) or 69 plus 275 (IRT-14), tazobactam was a more potent inhibitor than clavulanic acid. The effect of the Asn²⁷⁶→Asp substitution on the values of the kinetic constants and the concentration required to inhibit by 50% the hydrolysis of benzylpenicillin confirms that this single mutation is responsible for resistance to β-lactamase inhibitors. Molecular modeling of the Asn276Asp mutant shows that Asp²⁷⁶ can form two salt bonds with Arg²⁴⁴ close to the penicillin-binding cavity. The addition of the Asp²⁷⁶ mutation to that preexisting at position 69 confers a higher selective advantage to bacteria, as shown by the reduction in β-lactamase inhibitor efficiencies of the double variants. Therefore, the emergence of multiple mutations in TEM β-lactamases by virtue of the use of β-lactamase inhibitors increases selection pressure resulting in the convergent evolution of resistant strains.     

Inhibitor Resistance in the KPC-2 β-Lactamase,a Preeminent Property of This Class A β-Lactamase

As resistance determinants, KPC β-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type β-lactamases are often identified as resistant to standard β-lactam-β-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to β-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (k_cat/k_inact ratios, where k_inact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an sp²-hybridized C₃ carboxylate and a bicyclic R1 side chain (dihydropyrazolo[1,5-c][1,3]thiazole [penem 1] and dihydropyrazolo[5,1-c][1,4]thiazine [penem 2]) are potent inhibitors: K_m of penem 1, 0.06 ± 0.01 μM, and K_m of penem 2, 0.006 ± 0.001 μM. We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (k_cat/k_inact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor (k_inact/K_m = 6.5 ± 0.6 μM⁻¹ s⁻¹). We conclude that KPC-2 is unique among class A β-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type β-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current β-lactamase inhibitors.In Klebsiella pneumoniae, β-lactam resistance is mediated predominantly by class A SHV, TEM, and CTX-M β-lactamases (7, 35). Single amino acid substitutions in the SHV and TEM β-lactamases can drastically alter the substrate profiles of the enzymes and confer resistance to extended-spectrum cephalosporins and β-lactamase inhibitors (5, 12, 34, 36). β-Lactamases with altered substrate profiles (i.e., extended-spectrum or inhibitor-resistant β-lactamases) have significantly challenged the clinician''s approach to the treatment of serious infectious diseases (36). Thus, the search for effective mechanism-based inhibitors of novel β-lactamases merits significant effort (8, 9, 32).First identified in K. pneumoniae, KPC class A β-lactamases threaten the use of all current β-lactam antibiotics (57). These β-lactamase enzymes are present in an increasing number of bacterial genera, becoming the major carbapenemase expressed by Gram-negative pathogens (e.g., Enterobacter spp., Escherichia coli, Citrobacter freundii, Pseudomonas spp., Serratia marcescens, Proteus mirabilis, and Salmonella enterica) in the United States (3, 10, 11, 16, 17, 25, 37, 45, 49, 53, 59). Moreover, KPC β-lactamases are becoming geographically widespread (having been detected, e.g., in the United States, China, France, Colombia, Greece, Sweden, Norway, Argentina, the United Kingdom, Israel, Brazil, Puerto Rico, Canada, Ireland, Trinidad and Tobago, Poland, Italy, and Finland) (1, 2, 15, 23, 24, 29-31, 33, 38, 39, 42, 50, 51, 53, 55, 57). Evidence suggests that many K. pneumoniae strains in the United States harboring KPCs are genetically related (19).Why are KPC β-lactamases so problematic? KPC-2 has an overall structure similar to those of other class A enzymes, and interestingly, this β-lactamase has only 50% protein sequence conservation compared to CTX-M-1, 39% compared to SHV-1, and 35% compared to TEM-1. KPC-2 is more like other class A carbapenemases, having 55% identity to NmcA and Imi-1, 63% identity to Sfc-1, and 57% identity to Sme-1. The KPC-2 β-lactamase possesses a large and shallow active site, allowing it to accommodate “bulkier” β-lactams (26). As a result of these structural characteristics, KPC-2 is regarded as a versatile β-lactamase (37); it is a penicillinase, carbapenemase, and cephamycinase and an extended-spectrum β-lactamase (57, 58). Microbiologists and clinicians have observed that many bla_KPC-2-containing strains are resistant to β-lactam-β-lactamase inhibitor combinations (6, 19, 50, 54, 55, 59). According to Clinical and Laboratory Standards Institute (CLSI) breakpoints, bla_KPC-2-carrying clinical strains for which the MICs of amoxicillin-clavulanic acid are ≥32/16 mg/liter and those of piperacillin-tazobactam are ≥128/4 mg/liter are resistant (14, 58). These observations led us to examine the kinetic properties of the KPC-2 β-lactamase tested against commercially available and novel inhibitors.A β-lactamase inhibitor demonstrating an affinity in the nanomolar range for KPC-2 and other class A carbapenemases would be an important addition to our therapeutic armamentarium. Thus, we wondered if penem inhibitors that possess an sp²-hybridized C₃ carboxylate (a property resembling a characteristic of carbapenems), a complex and reactive R1 side chain, and inactivation chemistry different from that of clavulanic acid could be exploited to inhibit KPC enzymes (41). The methylidene inhibitors penem 1 and penem 2 have dihydropyrazolo[1,5-c][1,3]thiazole and dihydropyrazolo[5,1-c][1,4]thiazine moieties, respectively (see Fig. ​Fig.1).1). These penems demonstrate similar levels of in vivo efficacy in mice and have been shown to be effective inhibitors of several class A, C, and D β-lactamases (4, 43, 46-48, 52).Open in a separate windowFIG. 1.Chemical structures of the classical β-lactamase inhibitors, the novel penem β-lactamase inhibitors, cefotaxime, and imipenem.In this paper, we show why K. pneumoniae containing bla_KPC-2 and an E. coli laboratory strain harboring bla_KPC-2 are not susceptible to the commercially available β-lactamase inhibitors. Our results demonstrated that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by the KPC-2 β-lactamase. 6-Methylidene penems with complex fused bicyclic R1 side chains are better inhibitors because they possess greater affinity for the active site, have low K_ms, and act as mechanism-based inactivators.     

Boronic Acid Transition State Inhibitors Active against KPC and Other Class A β-Lactamases: Structure-Activity Relationships as a Guide to Inhibitor Design

Boronic acid transition state inhibitors (BATSIs) are competitive, reversible β-lactamase inhibitors (BLIs). In this study, a series of BATSIs with selectively modified regions (R1, R2, and amide group) were strategically designed and tested against representative class A β-lactamases of Klebsiella pneumoniae, KPC-2 and SHV-1. Firstly, the R1 group of compounds 1a to 1c and 2a to 2e mimicked the side chain of cephalothin, whereas for compounds 3a to 3c, 4a, and 4b, the thiophene ring was replaced by a phenyl, typical of benzylpenicillin. Secondly, variations in the R2 groups which included substituted aryl side chains (compounds 1a, 1b, 1c, 3a, 3b, and 3c) and triazole groups (compounds 2a to 2e) were chosen to mimic the thiazolidine and dihydrothiazine ring of penicillins and cephalosporins, respectively. Thirdly, the amide backbone of the BATSI, which corresponds to the amide at C-6 or C-7 of β-lactams, was also changed to the following bioisosteric groups: urea (compound 3b), thiourea (compound 3c), and sulfonamide (compounds 4a and 4b). Among the compounds that inhibited KPC-2 and SHV-1 β-lactamases, nine possessed 50% inhibitory concentrations (IC₅₀s) of ≤600 nM. The most active compounds contained the thiopheneacetyl group at R1 and for the chiral BATSIs, a carboxy- or hydroxy-substituted aryl group at R2. The most active sulfonamido derivative, compound 4b, lacked an R2 group. Compound 2b ({"type":"entrez-protein","attrs":{"text":"S02030","term_id":"83679","term_text":"pir||S02030"}}S02030) was the most active, with acylation rates (k₂/K) of 1.2 ± 0.2 × 10⁴ M⁻¹ s⁻¹ for KPC-2 and 4.7 ± 0.6 × 10³ M⁻¹ s⁻¹ for SHV-1, and demonstrated antimicrobial activity against Escherichia coli DH10B carrying bla_SHV variants and bla_KPC-2 or bla_KPC-3 and against clinical strains of Klebsiella pneumoniae and E. coli producing different class A β-lactamase genes. At most, MICs decreased from 16 to 0.5 mg/liter.     

Substrate Selectivity and a Novel Role in Inhibitor Discrimination by Residue 237 in the KPC-2 β-Lactamase

β-Lactamase-mediated antibiotic resistance continues to challenge the contemporary treatment of serious bacterial infections. The KPC-2 β-lactamase, a rapidly emerging Gram-negative resistance determinant, hydrolyzes all commercially available β-lactams, including carbapenems and β-lactamase inhibitors; the amino acid sequence requirements responsible for this versatility are not yet known. To explore the bases of β-lactamase activity, we conducted site saturation mutagenesis at Ambler position 237. Only the T237S variant of the KPC-2 β-lactamase expressed in Escherichia coli DH10B maintained MICs equivalent to those of the wild type (WT) against all of the β-lactams tested, including carbapenems. In contrast, the T237A variant produced in E. coli DH10B exhibited elevated MICs for only ampicillin, piperacillin, and the β-lactam-β-lactamase inhibitor combinations. Residue 237 also plays a novel role in inhibitor discrimination, as 11 of 19 variants exhibit a clavulanate-resistant, sulfone-susceptible phenotype. We further showed that the T237S variant displayed substrate kinetics similar to those of the WT KPC-2 enzyme. Consistent with susceptibility testing, the T237A variant demonstrated a lower k_cat/K_m for imipenem, cephalothin, and cefotaxime; interestingly, the most dramatic reduction was with cefotaxime. The decreases in catalytic efficiency were driven by both elevated K_m values and decreased k_cat values compared to those of the WT enzyme. Moreover, the T237A variant manifested increased K_is for clavulanic acid, sulbactam, and tazobactam, while the T237S variant displayed K_is similar to those of the WT. To explain these findings, a molecular model of T237A was constructed and this model suggested that (i) the hydroxyl side chain of T237 plays an important role in defining the substrate profile of the KPC-2 β-lactamase and (ii) hydrogen bonding between the hydroxyl side chain of T237 and the sp²-hybridized carboxylate of imipenem may not readily occur in the T237A variant. This stringent requirement for selected cephalosporinase and carbapenemase activity and the important role of T237 in inhibitor discrimination in KPC-2 are central considerations in the future design of β-lactam antibiotics and inhibitors.Antibiotic resistance is a critical challenge to clinicians treating complex bacterial infections. Moreover, the continued evolution of bacterial proteins responsible for mediating antibiotic resistance is alarming. The most notable resistance determinants in nature are the β-lactamases present in Gram-negative bacteria. Presently, the β-lactamases (EC 3.5.2.6) are classified into four distinct classes based on structural similarities (A, B, C, and D) or four groups based on hydrolytic profiles (1, 2, 3, and 4) (1, 6, 46, 53). Class A, C, and D β-lactamases use a serine as the nucleophile in the active site to hydrolyze the β-lactam, while class B β-lactamases employ either one or two reactive Zn²⁺ ions. In general, class A, C, and D β-lactamases hydrolyze β-lactams through a three-step reaction mechanism represented as follows: In this reaction scheme, E corresponds to the β-lactamase, S is the β-lactam substrate, E:S is the Michaelis complex, E-S is the acylated β-lactamase, P is the inactive product; k₁ and k₋₁ represent the on and off rates, k₂ is the acylation rate constant, and k₃ is the deacylation rate constant.Interestingly, single amino acid substitutions allow β-lactamases to expand their substrate profile and dramatically alter the ability of β-lactamases to hydrolyze β-lactams before they reach their targets, the penicillin binding proteins. Such β-lactamases include the extended-spectrum β-lactamases (ESBLs), inhibitor-resistant TEMs and SHVs (IRTs and IRSs), and extended-spectrum AmpCs (ESACs) (4, 11, 29, 30, 37, 39). As a result of this progression, each new, improved β-lactam is eventually greeted with a new β-lactamase that threatens the efficacy of the drug (31, 33).Resistance to carbapenems, which have been considered the last line of therapy for many types of infections, is one of the greatest threats in clinical medicine (23, 24). The serine class A β-lactamases responsible for carbapenem resistance include KPC-2-10, SME-1-3, IMI-1-2, SFC-1, BIC-1, NmcA, and GES-2,4-6 (14, 17, 18, 43, 45). Among these class A β-lactamases, KPC-2 is a clinically important and unique enzyme, as it is the most prevalent carbapenemase in enteric bacteria in the United States (12, 24, 45). In addition, the bla_KPC-2 gene is located on a mobile transposon designated Tn4401, which is rapidly spreading throughout the world (45). KPC-2 is also a class A β-lactamase with a very broad substrate profile, including penicillins, extended-spectrum cephalosporins, cephamycins, carbapenems, and even the β-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam (36, 58, 59). As the structural basis for β-lactam resistance mediated by KPC-2 is elusive, we endeavored to explore the sequence determinants of carbapenem resistance mediated by KPC-2.Comparing the amino acid sequence and crystal structure of KPC-2 to those of other class A β-lactamases (i.e., CTX-M-1, SHV-1, and TEM-1) shows that nine residues near or in the active site are unique and/or in distinctive positions in KPC-2 (e.g., W105, S130, N132, N170, R220, K234, T235, T237, and H274) (24). Five of these amino acids (S130, N132, N170, K234, and T235) are located in the following four conserved elements: the SSFK motif (Ambler positions 70 to 73), the SDN loop (residues 130 to 132), the omega loop (positions 164 to 179), and the KTG motif (amino acids 234 to 236) (Fig. ​(Fig.1)1) (22). In the crystal structure of KPC-2, a bicine molecule is trapped in the active site of the β-lactamase. This bicine molecule is observed to interact via its carboxyl group with conserved active-site residues S130, K234, T235, and T237, resembling the interactions of the β-lactam carboxylate moiety in the Michaelis complex (14). In particular, our attention was drawn to T237, as the backbone nitrogens of S70 and T237 form the oxyanion hole for the β-lactam carbonyl when binding in class A β-lactamases (35). This oxyanion hole, or electrophilic center, positions the β-lactam carbonyl such that the β-lactam bond can be hydrolyzed. In other class A β-lactamases, such as SHV-1 and CTX-M-9, position 237 is occupied by an alanine and a serine, respectively. In addition, a hydroxyl side chain at position 237 has been found to be important for extending the substrate spectra of certain class A β-lactamases (3, 5, 15, 16, 28, 52, 54).Open in a separate windowFIG. 1.Model of KPC-2 (PDB entry 2OV5) highlighting the four conserved regions (the SSFK motif [purple], the SDN loop [blue], the omega loop [green/pink], and the KTG motif [gray]) found in class A β-lactamases, the three main epitopes (A [light blue], B [pink], and C [orange]) of our polyclonal anti-KPC-2 antibody, and T237 (yellow).To investigate the role residue 237 plays in KPC-2, we conducted site saturation mutagenesis at position 237 in KPC-2. Our results demonstrate that this residue is necessary to maintain carbapenemase and selected cephalosporinase activities of the enzyme and is involved in the discrimination of clavulanic acid from sulfone inhibitors. In addition, our study suggests that a hydroxyl side chain is necessary at position 237 for hydrogen bonding to the carboxylate of imipenem.     

A Novel Extended-Spectrum β-Lactamase,SGM-1, from an Environmental Isolate of Sphingobium sp.

SGM-1 is a novel class A β-lactamase from an environmental isolate of Sphingobium sp. containing all of the distinct amino acid motifs of class A β-lactamases. It shares 77 to 80% amino acid sequence identity with putative β-lactamases that are present on the chromosome of all Sphingobium species whose genomes were sequenced and annotated. Thus, SGM-type β-lactamases are native to this genus. Antibiotic susceptibility testing classifies SGM-1 as an extended-spectrum β-lactamase, conferring the highest level of resistance to penicillins. Although SGM-1 contains the conserved cysteine residues characteristic of class A carbapenemases, it does not confer resistance to the carbapenem antibiotics imipenem, meropenem, or doripenem but does increase the MIC of ertapenem 8-fold. SGM-1 hydrolyzes penicillins and the monobactam aztreonam with similar catalytic efficiencies, ranging from 10⁵ to 10⁶ M⁻¹ s⁻¹. The catalytic efficiencies of SGM-1 for cefoxitin and ceftazidime were the lowest (10² to 10³ M⁻¹ s⁻¹) among the cephalosporins tested, while the catalytic efficiencies against all other cephalosporins varied from about 10⁵ to 10⁶ M⁻¹ s⁻¹. SGM-1 exhibited measurable but not significant activity toward the carbapenems tested. SGM-1 also showed high affinity for clavulanic acid, tazobactam, and sulbactam (K_i < 1 μM); however, only clavulanic acid significantly reduced the MICs of β-lactams.     

In Vitro Susceptibility of Characterized β-Lactamase-Producing Strains Tested with Avibactam Combinations

Avibactam, a broad-spectrum β-lactamase inhibitor, was tested with ceftazidime, ceftaroline, or aztreonam against 57 well-characterized Gram-negative strains producing β-lactamases from all molecular classes. Most strains were nonsusceptible to the β-lactams alone. Against AmpC-, extended-spectrum β-lactamase (ESBL)-, and KPC-producing Enterobacteriaceae or Pseudomonas aeruginosa, avibactam lowered ceftazidime, ceftaroline, or aztreonam MICs up to 2,048-fold, to ≤4 μg/ml. Aztreonam-avibactam MICs against a VIM-1 metallo-β-lactamase-producing Enterobacter cloacae and a VIM-1/KPC-3-producing Escherichia coli isolate were 0.12 and 8 μg/ml, respectively.     

Avibactam and Inhibitor-Resistant SHV β-Lactamases

β-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of β-lactam antibiotics to treat infections. A novel non-β-lactam β-lactamase inhibitor with activity against many class A and C and some class D β-lactamase variants, avibactam, is now available in the clinic in partnership with ceftazidime. Here, we explored the activity of avibactam against a variety of characterized isogenic laboratory constructs of β-lactamase inhibitor-resistant variants of the class A enzyme SHV (M69I/L/V, S130G, K234R, R244S, and N276D). We discovered that the S130G variant of SHV-1 shows the most significant resistance to inhibition by avibactam, based on both microbiological and biochemical characterizations. Using a constant concentration of 4 mg/liter of avibactam as a β-lactamase inhibitor in combination with ampicillin, the MIC increased from 1 mg/liter for bla_SHV-1 to 256 mg/liter for bla_{SHV S130G} expressed in Escherichia coli DH10B. At steady state, the k₂/K value of the S130G variant when inactivated by avibactam was 1.3 M⁻¹ s⁻¹, versus 60,300 M⁻¹ s⁻¹ for the SHV-1 β-lactamase. Under timed inactivation conditions, we found that an approximately 1,700-fold-higher avibactam concentration was required to inhibit SHV S130G than the concentration that inhibited SHV-1. Molecular modeling suggested that the positioning of amino acids in the active site of SHV may result in an alternative pathway of inactivation when complexed with avibactam, compared to the structure of CTX-M-15–avibactam, and that S130 plays a role in the acylation of avibactam as a general acid/base. In addition, S130 may play a role in recyclization. As a result, we advance that the lack of a hydroxyl group at position 130 in the S130G variant of SHV-1 substantially slows carbamylation of the β-lactamase by avibactam by (i) removing an important proton acceptor and donator in catalysis and (ii) decreasing the number of H bonds. In addition, recyclization is most likely also slow due to the lack of a general base to initiate the process. Considering other inhibitor-resistant mechanisms among class A β-lactamases, S130 may be the most important amino acid for the inhibition of class A β-lactamases, perhaps even for the novel diazabicyclooctane class of β-lactamase inhibitors.     

Substitutions at Position 105 in SHV Family β-Lactamases Decrease Catalytic Efficiency and Cause Inhibitor Resistance

Ambler position 105 in class A β-lactamases is implicated in resistance to clavulanic acid, although no clinical isolates with mutations at this site have been reported. We hypothesized that Y105 is important in resistance to clavulanic acid because changes in positioning of the inhibitor for ring oxygen protonation could occur. In addition, resistance to bicyclic 6-methylidene penems, which are interesting structural probes that inhibit all classes of serine β-lactamases with nanomolar affinity, might emerge with substitutions at position 105, especially with nonaromatic substitutions. All 19 variants of SHV-1 with variations at position 105 were prepared. Antimicrobial susceptibility testing showed that Escherichia coli DH10B expressing Y105 variants retained activity against ampicillin, except for the Y105L variant, which was susceptible to all β-lactams, similar to the case for the host control strain. Several variants had elevated MICs to ampicillin-clavulanate. However, all the variants remained susceptible to piperacillin in combination with a penem inhibitor (MIC, ≤2/4 mg/liter). The Y105E, -F, -M, and -R variants demonstrated reduced catalytic efficiency toward ampicillin compared to the wild-type (WT) enzyme, which was caused by increased K_m. Clavulanic acid and penem K_i values were also increased for some of the variants, especially Y105E. Mutagenesis at position 105 in SHV yields mutants resistant to clavulanate with reduced catalytic efficiency for ampicillin and nitrocefin, similar to the case for the class A carbapenemase KPC-2. Our modeling analyses suggest that resistance is due to oxyanion hole distortion. Susceptibility to a penem inhibitor is retained although affinity is decreased, especially for the Y105E variant. Residue 105 is important to consider when designing new inhibitors.     

BLIP-II Is a Highly Potent Inhibitor of Klebsiella pneumoniae Carbapenemase (KPC-2)

β-Lactamase inhibitory protein II (BLIP-II) is a potent inhibitor of class A β-lactamases. KPC-2 is a class A β-lactamase that is capable of hydrolyzing carbapenems and has become a widespread source of resistance to these drugs for Gram-negative bacteria. Determination of association and dissociation rate constants for binding between BLIP-II and KPC-2 reveals a very tight interaction with a calculated (k_off/k_on) equilibrium dissociation constant of 76 fM (76 × 10⁻¹⁵ M).     

Clavulanic Acid: a Beta-Lactamase-Inhibiting Beta-Lactam from Streptomyces clavuligerus

A novel β-lactamase inhibitor has been isolated from Streptomyces clavuligerus ATCC 27064 and given the name clavulanic acid. Conditions for the cultivation of the organism and detection and isolation of clavulanic acid are described. This compound resembles the nucleus of a penicillin but differs in having no acylamino side chain, having oxygen instead of sulfur, and containing a β-hydroxyethylidine substituent in the oxazolidine ring. Clavulanic acid is a potent inhibitor of many β-lactamases, including those found in Escherichia coli (plasmid mediated), Klebsiella aerogenes, Proteus mirabilis, and Staphylococcus aureus, the inhibition being of a progressive type. The cephalosporinase type of β-lactamase found in Pseudomonas aeruginosa and Enterobacter cloacae P99 and the chromosomally mediated β-lactamase of E. coli are less well inhibited. The minimum inhibitory concentrations of ampicillin and cephaloridine against β-lactamase-producing, penicillin-resistant strains of S. aureus, K. aerogenes, P. mirabilis, and E. coli have been shown to be considerably reduced by the addition of low concentrations of clavulanic acid.     

Beta-Lactamases Produced by a Pseudomonas aeruginosa Strain Highly Resistant to Carbenicillin

A Pseudomonas aeruginosa strain isolated at Besançon Hospital, France, proved to be highly resistant to carbenicillin and showed a high hydrolytic activity toward this antibiotic. We clearly demonstrated that two β-lactamases were synthetized: one of them, constitutive, has its enzymatic activity directed mainly toward penicillins, and carbenicillin appears to be its best substrate (higher V_max); thus, this β-lactamase is a “carbenicillinase” that differs from the well-known “TEM-like” enzymes. The isoelectric point of this carbenicillinase is 5.30 ± 0.03. The other one is an inducible cephalosporinase, very similar to the cephalosporinases usually found in these organisms. Its isoelectric point is 8.66 ± 0.04. These two enzymes have been separated by affinity chromatography and isoelectric focusing. The kinetic constants were measured by computerized microacidimetry.     

In Vitro Activities of Ceftazidime-Avibactam and Aztreonam-Avibactam against 372 Gram-Negative Bacilli Collected in 2011 and 2012 from 11 Teaching Hospitals in China

Ceftazidime-avibactam, aztreonam-avibactam, and comparators were tested by reference broth microdilution against 372 nonrepetitive Gram-negative bacilli (346 unselected plus 26 selected meropenem-nonsusceptible Enterobacteriaceae isolates) collected from 11 teaching hospitals in China in 2011 and 2012. Meropenem-nonsusceptible isolates produced extended-spectrum β-lactamases (ESBLs; e.g., CTX-M-14/3), AmpCs (e.g., CMY-2), and/or carbapenemases (e.g., KPC-2 and NDM-1). Avibactam potentiated the activity of ceftazidime against organisms with combinations of ESBLs, AmpCs, and KPC-2. Aztreonam-avibactam was active against all β-lactamase producers (including producers of NDM-1 and IMP-4/8) except bla_OXA-containing Acinetobacter baumannii and some Pseudomonas aeruginosa isolates.     

Interactions of OP0595, a Novel Triple-Action Diazabicyclooctane,with β-Lactams against OP0595-Resistant Enterobacteriaceae Mutants

OP0595 is a novel diazabicyclooctane which, like avibactam, inhibits class A and C β-lactamases. In addition, unlike avibactam, it has antibacterial activity, with MICs of 0.5 to 4 μg/ml for most members of the family Enterobacteriaceae, owing to inhibition of PBP2; moreover, it acts synergistically with PBP3-active β-lactams independently of β-lactamase inhibition, via an “enhancer effect.” Enterobacteriaceae mutants stably resistant to 16 μg/ml OP0595 were selected on agar at frequencies of approximately 10⁻⁷. Unsurprisingly, OP0595 continued to potentiate substrate β-lactams against mutants derived from Enterobacteriaceae with OP0595-inhibited class A and C β-lactamases. Weaker potentiation of partners, especially aztreonam, cefepime, and piperacillin—less so meropenem—remained frequent for OP0595-resistant Enterobacteriaceae mutants lacking β-lactamases or with OP0595-resistant metallo-β-lactamases (MBLs), indicating that the enhancer effect is substantially retained even when antibiotic activity is lost.     

Characterization of the New AmpC β-Lactamase FOX-8 Reveals a Single Mutation,Phe313Leu,Located in the R2 Loop That Affects Ceftazidime Hydrolysis

A novel class C β-lactamase (FOX-8) was isolated from a clinical strain of Escherichia coli. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) compared to FOX-3. Isogenic E. coli strains carrying FOX-8 showed an 8-fold reduction in resistance to ceftazidime relative to FOX-3. In a kinetic analysis, FOX-8 displayed a 33-fold reduction in k_cat/K_m for ceftazidime compared to FOX-3. In the FOX family of β-lactamases, the Phe313 residue located in the R2 loop affects ceftazidime hydrolysis and alters the phenotype of E. coli strains carrying this variant.     

Enzymatic and Immunological Characterization of a New Cephalosporinase from Enterobacter aerogenes

A hospital strain of Enterobacter aerogenes (MULB 250) isolated from a urinary tract infection was found to be cephalosporin and ampicillin resistant and carbenicillin susceptible. The β-lactamase produced by this strain was extracted and purified by means of affinity chromatography, using a cephalosporin C-bound Sepharose 4B column. The purified enzyme was tested for hydrolysis of penicillin and various cephalosporins. The K_m value is 11.8 μM for benzyl penicillin and 130 μM for cephalosporin C. The isoelectric point of the enzyme is 9.3, and its molecular weight is 29,500 ± 1,000. Rabbit antiserum obtained against this MULB 250 β-lactamase showed no cross-reaction with other penicillinases or cephalosporinases in neutralization tests. Comparisons of results obtained with other β-lactamases, particularly from Enterobacter cloacae P99, indicate that the Enterobacter MULB 250 enzyme presents a typical cephalosporinase profile. As far as we know, this type of enzyme is relatively rare.     

Novel chimeric beta-lactamase CTX-M-64, a hybrid of CTX-M-15-like and CTX-M-14 beta-lactamases, found in a Shigella sonnei strain resistant to various oxyimino-cephalosporins, including ceftazidime

The plasmid-mediated novel β-lactamase CTX-M-64 was first identified in Shigella sonnei strain UIH-1, which exhibited resistance to cefotaxime (MIC, 1,024 μg/ml) and ceftazidime (MIC, 32 μg/ml). The amino acid sequence of CTX-M-64 showed a chimeric structure of a CTX-M-15-like β-lactamase (N- and C-terminal moieties) and a CTX-M-14-like β-lactamase (central portion, amino acids 63 to 226), suggesting that it originated by homologous recombination between the corresponding genes. The introduction of a recombinant plasmid carrying bla_CTX-M-64 conferred resistance to cefotaxime in Escherichia coli, and the activities of cefotaxime and ceftazidime were restored in the presence of clavulanic acid. Of note, CTX-M-64 production could also confer consistent resistance to ceftazidime, which differs from the majority of CTX-M-type enzymes, which poorly hydrolyze ceftazidime. These results were consistent with the kinetic parameters determined with the purified CTX-M-64 enzyme. The bla_CTX-M-64 gene was flanked upstream by an ISEcp1 sequence and downstream by an orf477 sequence. The sequence of the 45-bp spacer region between the right inverted repeat (IRR) of ISEcp1 and bla_CTX-M-64 was exactly identical to that of ISEcp1-bla_{CTX-M-15-like}. Moreover, the presence of a putative IRR of ISEcp1 at the right end of truncated orf477 is indicative of an ISEcp1-mediated transposition event in the bla_CTX-M-64 gene. The emergence of CTX-M-64 by probable homologous recombination would suggest the natural potential of an alternative mechanism for the diversification of CTX-M-type β-lactamases.     

In Vitro Activity of Aztreonam-Avibactam against a Global Collection of Gram-Negative Pathogens from 2012 and 2013

The combination of aztreonam plus avibactam is being developed for use in infections caused by metallo-β-lactamase-producing Enterobacteriaceae strains that also produce serine β-lactamases. The in vitro activities of aztreonam-avibactam and comparator antimicrobials were determined against year 2012 and 2013 clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii using the broth microdilution methodology recommended by the Clinical and Laboratory Standards Institute (CLSI). A total of 28,501 unique clinical isolates were obtained from patients in 190 medical centers within 39 countries. MIC₉₀ values of aztreonam and aztreonam-avibactam against all collected isolates of Enterobacteriaceae (n = 23,516) were 64 and 0.12 μg/ml, respectively, with 76.2% of the isolates inhibited by ≤4 μg/ml of aztreonam (the CLSI breakpoint) and 99.9% of the isolates inhibited by ≤4 μg/ml of aztreonam-avibactam using a fixed concentration of 4 μg/ml of avibactam. The MIC₉₀ was 32 μg/ml for both aztreonam and aztreonam-avibactam against P. aeruginosa (n = 3,766). Aztreonam alone or in combination with avibactam had no in vitro activity against isolates of A. baumannii. PCR and sequencing were used to characterize 5,076 isolates for β-lactamase genes. Aztreonam was not active against most Enterobacteriaceae isolates producing class A or class C enzymes alone or in combination with class B metallo-β-lactamases. In contrast, >99% of Enterobacteriaceae isolates producing all observed Ambler classes of β-lactamase enzymes were inhibited by ≤4 μg/ml aztreonam in combination with avibactam, including isolates that produced IMP-, VIM-, and NDM-type metallo-β-lactamases in combination with multiple serine β-lactamases.     

Cloning and Sequencing of the Gene Encoding Toho-2, a Class A β-Lactamase Preferentially Inhibited by Tazobactam

Escherichia coli TUM1083, which is resistant to ampicillin, carbenicillin, cephaloridine, cephalothin, piperacillin, cefuzonam, and aztreonam while being sensitive to cefoxitin, moxalactam, cefmetazole, ceftazidime, and imipenem, was isolated from the urine of a patient treated with β-lactam antibiotics. The β-lactamase (Toho-2) purified from the bacteria hydrolyzed β-lactam antibiotics such as penicillin G, carbenicillin, cephaloridine, cefoxitin, cefotaxime, ceftazidime, and aztreonam and especially had increased relative hydrolysis rates for cephalothin, cephaloridine, cefotaxime, and ceftizoxime. Different from other extended-spectrum β-lactamases, Toho-2 was inhibited 16-fold better by the β-lactamase inhibitor tazobactam than by clavulanic acid. Resistance to β-lactams was transferred by conjugation from E. coli TUM1083 to E. coli ML4909, and the transferred plasmid was about 54.4 kbp, belonging to the incompatibility group IncFII. The cefotaxime resistance gene for Toho-2 was subcloned from the 54.4-kbp plasmid. The sequence of the gene was determined, and the open reading frame of the gene was found to consist of 981 bases. The nucleotide sequence of the gene (DDBJ accession no. {"type":"entrez-nucleotide","attrs":{"text":"D89862","term_id":"3142148","term_text":"D89862"}}D89862) designated as bla_toho was found to have 76.3% identity to class A β-lactamase CTX-M-2 and 76.2% identity to Toho-1. It has 55.9% identity to SHV-1 β-lactamase and 47.5% identity to TEM-1 β-lactamase. Therefore, the newly isolated β-lactamase designated as Toho-2 produced by E. coli TUM1083 is categorized as an enzyme similar to Toho-1 group β-lactamases rather than to mutants of TEM or SHV enzymes. According to the amino acid sequence deduced from the DNA sequence, the precursor consisted of 327 amino acid residues. Comparison of Toho-2 with other β-lactamase (non-Toho-1 group) suggests that the substitutions of threonine for Arg-244 and arginine for Asn-276 are important for the extension of the substrate specificity.β-Lactam antibiotics are widely used as front line agents in the clinical field. In the early 1980s, expanded-spectrum β-lactams, with stability for β-lactamase and good activity against gram-negative bacteria, were first used in the clinical setting. Not long after the beginning of wide use of the expanded-spectrum β-lactams, extended-spectrum β-lactamases were isolated in Europe and the United States and now have become a serious problem in the clinical field (29). In the late 1980s and early 1990s, those enzymes hydrolyzing the expanded-spectrum β-lactams were generally derived from TEM- or SHV-type β-lactamases through several mutations (6, 24). The mutations of Glu-104, Arg-164, and Glu-240 have been suggested to be important for the spectrum expansion (16, 24). In more recent years, non-TEM- or non-SHV-type β-lactamases such as Toho-1 (13), CTX-M-2 (5), and MEN-1 (3) have been identified. Those β-lactamases have high homology to the chromosomally encoded β-lactamase of Proteus vulgaris or Klebsiella oxytoca (2, 9, 23). In most cases, the β-lactamase-producing organisms show resistance to expanded-spectrum β-lactams such as cefotaxime and ceftazidime (6, 24). On the other hand, they are susceptible to carbapenems such as imipenem (6, 24). The main characteristic of those class A β-lactamases, except TEM-30 to TEM-40, is that they are sensitive to β-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam (6). The reaction mechanism and the amino acid residues associated with the spectrum expansion of β-lactamases are still under investigation. Ishii et al. (13) proposed that mutations at positions 244 and 276 are important for the substrate extension after performing sequence alignment of Toho-1 and other β-lactamases.The expanded-spectrum β-lactam-resistant strains isolated from several hospitals were surveyed and collected. We investigated those strains by enzymological and molecular biological methods and focused upon Escherichia coli TUM1083, a cefotaxime-resistant clinical isolate.In this report, we discuss a correlation between the mutation and the substrate specificity of the β-lactamase from E. coli TUM1083 based on the sequence alignment and a three-dimensional structure of a related β-lactamase of Bacillus licheniformis (17).